Nitric Oxide (NO) Endows Arylamine-Containing Block Copolymers with Unique Photoresponsive and Switchable LCST Properties

Article abstract of DOI:10.1021/acs.macromol.6b00054

The fabrication of materials that are responsive to endogenous gasotransmitter molecules (i.e., nitric oxide, hydrogen sulfide, and carbon monoxide) has emerged as an area of increasing research interest. In the case of nitric oxide (NO), o-phenylenediami

Full text of DOI:10.1021/acs.macromol.6b00054

Article
pubs.acs.org/Macromolecules
Nitric Oxide (NO) Endows Arylamine-Containing Block Copolymers
with Unique Photoresponsive and Switchable LCST Properties
Jinming Hu,†,‡ Michael R. Whittaker,‡ John F. Quinn,*,‡ and Thomas P. Davis*,‡,§
†CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and
Technology of China, Hefei, Anhui 230026, China
‡ARC Centre of Excellence in Convergent Bio-Nano Science & Technology, Monash Institute of Pharmaceutical Sciences, Monash
University, Parkville, VIC 3052, Australia
§Department of Chemistry, University of Warwick, Coventry ULCV4 7AL, U.K.
S
* Supporting Information
ABSTRACT: The fabrication of materials that are responsive
to endogenous gasotransmitter molecules (i.e., nitric oxide,
hydrogen sulfide, and carbon monoxide) has emerged as an
area of increasing research interest. In the case of nitric oxide
(NO), o-phenylenediamine derivatives have traditionally been
employed due to their ability to react with NO in the presence
of oxygen (O2) with the formation of benzotriazole residues.
Herein, we report the synthesis of a novel NO-responsive
polymer containing aromatic primary amine groups derived
from p-phenylenediamine groups (i.e., isomers of o-phenylene-
diamine). A new NO-responsive monomer, N-(4-amino-
phenyl)methacrylamide (p-NAPMA), was first synthesized via the amidation of one of the primary amine groups in the p-
phenylenediamine with methacrylic anhydride. Notably, the p-NAPMA monomer can efficiently react with NO in aqueous
solution in the presence of O2 with the generation of phenyldiazonium groups rather than benzotriazole moieties. While the
resultant phenyldiazonium residues were relatively stable in aqueous solution, they were highly sensitive to UV irradiation (i.e.,
λmax = 365 nm) which gave the formation of phenol derivatives. After incorporation into a thermoresponsive block copolymer
using reversible addition−fragmentation chain transfer (RAFT) polymerization, the resulting diblock copolymer, poly(ethylene
glycol)-b-(N-isopropylacrylamide-co-p-NAPMA) (PEG-b-P(NIPAM-co-p-NAPMA)), was rendered with unique NO- and UV-
responsive characteristics. Specifically, the NO-triggered transformation of p-NAPMA moieties into phenyldiazonium residues
dramatically elevated the lower critical solution temperature (LCST) of the block copolymer due to increased water solubility of
phenyldiazonium residues at neutral pH (i.e., pH 7.4). Further, subsequent UV irradiation significantly decreased the LCST due
to the formation of relatively hydrophobic phenol derivatives from the hydrophilic phenyldiazonium intermediate. These results
demonstrate, for the first time, that NO-responsive polymers can be synthesized without the necessity of incorporating o-
phenylenediamine groups and that a further solubility switch can be stimulated by irradiation with ultraviolet light.
lower than 1 μM,26 has been identified as one of the most
important endogenous transmitters in biological systems,
INTRODUCTION
■
The preparation of stimuli-responsive materials has been an
serving as a broad-spectrum signaling molecule throughout
area of increasing research focus in recent years due to their
the nervous and immune systems.27−29 The physiological
potential application in a number of fields.1−5 In addition to
importance of NO has spurred the invention of robust
strategies to probe NO in situ precisely and efficiently, leading
to the successful development of a variety of fluorescent probes
materials that exploit conventional stimuli such as changes in
pH value,6 temperature,7 redox conditions,8,9 or incident
light,10,11 or the presence of certain enzymes12 or electro-
magnetic fields,13,14 recent investigations have explored gaseous
that are highly sensitive to NO over other biologically derived
reactive oxygen species (ROS).30−37 Of these probes, the most
stimuli such as carbon dioxide (CO2)15−18 and oxygen
(O2).19,20 By taking advantage of chemical and/or physical
interactions between these gaseous stimuli and specific
functional groups, a variety of CO2- and O2-responsive
materials have been developed. Such materials have potential
popular design strategy has been to incorporate o-phenylene-
diamine moieties, which can react with NO in the presence of
O2 to yield benzotriazole derivatives and therefore turn on a
fluorescence emission due to a suppressed photoinduced
applications in controlled release, oil/water separation, and
water-solubility regulation.21−25
Received: January 9, 2016
Revised: March 3, 2016
Nitric oxide (NO), a diatomic, lipophilic free radical
synthesized in the body with a typical concentration range
© XXXX American Chemical Society
A
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Scheme 1. Schematic Representation of the Chemical Structure Transitions of Thermoresponsive PEG-b-P(NIPAM-co-p-
a
NAPMA) Diblock Copolymer in the Presence of NO Followed by UV Irradiation (λmax = 365 nm)
a
The aniline residues are transformed into phenyldiazonium when subjected to NO purging, these moieties being further converted to phenol
derivatives when subjected to UV irradiation.
Scheme 2. (a) Consecutive Transition of p-NAPMA Monomer in the Presence of NO and Subsequent UV Irradiation and (b)
Synthetic Route Employed for the Preparation of PEG-b-P(NIPAM-co-p-NAPMA) Diblock Copolymer via RAFT
Polymerization
electron transfer (PET) mechanism. Inspired by this intricate
chemistry, we have developed a category of NO-responsive
polymers and conducted preliminary investigations into their
potential application in NO-triggered self-assembly and anion
sensing.38−41 However, in comparison with the substantial
advances made in the development of NO probes based on
diverse fluorophores, the exploration of moieties that are highly
sensitive to NO stimulus is far less common. Indeed, the
identification of novel NO-reactive substances would not only
advance the field of NO sensing but also could enable better
understanding of the biological functions of NO.32,34
It is important to note that the reaction between NO and an
o-phenylenediamine derivative yields the benzotriazole via an
intramolecular nucleophilic displacement on the diazohydr-
oxide by the neighboring amino group.42 As such, the reaction
between o-phenylenediamine and NO initially involves only
one primary amine group with the formation of a phenyl-
diazonium intermediate. We thus hypothesized that a category
of novel NO-responsive materials might conceivably be derived
from aromatic compounds with only a single primary group.
Interestingly, there were a few early studies regarding the
reaction of aniline toward NO, and these reactions were
reported to proceed via a deamination mechanism in organic
solvents.43−45 Recently, the NO-induced deamination reaction
has also been performed in an aqueous milieu, and this
chemistry has been successfully employed to construct novel
NO probes using a fluorescein-based fluorophore.46
Herein, we report the fabrication of a new NO-responsive
material based on an aniline moiety which exhibits a unique
NO-responsive behavior in purely aqueous medium and which
does not proceed via the widely suggested deamination route.
Starting from p-phenylenediamine (an isomer of o-phenylene-
diamine), one of the primary amine groups was functionalized
with a polymerizable double bond by reaction with methacrylic
anhydride, yielding the target monomer N-(4-aminophenyl)-
methacrylamide (p-NAPMA) which contains an aromatic
primary amine. The p-NAPMA monomer was then incorpo-
rated into a polymeric system via reversible addition−
fragmentation chain transfer (RAFT) copolymerization with
N-isopropylacrylamide (NIPAM) using a poly(ethylene glycol)
B
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Figure 1. 1H NMR spectra recorded in D2O for (a) p-NAPMA monomer at pH 1, (b) p-NAPMA after reaction with NO (p-NDPMA), and (c) p-
NAPMA after reaction with NO followed by irradiation with UV 365 nm (p-NHPMA).
thioylthio)pentanoic acid using a similar protocol as reported
previously.47,48 NO was prepared according to a literature procedure,
and the concentration of NO solution was quantified by Griess
reagent.46
Synthesis. The preparation of N-(4-aminophenyl)methacrylamide
(p-NAPMA) monomer and subsequent chemical transitions in the
presence of NO and under UV irradiation are shown in Scheme 2a.
The synthesis of PEG-b-P(NIPAM-co-p-NAPMA) via RAFT polymer-
ization using a PEG45-based macromolecular RAFT agent is shown in
Scheme 2b.
(PEG)-based macromolecular RAFT agent, thus affording a
thermoresponsive diblock copolymer, PEG-b-P(NIPAM-co-p-
NAPMA). The presence of p-NAPMA moieties in the polymer
chain imparted the resulting block copolymer with distinct NO-
responsive behavior. Upon exposure to NO in aqueous
solution, the primary amine residues were efficiently trans-
formed into phenyldiazonium derivative (abbreviated to p-
NDPMA) which had long-term stability at room temperature.
However, under UV irradiation (λmax = 365 nm), the
phenyldiazonium moieties were readily converted to phenol
derivatives (abbreviated to as p-NHPMA) with the liberation of
nitrogen. Importantly, the NO-induced formation of phenyl-
diazonium residues followed by the generation of phenol
moieties under UV light irradiation dramatically switched the
lower critical solution temperature (LCST) of the block
copolymers, leading to increased and decreased LCST,
respectively (compared to the intact block copolymer without
NO treatment) (see Scheme 1).
Synthesis of N-(4-Aminophenyl)methacrylamide (p-NAPMA,
Scheme 2a). The preparation of p-NAPMA monomer was conducted
via the amidation reaction between p-phenylenediamine and
methacrylic anhydride. In a typical experiment, p-phenylenediamine
(5.4 g, 50 mmol) was dissolved in pyridine (150 mL), and methacrylic
anhydride (7.7 g, 50 mmol) was added into the solution dropwise via a
dropping funnel. After the addition, the reaction was stirred at room
temperature overnight. The insoluble salt was then removed via
vacuum filtration, and pyridine was removed under reduced pressure.
The solid residue was washed three times with diethyl ether and
further purified by column chromatography using ethyl acetate/
petroleum ether (v/v = 1:2) as the eluent. The target p-NAPMA
monomer was obtained as a white solid (6.78 g, yield: 77%). 1H NMR
(Figure 1a, D2O, δ, ppm): 7.56−7.40 (2H), 7.36−7.19 (2H), 5.69
(1H), 5.46 (1H), 1.86 (3H). 13C NMR (Figure S1, CDCl3, δ, ppm):
166.44, 143.43, 140.92, 129.12, 122.11, 119.43, 115.41, 18.82.
Treatment of p-NAPMA Monomer with NO To Generate p-
NDPMA Monomer (Scheme 2a). Typically, p-NAPMA monomer
(176 mg, 1 mmol) was dissolved in 20 mL of deionized water, and the
solution pH was adjusted to approximately 1 with hydrochloric acid
(HCl, 37%) to fully dissolve the p-NAPMA monomer. The solution
was then purged with NO gas for 1 h and subsequently stirred at room
temperature for 12 h. The resulting aqueous solution was lyophilized,
EXPERIMENTAL SECTION
■
Materials. p-Aminophenol, p-phenylenediamine, methacrylic anhy-
dride, methacryloyl chloride, sodium nitrite (NaNO2), and poly-
(ethylene glycol) methyl ether (PEG45-OH, Mn = 2 kDa) were
purchased from Sigma-Aldrich and used as received. N-Isopropyl-
acrylamide (NIPAM) was purchased from Sigma-Aldrich and purified
by crystallization from a mixture of toluene and petroleum ether (v/v
= 1:3) twice prior to use. Sulfuric acid (H2SO4, 98%) was obtained
from RCI Labscan Ltd. All solvents were purchased from EMSURE
and used as received unless otherwise noted. PEG45-based macro-
molecular RAFT agent was synthesized through the esterification
reaction between PEG45-OH and 4-cyano-4-(phenylcarbono-
1
and the resultant solid was collected without further purification. H
C
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NMR (Figure 1b, D2O, δ, ppm): 8.44−8.37 (2H), 8.06−7.96 (2H),
5.84 (1H), 5.67 (1H), 1.93 (3H).
bead-size guard column (50 × 7.8 mm) followed by three KF-805L
columns (300 × 8 mm, bead size: 10 μm, pore size maximum: 5000
Å), a SPD-20A ultraviolet detector, and an RID-10A differential
refractive index detector. The temperature of columns was maintained
at 40 °C using a CTO-20A oven, and the flow rate was kept at 1 mL/
min using a LC-10AT pump. A molecular weight calibration curve was
produced using commercial narrow molecular weight distribution
polystyrene standards with molecular weights ranging from 500 to 106
g/mol. Polymer solutions at 2−3 mg/mL were prepared in the eluent
and filtered through 0.45 μm filters prior to injection.
UV−Vis Spectrophotometry. All UV−vis spectra were acquired on
a Shimadzu UV-3600 UV−vis−NIR spectrophotometer. Quartz
cuvettes of 10 mm path length were employed for all the
measurements. For the turbidity measurements, the optical trans-
mittance of the aqueous PEG-b-P(NIPAM-co-p-NAPMA) solutions
(2.0 g/L in all cases) before and after reaction with NO and further
treatment with UV irradiation were acquired on the same
spectrophotometer equipped with a Shimadzu temperature controller
and Tm Analysis software. The recorded wavelength was set at 500 nm
with a heating rate of 0.5 °C/min. The LCST was defined as the
temperature leading to a 50% decrease in transmittance.39
Treatment of p-NDPMA Monomer with N,N-Dimethylaniline
(Figure S2). p-NDPMA monomer (205 mg, 1 mmol) was dissolved in
5 mL of N,N-dimethylaniline, and 15 mL of deionized water was then
added. The mixture was stirred at room temperature overnight, and
the successful transformation of p-NDPMA monomer into azoben-
zene-containing derivatives was evidenced by the formation of an
undissolved yellowish solid. After removing the water and excess N,N-
dimethylaniline by filtration, the solid was washed with an excess of
diethyl ether and was dried in a vacuum oven at room temperature,
1
yielding a yellowish solid. H NMR (Figure S2a, CDCl3, δ, ppm):
7.94−7.83 (4H), 7.75−7.67 (2H), 7.67−7.55 (1H), 6.79 (2H), 5.85
(1H), 5.52 (1H), 3.11 (6H), 2.11 (3H). 13C NMR (Figure S2b,
CDCl3, δ, ppm): 166.49, 152.32, 149.67, 143.64, 140.87, 138.88,
124.88, 123.16, 120.09, 120.05, 111.59, 40.34, 18.78.
Exposure of p-NDPMA Monomer to UV Irradiation (λmax = 365
nm) To Generate p-NHPMA Monomer (Scheme 2a). The p-NDPMA
monomer (41 mg, 0.2 mmol) was dissolved in 5 mL of deionized
water, and the resulting solution was irradiated with UV light (λmax
=
365 nm) in a 10 mL glass vial for a predetermined time. After
irradiation, the resulting solid was recovered by lyophilization and was
Fluorescence Spectroscopy. All fluorescence spectra were acquired
on a Shimadzu RF-5301 fluorescence spectrometer. The excitation
wavelength was set at 495 nm and the slit widths for both excitation
and emission were 3 nm.
Attenuated Total Reflectance−Fourier Transform Infrared Spec-
troscopy (ATR-FTIR). ATR-FTIR measurements were performed using
a Shimadzu IRTracer-100 Fourier transform infrared spectrometer by
averaging 128 scans with a resolution of 4 cm−1.
1
analyzed by NMR. H NMR (Figure 1c, D2O, δ, ppm): 7.24 (2H),
6.81 (2H), 5.69 (1H), 5.43 (1H), 1.89 (3H).
Synthesis of N-(4-Hydroxyphenyl)methacrylamide (p-NHPMA,
Figure S7). p-Aminophenol (5.46 g, 50 mmol) and K2CO3 (13.8 g,
100 mmol) were dissolved in 150 mL of dry THF (dried over
molecular sieves), and the solution was cooled to 0 °C in an ice−water
bath for 10 min. To the cooled 4-aminophenol/K2CO3 solution was
added dropwise a THF solution (20 mL) of methacryloyl chloride
(4.84 mL, 50 mmol). After the addition, the reaction was gradually
warmed up to room temperature and stirred overnight at room
temperature. After removing insoluble salt by filtration, THF was
removed under vacuum, and the crude product was purified by column
chromatography using ethyl acetate/petroleum ether (v/v = 1:2) as
the eluent. The target p-NHPMA monomer was obtained as a white
RESULTS AND DISCUSSION
■
NO-responsive monomer containing a single primary amine, p-
NAPMA, was first synthesized via the monoamidation reaction
between p-phenylenediamine and methacrylic anhydride in a
molar ratio of 1:1. The chemical structure of p-NAPMA
monomer was confirmed by both 1H and 13C NMR
spectroscopy analysis (Figure 1a and Figure S1). Subsequently,
the reactivity of p-NAPMA monomer toward NO was
evaluated by monitoring the UV−vis absorption changes of
p-NAPMA in the presence of varying amounts of NO. Initially,
the p-NAPMA monomer in aqueous solution in its non-
protonated form possessed a maximum absorption peak at 273
nm. Upon progressive addition of NO to the solution, the
absorption peak initially blue-shifted to 261 nm in the presence
of 0.125 equiv of NO (relative to p-NAPMA monomer). It
should be noted that this initial hypochromic shift was ascribed
to the lowering of the solution pH and the resulting
protonation of p-NAPMA moieties, since NO is easily oxidized
to nitric acid and nitrous acid in air.40 Subsequently, the
absorption intensity at 261 nm gradually dropped, while a new
absorption peak centered at 340 nm appeared and steadily
intensified upon increasing the NO concentration in the range
of 0.25−1.5 equiv (relative to p-NAPMA moieties) (Figure
2a,b). The absorption intensities stabilized when the NO
concentration was greater than 1.5 equiv of p-NAPMA
monomer, suggesting a high reactivity of p-NAPMA toward
NO in aqueous solution. This high reactivity was comparable to
that observed for the N-(2-aminophenyl)methacrylamide
counterpart, which possess the typical o-phenylenediamine
motif frequently used in NO reactive materials.38 However, for
the N-(2-aminophenyl)methacrylamide monomer, only a slight
shift in the UV−vis absorbance spectra (from 288 to 302 nm)
was observed upon NO addition as a result of the formation of
amide-functionalized benzotriazole moieties.38 Therefore, this
unique variation in the UV−vis absorption not only revealed a
high reactivity of p-NAPMA monomer toward NO but also
1
crystal (3.01 g, yield: 34%). H NMR (Figure S7b, D2O, δ, ppm):
7.31−7.08 (2H), 6.91−6.73 (2H), 5.73 (1H), 5.47 (1H), 1.93 (3H).
Synthesis of Thermoresponsive PEG-b-P(NIPAM-co-p-NAPMA)
Diblock Copolymer (Scheme 1b). PEG-b-P(NIPAM-co-p-NAPMA)
diblock copolymer was synthesized via RAFT copolymerization of
NIPAM and p-NAPMA monomers using a PEG-based macro-
molecular RAFT agent. Typically, PEG45-based macromolecular
RAFT agent (22.6 mg, 0.1 mmol), NIPAM (905 mg, 8 mmol), p-
NAPMA (352 mg, 2 mmol), AIBN (2 mg, 12 μmol), and DMF (3
mL) were charged into a 20 mL polymerization vial with a rubber
septum. The mixture was deoxygenated via purging with nitrogen for
30 min. The polymerization vial was then transferred to a hot plate set
at 70 °C to commence the polymerization. After 16 h polymerization,
the reaction was quenched into an ice−water bath, and the obtained
diblock copolymer was purified by precipitating three times into an
excess amount of diethyl ether. After drying in a vacuum oven
overnight, the diblock copolymer was obtained as a pale red solid
(0.54 g). The molecular composition of the diblock copolymer was
determined to be PEG45-b-P(NIPAM0.74-co-p-NAPMA0.26
)
as
30
determined by analysis of the 1H NMR spectrum (Figure S10a).
The number-average molecular weight (Mn) and polydispersity (Mw/
Mn) of the diblock copolymer were determined to be 12.2 kDa and
1.26, respectively, based on GPC measurement using N,N-dimethyl-
acetamide (DMAc) as the eluent (Figure 4C) and PS calibration
standards.
Characterization. Nuclear Magnetic Resonance (NMR) Spectra.
1H and 13C NMR spectra were recorded on a Bruker AC400F (400
MHz) spectrometer. Deuterated chloroform (CDCl3) and deuterium
oxide (D2O) were used as the solvents, depending on the particular
substances being analyzed.
Gel Permeation Chromatography (GPC). GPC analyses of
polymer samples were performed in N,N-dimethylacetamide (DMAc
with 0.03% w/v LiBr using a Shimadzu modular system comprising a
DGU-12A degasser, an SIL-10AD automatic injector, and a 5.0 μm
D
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vinylic protons. This result suggested that the reaction between
p-NAPMA monomer and NO was highly selective and was
compatible with other functional groups (e.g., polymerizable
double bonds). Although we could safely conclude that the p-
NAPMA monomer, after reaction with NO, afforded a new
compound with a para-substituted phenyl ring structure,
unravelling the exact chemical structure was not straightforward
1
using only the H NMR result.
It should be noted that in previous studies the reaction
between aniline derivatives and NO has been performed in
various organic solvents such as tetrahydrofuran and chloro-
form. In these studies the result of NO treatment was the
formation of deaminated products, likely through an aryl radical
mechanism. In these cases an unstable intermediate product (a
phenyldiazonium salt) was suggested, which can be further
transformed into an aryl radical in the presence of NO, after
which proton abstraction from the solvents was suggested to
give the deaminated product.44,45 However, this deamination
mechanism cannot be reconciled with the current experimental
results because the 1H NMR spectrum clearly demonstrated the
generation of para-substituted product (Figure 1b). This
discrepancy could be a result of the differing solvents used.45
We postulated that the aqueous milieu may impede the
deamination reaction of p-NAPMA monomer in the presence
of NO. However, after treating the NO-reacted product
recovered from water with N,N-dimethylaniline, a yellowish
solid was obtained and its chemical structure was identified by
1H and 13C NMR spectroscopy (Figure S2).49 The formation
of azo-containing derivatives revealed that the product of
reacting p-NAPMA with NO was a phenyldiazonium derivative,
which was in accord with the previously assumed intermediate
product achieved in organic solvents.45 On the basis of the
above results, we concluded that the p-NAPMA monomer
comprising an aromatic primary amine could efficiently react
with NO to generate a phenyldiazonium product in aqueous
solution (Scheme 2a). Although the phenyldiazonium product
Figure 2. (a, c) Normalized UV−vis absorbance spectra and (b, d)
relative absorption intensity changes of (a, b) p-NAPMA monomer
(40 μM) in the presence of varying amounts of NO and (c, d) p-
NAPMA monomer after NO treatment (p-NDPMA) under UV 365
nm irradiation. Note: UV−vis spectra were recorded immediately after
the NO introduction without additional standing time.
implied a distinct reaction mechanism between p-NAPMA and
NO compared to that of the N-(2-aminophenyl)-
methacrylamide analogue with an o-phenylenediamine motif.38
To identify the resulting product obtained by treating p-
NAPMA monomer with NO and elucidate the underlying
reaction mechanism, the product was recovered from aqueous
1
solution through lyophilization and was analyzed by H NMR
spectroscopy (Figure 1b). In comparison with the initial p-
NAPMA monomer, the signals originating from the phenyl ring
shifted to downfield after NO treatment, but the reaction did
not significantly affect the chemical shifts corresponding to the
1
Figure 3. H NMR spectra recorded in D2O for p-NDPMA monomer (10 mg/mL) under UV 365 nm irradiation for varying times (0−70 min).
E
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was reported to be labile in organic solvent,44,45 we found that
it was relatively stable in aqueous solution. Specifically, there
were no noticeable changes in the absorbance spectra after 15 h
standing in PBS buffer solution at pH 7.4 at room temperature
(Figure S3), which was much longer than that needed to obtain
the deaminated product in organic solvents.45 It is worth noting
that this characteristic is quite unique as the phenyldiazonium
salts are typically unstable at room temperature with the
liberation of nitrogen.
In spite of the unexpected stability at room temperature, we
found the as-synthesized phenyldiazonium monomer was
highly sensitive to UV irradiation. Upon UV irradiation with
a hand-held UV light (λmax = 365 nm), the UV−vis absorbance
spectra underwent a dramatic change within only 3 min.
Specifically, the absorbance assigned to the phenyldiazonium
salt (at 339 nm) gradually attenuated, while a new absorption
peak at 263 nm steadily increased (Figure 2c,d). Moreover,
there were three isosbestic points observed at 220, 240, and 288
nm for the spectra measured during the UV irradiation (Figure
2c). The UV light-responsive feature was further confirmed by
examining the time-dependent evolution of the 1H NMR
spectrum (Figure 3). Upon constant UV irradiation, the signals
due to the protons on the phenyl ring of the phenyldiazonium
gradually shifted to higher field over 70 min. The
corresponding conversion of the phenyldiazonium could be
quantified from the NMR analysis, and this data is shown in
Figure S4. This UV-induced conversion of phenyldiazonium
be nitrogen (N2) rather than NO under UV irradiation. As a
result, the final product subjected to UV irradiation was
postulated to be N-(4-hydroxyphenyl)methacrylamide (p-
NHPMA) (Scheme 2a).
To verify this hypothesis, p-NHPMA was prepared through
the amidation reaction between p-aminophenol and meth-
1
acryloyl chloride, and the H NMR spectrum of the product
formed is shown in Figure S7b. The spectrum obtained
concurred quite well with the 1H NMR spectrum of the
phenyldiazonium derivative subjected to UV irradiation.
Further, the absorbance spectrum of as-synthesized p-
NHPMA was identical to that of the product obtained from
the phenyldiazonium salt after UV irradiation (Figure S8).
These results confirmed that the final photodegraded product
yielded from the phenyldiazonium derivative after UV light
irradiation was p-NHPMA (Scheme 2a). Further, the
consecutive transition of p-NAPMA monomer in the presence
of NO and UV light irradiation to form phenyldiazonium (p-
NDPMA) and phenol (p-NHPMA) derivatives was further
confirmed by ATR-FTIR spectroscopy (Figure S9). It should
be noted that although we did not visually discern the changes
in water solubility after the formation of the phenyldiazonium
derivative, the UV light irradiation of the phenyldiazonium
derivative led to significantly decreased water miscibility, which
was evident by the appearance of insoluble solid that was clearly
visible to the naked eye. We thus inferred that the cascade
reaction of p-NAPMA monomer caused by NO and UV light
irradiation could lead to a drastic transition in the water
solubility if the monomer was incorporated into a thermores-
ponsive polymeric system. In other words, the temperature-
induced volume phase transition (VPT) of p-NAPMA
containing thermoresponsive polymers could potentially be
manipulated by both NO gas and UV irradiation.
derivatives followed first-order kinetics, and the half-life (τ1/2
)
was determined to be approximately 17.3 min (Figure S4b).
Interestingly, there were no changes in the signals associated
with the vinylic protons, which were similar to those observed
after NO addition. That said, it is acknowledged that the double
bonds are potentially susceptible to UV irradiation and UV-
mediated coupling reactions could occur.50 The current very
clean result can be attributed to the very low UV irradiation
dosage applied (hand-held UV lamp; ∼1.5 mW/cm2) and
indicates that the phenyldiazonium is highly sensitive toward
UV irradiation. It is worth noting that the UV irradiation time
As a proof-of-principle, p-NAPMA monomer was incorpo-
rated into a thermoresponsive diblock copolymer, PEG-b-
P(NIPAM-co-p-NAPMA), through RAFT polymerization using
a PEG-based macromolecular RAFT agent (Scheme 2b).
Importantly, the incorporation into a polymeric system did
not compromise the NO-responsive behavior of p-NAPMA
1
applied for the H NMR analysis was significantly longer than
1
for the UV−vis absorption measurement, and this variation was
moieties as evidenced by H NMR spectroscopy. As shown in
unsurprising due to the different monomer concentrations used
Figure S10, the signals corresponding to the phenyl ring
protons shifted to downfield after NO treatment, presumably
due to the formation of phenyldiazonium moieties (Figure
S10b), as was observed for the p-NAPMA monomer (Figure
1). Interestingly, even with an extra 15 h standing in aqueous
solution, there were no evident changes in 1H NMR spectra of
PEG-b-P(NIPAM-co-p-NAPMA) block copolymer after reac-
tion with NO, suggesting the stability of newly generated
phenyldiazonium moieties within the polymeric chains (Figure
S11). This result concurred quite well with the stability of p-
NAPMA monomer after NO treatment (Figure S3).
Importantly, this result demonstrated that the aniline
derivative-containing monomer can be successfully polymerized
via RAFT polymerization without loss of the NO reactivity.
Further, the resulting diblock copolymer after NO treatment
underwent a comparable UV−vis spectral evolution to the
monomer when exposed to UV irradiation at λmax = 365 nm
(Figure 4A). The absorption corresponding to phenyl-
diazonium moieties at 336 nm gradually decreased, while the
absorption of corresponding to phenol residues (i.e., at 247
nm) steadily increased within 360 s of UV irradiation (Figure
4A,B). The extended irradiation time required for the diblock
copolymer (compared to that of the p-NAPMA small
1
in each experiment (40 μM for UV−vis vs 48.7 mM for H
NMR). Importantly, the initial p-NAPMA monomer with a free
primary amine group (i.e., without NO treatment) was
essentially inert to the UV irradiation. With the same time
period of UV irradiation (i.e., 70 min), no shifts in the 1H NMR
spectrum were evident (Figure S5). Therefore, the emergence
of UV light-responsive behavior for the p-NAPMA monomer
after NO treatment is considered to be a consequence of
forming a phenyldiazonium derivative.
From the 1H NMR spectra (Figures 1c and 3f), it is apparent
that a new product with another para-substituted phenyl ring
structure was achieved after UV irradiation. Interestingly,
bubbles were observed during the UV irradiation, suggesting
that a gas was produced and released from the aqueous solution
due to the UV exposure. To confirm that the gas was not NO,
the fluorescence spectra of a mixture of the phenyldiazonium
and 4,5-diaminofluorescein (DAF-2, a fluorescent probe of
NO)33 was irradiated with the UV lamp. This experiment
revealed no significant fluorescence turn-on under UV light
irradiation (Figure S6), demonstrating that the generated gas
was not NO. Given the chemical structure of the phenyl-
diazonium derivative, we surmised that the generated gas could
F
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Article
hydrophilic PEG block.39,53 However, after reaction with NO,
the LCST was switched to approximately 57 °C due to the
formation of water-soluble phenyldiazonium moieties. This
1
result was in accordance with the H NMR result (Figure
S10b) and the significant bathochromic shift of the maximum
absorbance from 258 to 336 nm in the UV−vis absorption
spectra (Figure S12). Upon UV irradiation, a pronounced drop
in the LCST from 57 to 33 °C was observed as a result of the
formation of relatively hydrophobic phenol residues and
hydrogen bonding interaction between phenol and amide
groups.51,52 This pronounced transition in the LCST was
accompanied by a hypochromatic shift in the maximum
absorption peaks from 336 to 246 nm (Figure 4A and Figure
S12).
CONCLUSIONS
■
In summary, we have successfully prepared a new NO-
responsive monomer starting from p-phenylenediamine rather
than conventional o-phenylenediamine. After converting to a
polymerizable form with methacrylic anhydride, the resulting p-
NAPMA monomer can selectively react with NO with the
formation of a phenyldiazonium derivative instead of a
benzotriazole (as observed with o-phenylenediamine deriva-
tives). Moreover, although the phenyldiazonium residues were
unexpectedly stable in aqueous solution at room temperature,
they were inherently responsive to UV light irradiation, with
the generation of p-NHPMA monomer. This cascade transition
under NO treatment and UV light irradiation was characterized
by a significant shift in the water solubility, thus providing the
opportunity to regulate the VPTs of thermoresponsive
polymers. After incorporating p-NAPMA monomer into a
diblock copolymer (PEG-b-P(NIPAM-co-p-NAPMA)), the
LCST of the copolymer could be regulated by exposure to
NO and subsequent UV light irradiation. This work opens a
new window toward fabricating novel gasotransmitter-respon-
sive materials by exploiting the unique reaction of primary
amines with NO. The exploration of the potential application
of this novel dual-responsive material in a biological milieu is
currently underway.
Figure 4. (A) UV−vis spectra and (B) relative absorbance intensity
changes of 0.1 g/L of PEG-b-P(NIPAM-co-p-NAPMA) in PBS buffer
solution (0.1 mM, pH 7.4) after treatment with NO and UV 365 nm
irradiation. (C) GPC elution traces for (a) PEG45-based macroRAFT
agent and PEG-b-P(NIPAM-co-p-NAPMA) diblock copolymer (b)
before and (c) after reaction with NO and (d) after reaction with NO
followed by UV 365 nm irradiation. (D) Temperature-dependent
transmittance at 500 nm for an aqueous solution of PEG-b-P(NIPAM-
co-p-NAPMA) diblock copolymer (2.0 g/L in PBS buffer, pH 7.4)
before and after reaction with NO and after reaction with NO followed
by UV 365 nm irradiation.
molecule) could be attributed to macromolecular effects
deriving from a relatively high local concentration of p-
NAPMA moieties. Moreover, the blue-shift of the maximum
absorbance associated with the phenol derivatives in the
polymer chain was tentatively attributed to the hydrogen
bonding interactions among pendant phenol and acrylamide
moieties.51,52
1
After NO treatment followed by UV irradiation, the H
NMR spectrum of PEG-b-P(NIPAM-co-p-NAPMA) diblock
copolymer revealed a shift to high-field for the phenyl ring
protons, which is in accord with the result for the p-NAPMA
monomer after UV irradiation (Figure S10c). Despite using the
same concentration (i.e., 20 mg/mL), the signal intensities
corresponding to the protons of the phenyl rings significantly
dropped when subjected to UV irradiation compared to those
observed before UV light irradiation. This is likely a result of
the decreased solubility of the phenol derivatives in aqueous
solution and is consistent with the formation of insoluble solid
after UV irradiation for the p-NAPMA small molecule.
Moreover, the changes in chemical composition in the presence
of NO and after UV irradiation could also be evidenced by the
GPC elution traces (Figure 4C), in which a much boarder
elution trace was achieved for the diblock copolymer after both
NO and UV irradiation.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.macro-
mol.6b00054.
Additional 1H NMR spectra, UV−vis spectra, and
fluorescence spectra (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail Thomas.p.davis@monash.edu (T.P.D).
*E-mail john.f.quinn@monash.edu (J.F.Q.).
Finally, the temperature-induced VPT of the thermores-
ponsive block copolymer was further examined by following
turbidity changes with variation in temperature. Specifically,
prior to NO treatment and UV irradiation, the lower critical
solution temperature (LCST) was determined to be 43 °C (if
the LCST is defined as the temperature corresponding to a 50%
decrease in the transmittance at the wavelength of 500 nm).
The LCST of the current diblock copolymer was higher than
for PNIPAM homopolymers due to the presence of the
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was conducted within the Australian Research
Council Centre of Excellence in Convergent Bio-Nano Science
and Technology (Project CE140100036). T.P.D. gratefully
acknowledges the award of an Australian Laureate Fellowship.
G
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Selective Release, Separation, and Reaction. Angew. Chem., Int. Ed.
2013, 52, 5070−5073.
(24) Yan, Q.; Zhao, Y. Polymeric Microtubules That Breathe: CO2-
Driven Polymer Controlled-Self-Assembly and Shape Transformation.
Angew. Chem., Int. Ed. 2013, 52, 9948−9951.
REFERENCES
■
(1) Hoffman, A. S. Stimuli-Responsive Polymers: Biomedical
Applications and Challenges for Clinical Translation. Adv. Drug
Delivery Rev. 2013, 65, 10−16.
(2) Hu, J. M.; Liu, S. Y. Responsive Polymers for Detection and
Sensing Applications: Current Status and Future Developments.
Macromolecules 2010, 43, 8315−8330.
(3) Stuart, M. A. C.; Huck, W. T. S.; Genzer, J.; Muller, M.; Ober, C.;
Stamm, M.; Sukhorukov, G. B.; Szleifer, I.; Tsukruk, V. V.; Urban, M.;
Winnik, F.; Zauscher, S.; Luzinov, I.; Minko, S. Emerging Applications
of Stimuli-Responsive Polymer Materials. Nat. Mater. 2010, 9, 101−
113.
(4) Roy, D.; Cambre, J. N.; Sumerlin, B. S. Future Perspectives and
Recent Advances in Stimuli-Responsive Materials. Prog. Polym. Sci.
2010, 35, 278−301.
(5) Theato, P.; Sumerlin, B. S.; O’Reilly, R. K.; Epps, T. H. Stimuli
Responsive Materials. Chem. Soc. Rev. 2013, 42, 7055−7056.
(6) Hu, J. M.; Zhang, G. Y.; Ge, Z. S.; Liu, S. Y. Stimuli-Responsive
Tertiary Amine Methacrylate-Based Block Copolymers: Synthesis,
Supramolecular Self-Assembly and Functional Applications. Prog.
Polym. Sci. 2014, 39, 1096−1143.
(7) Schmaljohann, D. Thermo- and pH-Responsive Polymers in
Drug Delivery. Adv. Drug Delivery Rev. 2006, 58, 1655−1670.
(8) Huo, M.; Yuan, J.; Tao, L.; Wei, Y. Redox-Responsive Polymers
for Drug Delivery: from Molecular Design to Applications. Polym.
Chem. 2014, 5, 1519−1528.
(25) Han, D. H.; Tong, X.; Boissiere, O.; Zhao, Y. General Strategy
for Making CO2-Switchable Polymers. ACS Macro Lett. 2012, 1, 57−
61.
(26) Coneski, P. N.; Schoenfisch, M. H. Nitric Oxide Release: Part
III. Measurement and Reporting. Chem. Soc. Rev. 2012, 41, 3753−
3758.
(27) Vasudevan, D.; Thomas, D. D. Insights into the Diverse Effects
of Nitric Oxide on Tumor Biology. Vitam. Horm. 2014, 96, 265−298.
(28) Vitturi, D. A.; Patel, R. P. Current Perspectives and Challenges
in Understanding the Role of Nitrite as an Iintegral Player in Nitric
Oxide Biology and Therapy. Free Radical Biol. Med. 2011, 51, 805−
812.
(29) Wink, D. A.; Mitchell, J. B. Chemical Biology of Nitric Oxide:
Insights into Regulatory, Cytotoxic, and Cytoprotective Mechanisms
of Nitric Oxide. Free Radical Biol. Med. 1998, 25, 434−456.
(30) Yu, H. B.; Xiao, Y.; Jin, L. J. A Lysosome-Targetable and Two-
Photon Fluorescent Probe for Monitoring Endogenous and
Exogenous Nitric Oxide in Living Cells. J. Am. Chem. Soc. 2012,
134, 17486−17489.
(31) Sun, Y. Q.; Liu, J.; Zhang, H. X.; Huo, Y. Y.; Lv, X.; Shi, Y. W.;
Guo, W. A Mitochondria-Targetable Fluorescent Probe for Dual-
Channel NO Imaging Assisted by Intracellular Cysteine and
Glutathione. J. Am. Chem. Soc. 2014, 136, 12520−12523.
(32) Nagano, T.; Yoshimura, T. Bioimaging of Nitric Oxide. Chem.
Rev. 2002, 102, 1235−1269.
(9) Cheng, R.; Feng, F.; Meng, F. H.; Deng, C.; Feijen, J.; Zhong, Z.
Y. Glutathione-Responsive Nano-Vehicles as a Promising Platform for
Targeted Intracellular Drug and Gene Delivery. J. Controlled Release
2011, 152, 2−12.
(10) Katz, J. S.; Burdick, J. A. Light-Responsive Biomaterials:
Development and Applications. Macromol. Biosci. 2010, 10, 339−348.
(11) Zhao, Y. Light-Responsive Block Copolymer Micelles. Macro-
molecules 2012, 45, 3647−3657.
(12) Hu, J. M.; Zhang, G. Q.; Liu, S. Y. Enzyme-Responsive
Polymeric Assemblies, Nanoparticles and Hydrogels. Chem. Soc. Rev.
2012, 41, 5933−5949.
(33) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino,
Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Detection and Imaging of
Nitric Oxide with Novel Fluorescent Indicators: Diaminofluoresceins.
Anal. Chem. 1998, 70, 2446−2453.
(34) Miller, E. W.; Chang, C. J. Fluorescent Probes for Nitric Oxide
and Hydrogen Peroxide in Cell Signaling. Curr. Opin. Chem. Biol.
2007, 11, 620−625.
(35) Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. Highly
Sensitive Fluorescence Probes for Nitric Oxide Based on Boron
Dipyrromethene Chromophore-Rational Design of Potentially Useful
Bioimaging Fluorescence Probe. J. Am. Chem. Soc. 2004, 126, 3357−
3367.
(36) Takakura, H.; Kojirna, R.; Kamiya, M.; Kobayashi, E.; Komatsu,
T.; Ueno, T.; Terai, T.; Hanaoka, K.; Nagano, T.; Urano, Y. New Class
of Bioluminogenic Probe Based on Bioluminescent Enzyme-Induced
Electron Transfer: BioLeT. J. Am. Chem. Soc. 2015, 137, 4010−4013.
(37) Yang, Y. J.; Seidlits, S. K.; Adams, M. M.; Lynch, V. M.; Schmidt,
C. E.; Anslyn, E. V.; Shear, J. B. A Highly Selective Low-Background
Fluorescent Imaging Agent for Nitric Oxide. J. Am. Chem. Soc. 2010,
132, 13114−13116.
(38) Hu, J. M.; Whittaker, M. R.; Duong, H.; Li, Y.; Boyer, C.; Davis,
T. P. Biomimetic Polymers Responsive to a Biological Signaling
Molecule: Nitric Oxide Triggered Reversible Self-assembly of Single
Macromolecular Chains into Nanoparticles. Angew. Chem., Int. Ed.
2014, 53, 7779−7784.
(39) Hu, J. M.; Whittaker, M. R.; Yu, S. H.; Quinn, J. F.; Davis, T. P.
Nitric Oxide (NO) Cleavable Biomimetic Thermoresponsive Double
Hydrophilic Diblock Copolymer with Tunable LCST. Macromolecules
2015, 48, 3817−3824.
(40) Hu, J. M.; Whittaker, M. R.; Li, Y.; Quinn, J. F.; Davis, T. P. The
Use of Endogenous Gaseous Molecules (NO and CO2) to Regulate
the Self-Assembly of a Dual-Responsive Triblock Copolymer. Polym.
Chem. 2015, 6, 2407−2415.
(41) Hu, J. M.; Whittaker, M. R.; Davis, T. P.; Quinn, J. F.
Application of Heterocyclic Polymers in the Ratiometric Spectropho-
tometric Determination of Fluoride. ACS Macro Lett. 2015, 4, 236−
241.
(13) Thevenot, J.; Oliveira, H.; Sandre, O.; Lecommandoux, S.
Magnetic Responsive Polymer Composite Materials. Chem. Soc. Rev.
2013, 42, 7099−7116.
(14) Murdan, S. Electro-Responsive Drug Delivery from Hydrogels.
J. Controlled Release 2003, 92, 1−17.
(15) Lin, S. J.; Theato, P. CO2-Responsive Polymers. Macromol.
Rapid Commun. 2013, 34, 1118−1133.
(16) Yan, Q.; Zhao, Y. Block Copolymer Self-Assembly Controlled
by the “Green” Gas Stimulus of Carbon Dioxide. Chem. Commun.
2014, 50, 11631−11641.
(17) Schattling, P.; Pollmann, I.; Theato, P. Synthesis of CO2-
Responsive Polymers by Post-Polymerization Modification. React.
Funct. Polym. 2014, 75, 16−21.
(18) Zhou, K. J.; Li, J. F.; Lu, Y. J.; Zhang, G. Z.; Xie, Z. W.; Wu, C.
Re-examination of Dynamics of Polyeletrolytes in Salt-Free Dilute
Solutions by Designing and Using a Novel Neutral-Charged-Neutral
Reversible Polymer. Macromolecules 2009, 42, 7146−7154.
(19) Zhang, Q.; Zhu, S. P. Oxygen and Carbon Dioxide Dual
Responsive Nanoaggregates of Fluoro- and Amino-Containing
Copolymer. ACS Macro Lett. 2014, 3, 743−746.
(20) Choi, J. Y.; Kim, J. Y.; Moon, H. J.; Park, M. H.; Jeong, B. CO2-
and O2-Sensitive Fluorophenyl End-Capped Poly(ethylene glycol).
Macromol. Rapid Commun. 2014, 35, 66−70.
(21) Yan, Q.; Zhao, Y. CO2-Stimulated Diversiform Deformations of
Polymer Assemblies. J. Am. Chem. Soc. 2013, 135, 16300−16303.
(22) Che, H. L.; Huo, M.; Peng, L.; Fang, T.; Liu, N.; Feng, L.; Wei,
Y.; Yuan, J. Y. CO2-Responsive Nanofibrous Membranes with
Switchable Oil/Water Wettability. Angew. Chem., Int. Ed. 2015, 54,
8934−8938.
(23) Yan, Q.; Wang, J. B.; Yin, Y. W.; Yuan, J. Y. Breathing
Polymersomes: CO2-Tuning Membrane Permeability for Size-
H
DOI: 10.1021/acs.macromol.6b00054
Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
(42) Uppu, R. M.; Pryor, W. A. Nitrosation of 1,2-Phenylenediamine
by Peroxynitrite/CO2: Evidence for a Free Radical Mechanism. J. Am.
Chem. Soc. 1999, 121, 9738−9739.
(43) Itoh, T.; Matsuya, Y.; Maeta, H.; Miyazaki, M.; Nagata, K.;
Ohsawa, A. Reaction of Secondary and Tertiary Amines with Nitric
Oxide in the Presence of Oxygen. Chem. Pharm. Bull. 1999, 47, 819−
823.
(44) Itoh, T.; Matsuya, Y.; Nagata, K.; Ohsawa, A. Reductive
Deamination of Aromatic Amines with Nitric Oxide (NO).
Tetrahedron Lett. 1996, 37, 4165−4168.
(45) Nagano, T.; Takizawa, H.; Hirobe, M. Reactions of Nitric-Oxide
with Amines in the Presence of Dioxygen. Tetrahedron Lett. 1995, 36,
8239−8242.
(46) Shiue, T. W.; Chen, Y. H.; Wu, C. M.; Singh, G.; Chen, H. Y.;
Hung, C. H.; Liaw, W. F.; Wang, Y. M. Nitric Oxide Turn-on
Fluorescent Probe Based on Deamination of Aromatic Primary
Monoamines. Inorg. Chem. 2012, 51, 5400−5408.
(47) Khanal, A.; Yusa, S.; Nakashima, K. Fabrication of Nano-
aggregates of a Triple Hydrophilic Block Copolymer by Cetyltrime-
thylammonium Chloride Binding. Langmuir 2007, 23, 10511−10517.
(48) Wang, L.; Liu, G. H.; Wang, X. R.; Hu, J. M.; Zhang, G. Y.; Liu,
S. Y. Acid-Disintegratable Polymersomes of pH-Responsive Amphi-
philic Diblock Copolymers for Intracellular Drug Delivery. Macro-
molecules 2015, 48, 7262−7272.
(49) Westheimer, F. H.; Segel, E.; Schramm, R. The Mechanism of
the Oxynitration of Benzene. J. Am. Chem. Soc. 1947, 69, 773−785.
(50) Lendlein, A.; Jiang, H. Y.; Junger, O.; Langer, R. Light-Induced
Shape-Memory Polymers. Nature 2005, 434, 879−882.
(51) Chen, S. C.; Kuo, S. W.; Liao, C. S.; Chang, F. C. Syntheses,
Specific Interactions, and pH-Sensitive Micellization Behavior of Poly
[vinylphenol-b-2-(dimethylamino)ethyl methacrylate] Diblock Co-
polymers. Macromolecules 2008, 41, 8865−8876.
(52) Kuo, S. W.; Tung, P. H.; Chang, F. C. Syntheses and the Study
of Strongly Hydrogen-Bonded Poly(vinylphenol-b-vinylpyridine) Di-
block Copolymer Through Anionic Polymerization. Macromolecules
2006, 39, 9388−9395.
(53) Schild, H. G. Poly-N-Isopropylacrylamide)-Experiment, Theory
and Application. Prog. Polym. Sci. 1992, 17, 163−249.
I
DOI: 10.1021/acs.macromol.6b00054
Macromolecules XXXX, XXX, XXX−XXX