Oxidation-responsive poly(amino ester)s containing arylboronic ester and self-immolative motif: Synthesis and degradation study

Article abstract of DOI:10.1021/ma401656t

We report the synthesis of a new type of amphiphilic poly(amino ester)s which can be completely degraded in aqueous media via H₂O₂ oxidation. The polymers were prepared by the controlled Michael-type addition polymerization of a phenylboronic pinacol ester-containing diacrylate and N-aminoethylpiperazine, followed by postmodification with mPEG5K-succinimide ester. Upon oxidation, the side chain phenylboronic esters will be transformed into phenol groups which can trigger the sequential self-immolative process to degrade the polymer main chain. Meanwhile, the amino groups on the polymer main chain are capable of trapping the highly active quinone methides generated in situ during the oxidative degradation of the polymers. Based on the detailed oxidation kinetics and products of several model compounds, the H ₂O₂-triggered degradation of nanoparticles of these copolymers was investigated by NMR spectroscopy, GPC, and Nile red fluorescence probe. The results demonstrate that the poly(amino ester) backbones were completely degraded by H₂O₂, resulting in the dissociation of nanoparticles. Oxidative degradation rates of the nanoparticles could be accelerated by increasing the concentration of H₂O₂, the PEGylation degree, or the pH of the buffer. Interestingly, the in situ formed quinone methides could be captured by secondary amines due to their higher nucleophilicity than H₂O. Of potential importance, these amphiphilic oxidation-responsive copolymers are sensitive to stimulation of 200 μM H ₂O₂; therefore, they may find application in the field of intelligent drug/gene delivery systems.

Full text of DOI:10.1021/ma401656t

Article
pubs.acs.org/Macromolecules
Oxidation-Responsive Poly(amino ester)s Containing Arylboronic
Ester and Self-Immolative Motif: Synthesis and Degradation Study
Cheng-Cheng Song, Ran Ji, Fu-Sheng Du,* and Zi-Chen Li*
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Polymer Chemistry and Physics of Ministry of Education,
College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, People’s Republic of China
S
* Supporting Information
ABSTRACT: We report the synthesis of a new type of
amphiphilic poly(amino ester)s which can be completely
degraded in aqueous media via H2O2 oxidation. The polymers
were prepared by the controlled Michael-type addition
polymerization of a phenylboronic pinacol ester-containing
diacrylate and N-aminoethylpiperazine, followed by post-
modification with mPEG5K-succinimide ester. Upon oxida-
tion, the side chain phenylboronic esters will be transformed
into phenol groups which can trigger the sequential self-
immolative process to degrade the polymer main chain.
Meanwhile, the amino groups on the polymer main chain are
capable of trapping the highly active quinone methides
generated in situ during the oxidative degradation of the polymers. Based on the detailed oxidation kinetics and products of
several model compounds, the H2O2-triggered degradation of nanoparticles of these copolymers was investigated by NMR
spectroscopy, GPC, and Nile red fluorescence probe. The results demonstrate that the poly(amino ester) backbones were
completely degraded by H2O2, resulting in the dissociation of nanoparticles. Oxidative degradation rates of the nanoparticles
could be accelerated by increasing the concentration of H2O2, the PEGylation degree, or the pH of the buffer. Interestingly, the in
situ formed quinone methides could be captured by secondary amines due to their higher nucleophilicity than H2O. Of potential
importance, these amphiphilic oxidation-responsive copolymers are sensitive to stimulation of 200 μM H2O2; therefore, they may
find application in the field of intelligent drug/gene delivery systems.
ROSs in vivo.4d,6 A series of small molecules used as H2O2-
specific fluorescent probes and H2O2-responsive prodrugs were
INTRODUCTION
■
Reactive oxygen species (ROS) at moderate concentrations
play an important role in several physiological processes such as
developed using arylboronic acid or ester as the protecting
group.4d,7 Furthermore, phenylboronic ester was also applied as
cell signaling, apoptosis, or proliferation and in the fight against
foreign objects.1 However, if ROS is overproduced, it may
a trigger to construct self-immolative dendritic fluorescent
probes. Upon exposure to one molecule of H2O2, the dendritic
damage biomolecules such as lipids, proteins, and DNA. A
probe decomposed in a domino manner and released all three
number of evidences reveal that oxidative stress caused by
overproduction of ROS is closely associated with various
fluorescent reporters, showing an amplifying effect.8 Oxidation-
responsive polymers based on phenylboronic pinacol ester were
also designed and synthesized by using different synthetic
routes. These polymers may find applications in the fields of
polymer-based vaccine, H2O2-detection, in vivo imaging, and
pathological disorders including cancer and inflammatory or
neurodegenerative diseases.2 Oxidative stress plays a pivotal
role in initiation, progression, and metastasis of cancer cells;
chemoresistance of some cancer cells may also be related to
polymer−drug conjugates with on-demand release property.9
All of the aforementioned phenylboronic ester-containing
compounds or polymers generate quinone methide intermedi-
ates upon H2O2 stimulation. Quinone methides are highly
reactive electrophiles that react efficiently with biomolecules
and have been reported to mediate cytotoxicity of some
anticancer drugs or alkylphenol derivatives.10,11 Although
quinone methide intermediates can be quenched by water
overproduced ROS.3 These biological features inspired
scientists to exploit various probes or sensors for the detection
of different reactive oxygen species or in vivo diagnosis and to
develop biomaterials for site-specific drug/gene delivery.4 For
example, polysulfides, poly(thioketal), selenium-containing
polymers, polyoxalates, and proline-containing networks have
been prepared for particulate vaccine, delivery of siRNA, DNA,
or anticancer drug, in vivo imaging of hydrogen peroxide
(H2O2), or as coating materials.5
Regarding ROS-sensitive sensors or materials, arylboronic
esters have attracted great attention due to their specificity and
high sensitivity to H2O2 that is one of the most representative
Received: August 7, 2013
Revised: October 8, 2013
Published: October 18, 2013
© 2013 American Chemical Society
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a
Scheme 1. Synthesis of Polymers of the F Series and the PF Series
a
The present drawing does not mean the polymers possess a head-to-tail structure.
a
Scheme 2. Synthesis of Monomer A1
a
Reagents and conditions: (i) TBDSCl, imidazole, DMF, 0 °C to rt, 12 h (82%); (ii) NaI, TMSCl, ACN, 0 °C to rt, 12 h (87%); (iii) DMF, K2CO3,
0 to 35 °C, 12 h (76%); (iv) p-toluenesulfonic acid, methanol, 30 °C, 2.5 h (90%); (v) acryloyl chloride, triethylamine, DCM, 0 °C to rt, 12 h (41%).
30 wt % Beijing Chemical Works) were used as received.
Dichloromethane (DCM) and chloroform were distilled over CaH2
after refluxing for ∼6 h. Deuterated phosphate buffer with pH 7.4 was
prepared from NaOD (40 wt % in D2O, Alfa) and deuterated
phosphoric acid (85 wt % in D2O).
Synthesis of Monomer A1 and Model Molecules A2 and A3.
Diacrylate monomer A1 was synthesized according to Scheme 2. Two
model molecules, A2 and A3, were prepared by the Michael-type
addition reaction of mOEG4-NH2 to 4-(4,4,5,5-tetramethyl- 1,3,2-
dioxaborolan-2-yl)benzyl acrylate and A1, respectively. All of the
detailed synthetic processes and characterization data are shown in the
Supporting Information.
molecules to form stable and safe hydroxymethylphenol
derivatives upon in vitro incubation in aqueous buffers, they
might be harmful when the arylboronic ester-containing
polymers are used in vivo as carrier materials.9a,d,12 Thus, it is
interesting to develop arylboronic ester-based oxidation-
responsive polymers with built-in nucleophilic groups capable
of catching quinone methide intermediates upon H2O2-
triggered degradation of the polymers. In this work, phenyl-
boronic pinacol ester-containing poly(amino ester)s with
different kinds of amino groups have been prepared by
Michael-type addition polymerization of the oxidation-cleavable
diacrylate and N-aminoethylpiperazine (AEPZ) (Scheme 1).
The built-in primary or secondary amino groups can be used
for PEGylation to afford amphiphilic copolymers. More
importantly, they are able to scavenge the in situ produced
quinone methides upon H2O2 oxidation. In addition, the
(co)polymers are pH-sensitive due to the presence of
secondary or tertiary amino groups in the backbones. This
type of oxidation-degradable, pH-sensitive (co)polymers may
find applications as drug carriers for curing cancer or
inflammatory diseases.
Synthesis and Fractionation of Polymer F. Monomer A1 (503
mg, 1.02 mM) and AEPZ (132 mg, 1.02 mM) were dissolved in
anhydrous chloroform (1.2 mL) in a Schlenk flask, and the reaction
mixture was stirred at 30 °C under a nitrogen atmosphere. The
1
polymerization was monitored with H NMR and GPC. After 4 days,
the polymerization was quenched by adding 80 μL of AEPZ (78.8 mg,
0.61 mM). The end-capping reaction was completed in 60 min as
confirmed by 1H NMR spectroscopy. The end-capped polymer F was
obtained by precipitation from ethyl ether (Et2O)/petroleum ether
(1/1, v/v) and dried in a vacuum for 24 h at ambient temperature.
Polymer F was further fractionated by using THF as the good solvent
and Et2O as the poor solvent at ambient temperature. Briefly, F (600
mg) was dissolved in 10 mL of THF, and 20 mL of Et2O was slowly
added to precipitate the highest molecular weight fraction F1. The
supernatant was concentrated and reprecipitated in THF/Et2O (10
mL/25 mL) mixed solvent to get fraction F2. By repeating the above
procedure using different THF/Et2O volume ratios, five narrowly
distributed polymers were obtained (F1−F5, Table 1).
EXPERIMENTAL SECTION
■
Materials. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl
acrylate was synthesized as reported in the literature.12 N-Amino-
ethylpiperazine (AEPZ, Alfa), mOEG4-NH2 (Beijing Isomersyn
Technology Co.), mPEG5K-succinimide (mPEG5K-SCM, Jenkem
Technology), Nile red (NR, Aldrich), and hydrogen peroxide (H2O2,
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of A2 disappeared completely. The other oxidation experiments of A2
or A3 in different media were carried out following the same
procedure.
Table 1. Molecular Weights and PDIs of Polymer F and the
Five Fractions
a
a
a
Mn
Mp
PDI
yield (%)
H2O2-Triggered Degradation of Copolymers of the PF
Series. The oxidative degradation of polymers of the PF series was
performed in D2O at 37 °C. Taking PF4a as an example, polymer
PF4a (3.6 mg) was fully dissolved in 250 μL of CDCl3 in an NMR
tube. Then, CDCl3 was removed by evaporation under reduced
pressure, and a thin film was formed in the tube. After 500 μL of D2O
was added, the tube was incubated at ∼4 °C for 12 h. For the
oxidation experiments, 3.5 μL of H2O2 (1.0 wt %) was added into the
F
11 700
91 700
66 100
22 700
12 700
5 300
3.52
1.43
1.48
1.71
1.61
1.36
95
5
F1
F2
F3
F4
F5
142 000
108 000
38 300
20 500
6 900
11
29
18
27
1
a
tube which was incubated at 37 °C for 24 h before collecting the H
Measured by GPC using polystyrene as standards in THF.
NMR spectrum. Degradation of the other polymers was conducted in
a similar way. The polymer concentration was calculated based on the
formula (621/492) × Pox (mg/mL), where Pox denotes the mass
percentage of the oxidation-responsive segment in the PF series of
polymers as listed in Table 2.
Degradation of Copolymers of the PF Series Was Also
Monitored by GPC. All the polymers were degraded under the same
conditions as the NMR measurements. After degradation for 1 day, the
mixture was lyophilized to afford a white powder and was analyzed by
GPC.
Degradation of Copolymer Nanoparticles As Followed by
NR Fluorescence Probe. The dispersions of copolymers of the PF
series were prepared following the film-rehydration procedure used for
preparation of the samples in the NMR measurement. To 10 mL of
the dispersion, 50 μL of NR in ethanol (1.0 mM) was added, and the
mixture was equilibrated under stirring for 12 h at room temperature.
The degradation of PF4a nanoparticles is here reported as an example.
The PF4a dispersion (1 mL) was adjusted to pH 7.4 (100 mM) by
adding 100 μL of concentrated phosphate buffer (PB, pH 7.4, 1.0 M)
and incubated for 10 min at 37 °C before the fluorescence spectrum at
0 time point was collected. Then, 7.0 μL of H2O2 (1.0 wt %) was
added, and the fluorescence spectra of the dispersion were recorded at
37 °C at desired time points. For the other degradation experiments,
different phosphate buffers or varied amounts of H2O2 were used. The
fluorescence spectra were recorded from 560 to 700 nm on a Hitachi
F-4500 spectrometer with the excitation wavelength of 545 nm and a
scanning rate of 240 nm/min.
PEGylation of Polymers of the F Series. The PEGylated
amphiphilic copolymers (PF series) were prepared from the F series
precursors (F3−F5) according to Scheme 1. Using PF4a in Table 2 as
an example, polymer F4 (72 mg, ∼0.12 mmol of reactive amino
groups) and mPEG5K-SCM (300 mg, ∼0.06 mmol) were dissolved in
3 mL of anhydrous DCM. The reaction was carried out at 30 °C for 3
days under stirring. After precipitation in Et2O, a white powder (81%
yield) was obtained. This crude polymer was further purified using
preparative GPC (Japan Analytical Industry Co. Ltd., LC-9210 NEXT;
CHCl3 as the eluent) to remove the unconjugated PEG, affording the
final polymer PF4a. The other PEGylated copolymers were prepared
in a similar manner with the feed ratios listed in Table 2. Copolymers
PF3a and PF5a were also purified using preparative GPC, while PF4b
and PF4c could be simply purified by precipitation using DCM as the
good solvent and Et2O as the poor solvent.
NMR Measurements. The degradation kinetics of the monomer,
model compounds, and the polymers were studied at 37 or 60 °C by
1
monitoring the H NMR spectra on the Bruker Avance III 400 MHz
spectrometer.
Gel Permeation Chromatography (GPC). The GPC measure-
ments were carried out on equipment consisting of a Waters 1525
binary HPLC pump, a Waters 2414 refractive index detector, and three
columns (Styragel HT2, HT3, and HT4) at 35 °C with THF as the
eluent (1.0 mL/min). A series of narrow dispersed polystyrenes were
used for calibration. Molecular weight and polydispersity index (PDI)
were calculated using the Millennium 32 software.
Oxidation of A1. Compound A1 (2.07 mg, 4.2 μmol) was
RESULTS AND DISCUSSION
■
dissolved in 220 μL of acetonitrile-d3 (ACN-d3). After the addition of
1
Synthesis and Characterization of Monomer A1 and
Polymers of the F Series. The oxidation-sensitive diacrylate
monomer A1 was synthesized via the sequential steps as shown
in Scheme 2. Compound S6 was synthesized following the
reported procedure.9d Reaction between S6 and acryloyl
chloride afforded the desired monomer A1, the structure of
which was confirmed by NMR, FT-IR, MS spectra, and
elemental analysis (Figure S2).
200 μL of D2O and thorough mixing, a H NMR spectrum of the
homogeneous solution was immediately recorded and used for the 0
time point. Then, 1 μL of H2O2 (30 wt %) was added to the tube to
initiate the oxidation reaction at 60 °C. 1H NMR spectra were
collected at desired time points. Control experiment of A1 without
H2O2 was carried out synchronously at 60 °C.
Oxidation of Model Compounds A2 and A3. Taking the
oxidation of A2 in D2O as an example, compound A2 (4.0 mg in ∼0.5
mL CDCl3) was charged into an NMR tube. After removing CDCl3 by
evaporation, 500 μL of D2O was added to dissolve A2 (16 mM). Upon
addition of H2O2 (16 mM), the oxidation reaction was conducted at
37 °C and monitored in situ by 1H NMR spectroscopy until the signals
Michael-type addition polymerization of A1 and AEPZ was
carried out at 30 °C in anhydrous chloroform (Scheme 1). The
1
polymerization was monitored by H NMR spectroscopy and
Table 2. Characterization of the PEGylated Polymers of PF Series
a
b
c
d
d
e
e
e
F series
Mn
DP (%)
PEG/N
PF series
Mn
PDI
Mn
DS (%)
Pox (%)
f
f
g
F3
F4
22 700
12 700
nd
1/1.8
1/2.0
1/8.0
1/35
1/1.0
PF3a
PF4a
PF4b
PF4c
PF5a
59 300
48 400
19 500
13 000
45 300
1.20
1.17
1.30
1.40
1.10
nd
66
57
8.7
3.4
93
16
22
80 000
23 700
17 700
70 900
17
58
78
11
F5
5 300
12
a
b
Molecular weight of the F series polymers measured by GPC using polystyrenes as standards in THF. Average degree of polymerization (DP) of
c
F4 or F5 calculated according to the 1H NMR spectra of their PEGylated derivatives PF4a and PF5a. Molar ratio of mPEG5K-SCM to the reactive
d
(primary and secondary) amino groups in feed. Molecular weight and polydispersity index of the PF series of copolymers measured by GPC.
e
Molecular weight (Mn), percent degree of PEG substitution (DS), and mass percentage of the oxidation-responsive segment (Pox) of the PF series
f
1
1
of amphiphilic copolymers calculated by the H NMR spectra as shown in Figure 2. Not calculated due to the poor resolution of the H NMR
spectrum of PF3a. Estimated by the formula (621/(621 + 5000 × DS)) × 100%.
g
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1
Figure 1. H NMR and 13C NMR spectra of polymer F.
GPC measurement (Figures S3 and S4), and the results are
summarized in Table S1. After 4 days, the conversion of the
acrylic double bond reached ∼98%, and the Mn of the polymers
reached 8900 with a PDI of 4.1. Statistically, the polymer ends
may contain residual vinyl groups; therefore, we added an
excess of AEPZ after the polymerization to end-cap the
polymer. Since the secondary amino group of AEPZ is much
more reactive than the primary one toward the Michael-type
addition reaction with the acrylic double bond,13 both ends of
the capped polymer are expected to be primary amino groups.
The end-capped polymer was purified by precipitation method
to afford polymer F, the chemical structure of which was
and subsequent self-immolative elimination of polymer F,10b
which is beneficial for the polymer considering its potential in
vivo use.
Polymer F has a broad molecular weight distribution due to
the step polymerization mechanism. We carefully fractionated F
by using THF as the good solvent and ethyl ether as the poor
solvent, and five fractions with different molecular weights and
relatively low PDIs were obtained (Figure S7 and Table 1).
These polymers were insoluble in water; F3−F5 were used for
further modification and property study.
PEGylation of Polymers of the F Series. Highly reactive
water-soluble mPEG5K-SCM was used to modify F3−F5
(Scheme 1). Although both primary and secondary amines in
these polymers can react with N-hydroxysuccinimide esters, the
former is much more reactive (10−1000 times) than the latter
mainly due to the steric effect.16 It is rationally speculated that
the PEGylation occurs preferentially at the chain ends of the F
series of polymers, and the degree of PEG substitution (DS)
can be easily tuned by varying the feed ratio of mPEG5K-SCM
to the precursor polymers. The model reactions confirmed this
speculation (Figures S8 and S9). The DS of the PEGylated
1
confirmed by its H and 13C NMR spectra (Figure 1).
Regarding the Michael-type addition to the acrylic double
bond, the acyclic secondary amine formed by the Michael-type
addition of the AEPZ primary amino group to the acrylic
double bond is much less reactive than the original cyclic
secondary or the primary amino groups at 40 °C in
chloroform.13 Our model reaction also revealed the linear
polymer with negligible branching points was obtained at 30
°C, and the branched structure was observed only at the higher
temperature, for example 60 °C (Figures S5 and S6). For
polymer F, its 13C NMR spectrum clearly demonstrated a linear
structure as proven by the strong peaks at ∼46 ppm (protons 1
and 22) and negligible branching signals at ∼51 ppm. These
results clearly indicate that polymer F has a linear topology with
the primary amino groups at both ends and numerous
secondary and tertiary amino groups in the backbone. The
primary or secondary amino groups can be used for further
functionalization of the polymer;14 also, the amino groups
render the polymer sensitive to pH.15 More importantly, the
primary or secondary amines can react quickly with the
intermediate quinone methides produced by H2O2 oxidation
1
polymers (PF series) was determined by the H NMR spectra
(Figure 2).
Using the characterization of PF4a as an example, this
polymer was obtained with a high molar feed ratio (1:2) of
mPEG5K-SCM to the reactive amino groups (primary and
secondary) of the precursor polymer F4. Therefore, it is
reasonable to expect that all of the primary amino groups at the
1
end of polymer F4 were consumed. In the H NMR spectrum
of PF4a, the two peaks c (∼4.0 ppm) and c′ (∼4.2 ppm) are
assigned to the carbonyl-adjacent methylene signals of the PEG
chains conjugated to the F4 chain ends and the polymer
backbone, respectively. By comparing the integration intensities
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1
Figure 2. H NMR spectra of the PF series copolymers in CDCl3. Average degree of polymerization (DP) of the parent polymers F4 and F5 is
calculated by the formula: DP = I6 × 2/Ic, where I6 and Ic denote the integration intensity of peak c (∼4.0 ppm) and peak 6 (∼4.9 ppm) in the
spectra of PF4a and PF5a, respectively. Percent degree of PEG substitution (DS) in the PF series copolymers except PF3a is calculated by the
formula DS = ((Ic + Ic′)/I6) × (DP/(DP + 1)) × 100%, where Ic′ denotes the peak intensity of c′ (∼4.2 ppm). For PF3a, DS is estimated by the
formula DS = ((Ic + Ic′)/I6) × 100%. The number-average molecular weight (Mn) of the PF series copolymers is calculated by the formula Mn = DP
× 621 + (DP + 1) × DS × 5000. Pox of the copolymers PF4a−4c and PF5a is calculated by the formula Pox = ((DP × 621)/Mn) × 100%.
1
Figure 3. (a) Oxidation mechanism of A1 triggered by H2O2. (b) H NMR spectra and (c) oxidation kinetics of A1 (5.0 mg/mL, 10 mM) in the
mixed solvent of D2O/ACN-d3 (1/1.1, v/v) with H2O2 (21 mM) at 60 °C. The instantaneous percent contents of compounds A1 and S1 and other
intermediates were calculated by the formulas: [A1]% = [I6/(I6 + I6i + I6′)] × 100%; [I]% = [I6i/(I6 + I6i + I6′)] × 100%; [S1]% = [I3′/(I3 + I3i + I3′)]
× 100%; ([II] + [III] + [IV])% = 100% − [A1]% − [I]% − [S1]%.
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Figure 4. 1H NMR spectra of A2 (7.9 mg/mL, 16 mM) after complete oxidation by H2O2 (16 mM) in ACN/D2O (1.1/1, v/v), D2O, and PB (pH
7.4, 300 mM) at 37 °C. The trapping efficiency is calculated by the formula (I7″/(I7′ + I7″)) × 100%, where I7′ and I7″ denote the integration
intensities of peaks 7′ and 7″.
of peaks c, c′, and 6 (∼4.9 ppm), the average degree of
polymerization (DP) of the precursor polymer F4 and the DS
of copolymer PF4a were estimated to be 22 and 57%,
respectively. This indicates that besides the two primary amino
groups at the chain ends, ∼50% of the secondary amino groups
in the F4 backbone were modified by the PEG chains. PF4b
was obtained in a 1:8 feed ratio where only peak c, but no peak
c′, was detected in the NMR spectrum; the corresponding DP
was 22 and the DS was 8.7%. Therefore, only the two ends of
PF4b have been PEGylated, and it can be considered a PFP-
type triblock copolymer. By further decreasing the feed ratio to
1:35, PF4c was obtained with a DS of 3.4%, implying that one
polymer chain averagely contains ∼0.8 PEG. In other words,
PF4c is a mixture of the unPEGylated F4, diblock, and
probably triblock copolymers.
hydrolyzed to boronic acid and pinacol. Afterward, compound I
decomposes to p-hydroxymethylphenol, compound S1, and
acrylic acid via the sequential steps of 1,6- and 1,4-elimination
1
(Figure 3a). This mechanism was confirmed by the H NMR
spectra shown in Figure 3b. The signals of the intermediate
compound I were clearly resolved during the oxidation process.
The kinetic curves of A1 oxidation (step 1), decomposition
of compound I (step 2), and the formation of compound S1
(step 3) were obtained from these spectra and are shown in
Figure 3c. The rapid decomposition of monomer A1 with a
significant accumulation of compound I at the initial stage
indicates the faster kinetics of step 1 than step 2. The amounts
of the intermediates II, III, and IV were negligible, suggesting
that the decomposition of compound I (step 2) is the rate-
determining step in the whole process. In the absence of H2O2,
A1 could not be oxidized, but the pinacol unmasking reaction
occurred due to the dynamic covalent chemistry (Figure
S11).17
Oxidation of A1 by H2O2 was also studied in a mixture of
deuterated PB (pH 7.4, 100 mM) and ACN-d3 at 60 °C (Figure
S12). As compared with the kinetics in D2O/ACN-d3, A1 was
oxidized and decomposed much faster into the final products in
PB/ACN-d3. Both A1 and compound I completely disap-
peared, and acrylic acid was generated quantitatively within 6 h.
These results can be explained by the pH difference of the
media. It is well-known that both the oxidation of phenyl-
boronic ester/acid and the subsequent self-immolative
eliminations are pH-dependent, undergoing a faster reaction
at a relatively higher pH.6,18 It is noted that compound S1 has a
poor solubility in PB/ACN-d3, resulting in the relatively lower
Similarly, in the 1H NMR spectrum of copolymer PF5a, both
peak c and peak c′ are clearly observable. More than 90% of the
reactive amines (primary or secondary) were PEGylated
because of the high feed ratio (1:1). The DS of PF3a was
only roughly estimated (Figure 2) for copolymer PF3a because
the 1H NMR spectrum was not finely resolved due to the high
molecular weight of its precursor F3, making it difficult to
calculate the DP of F3. The characterization results are
summarized in Table 2.
Oxidation of Monomer A1 by H2O2. Monomer A1 is
hydrophobic and not soluble in water. The H2O2-triggered
oxidation of A1 was carried out in a mixture of D2O and ACN-
d3 at 60 °C. According to the literature,8,9d upon oxidation, A1
will first be transformed to the phenolic compound I (Figure
3a), releasing the boronic pinacol ester which is quickly
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Figure 5. 1H NMR spectra of A3 (8.5 mg/mL, 9.3 mM) after completely oxidation triggered by H2O2 (9.3 mM) at 37 °C in ACN/D2O and D2O.
1H NMR spectra of A3 oxidized by H2O2 (Figure 5). In both
proton signals as compared with p-hydroxymethylphenol or
acrylic acid. When the oxidation of A1 was carried out at 37 °C
in PB/ACN-d3, we observed various phosphate intermediates
produced by the addition of phosphate anions to the highly
reactive quinone methides (Scheme S1 and Figure S13). These
organophosphates gradually hydrolyzed.
ACN/D2O and D2O, the proton signals (peaks 3″−5″ and 3‴−
5‴) of the addition products derived from o-quinone methides
were clearly detected (Figure S16).
Oxidative Degradation of Polymers of the PF Series.
The amphiphilic copolymers of the PF series (PF3a−5a and
PF4b) can self-assemble into stably dispersed nanoparticles in
aqueous solution (Table S2 and Figure S18). The proton
signals of the PEG chains in the nanoparticles were clearly
observed in the absence of H2O2, but the oxidation-responsive
segment was hardly detectable due to its hydrophobic feature
(Figure S19). These nanoparticles were oxidized by H2O2 in
Oxidation of A2 and A3. In order to clarify if the
secondary amines react preferentially with the in situ formed
quinone methides during the oxidation process, two model
compounds A2 and A3 that are well dispersible in water were
synthesized by the Michael-type addition of mOEG4-NH2 to
the corresponding acrylate precursors (Figure S14). These two
compounds were oxidized by H2O2 at 37 °C in different media,
1
pure water at 37 °C for 24 h, and their H NMR spectra were
1
and the oxidation products were analyzed by H NMR. As
recorded as shown in Figure 6. For the highly PEGylated PF5a
with few secondary amino groups, only p-hydroxymethylphenol
and compound S1 are the aromatic degradation products; no
other aromatic compounds derived from the quinone methides
were detected. Also, the proton signals c (∼4.0 ppm) and c′
(∼4.3 ppm) of the detached PEG are clearly observable. In the
case of PF3a, PF4a, and PF4b, the aromatic adducts of the
secondary amino groups and the quinone methides were
produced as indicated by the proton signals 7″, 8″, 5″, and 4″
(Figure 6 and Figure S20). With decreasing degree of PEG
substitution (PF4a vs PF4b), more in situ formed quinone
methides were captured by the unPEGylated secondary amines.
When PF4a nanoparticles were incubated in water at 37 °C for
24 h in the absence of H2O2, negligible degradation products
were observed (Figure S22). In PB (pH 7.4), PF4a can also be
oxidized quickly to produce p-hydroxymethylphenol phosphate
shown in Figure 4 for compound A2, the proton signals (peaks
6′, 7′, and 8′) of p-hydroxymethylphenol are clearly observable.
Besides, the proton signals 6″, 7″, and 8″ can be assigned to the
addition product of the in situ formed secondary amine and p-
quinone methide. By comparing the integration intensities of
peak 7″ (∼7.3 ppm) and peak 7′ (∼7.2 ppm), the trapping
efficiencies of p-quinone methide by the amine can be
calculated as 77% (ACN/D2O), 62% (D2O), and 57% (PB,
pH 7.4), respectively. The lowest efficiency in PB was partly
attributed to the competitive addition of the phosphate anion
to p-quinone methide (Figure S15).
For the oxidation of A3 in the presence of H2O2, we
speculate that besides the intermediate p-quinone methide, the
in situ formed o-quinone methides also react quickly with the
secondary amino groups (Scheme S2). This is confirmed by the
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1
Figure 6. H NMR spectra of PF series copolymer nanoparticles degraded in D2O triggered by H2O2 (2 mM); 24 h, 37 °C.
that hydrolyzed gradually into p-hydroxymethylphenol. As
mentioned in the previous section, compound S1 is poorly
soluble in the phosphate buffer, resulting in its weak proton
signals (Figures S23 and S24).
The H2O2-triggered degradation of copolymers of the PF
series was further confirmed by GPC (Figure 7). After
reduced in aqueous buffer.19 NR was used as a fluorescence
probe to study the dissociation of various pH-sensitive
nanoparticles.12,20 In this work, the degradation profiles of
the PF nanoparticles were also studied at 37 °C using NR as a
probe. As shown in Figure 8a, the fluorescence intensity of NR
remained constant in the absence of H2O2 at pH 7.4, indicating
no obvious degradation of the PF4a nanoparticles in the tested
period. Addition of H2O2 caused a drastic decrease in NR
fluorescence; the decreasing magnitude was greatly dependent
on the H2O2 concentration. The intensity of NR fluorescence
decreased ∼30% of its initial value in the presence of 0.2 mM
H2O2, that is approximately one-tenth of the arylboronic ester
units in the polymer, indicating the partial degradation of the
nanoparticles. With increasing H2O2 concentration to 2.0 mM,
the intensity dropped rapidly to <10% of the initial value.
Further increasing H2O2 concentration to 5.0 mM did not
obviously accelerate the degradation kinetics. These results
demonstrate that PF4a nanoparticles are sufficiently sensitive to
bio-related oxidation environments.9a
The pH-dependent degradation results of PF4a nano-
particles monitored by NR are shown in Figure 8b. As
expected, in the absence of H2O2, the normalized intensity
gradually decreased with decreasing pH because of the
protonation of the amino groups and the consequential
swelling or partial dissociation of the nanoparticles at lower
pH. When 2.0 mM H2O2 was added, the intensity quickly
decreased to ∼8% of the initial value, indicating rapid
degradation of the nanoparticles. Interestingly, the degradation
rate was slightly slower at pH 5.2 than at pH 7.4 or 6.5, which
can be ascribed to the combined result of protonation of the
amines and oxidation triggered cleavage of the polymer
backbone. A lower pH is beneficial for the protonation of
Figure 7. GPC curves of the copolymers PF3a−PF5a before (solid
line) and after (dashed line) degradation in water. The dash-dotted
line (PF4a′) denotes the degraded sample of PF4a in PB (pH 7.4, 300
mM). H2O2 concentration: 2.0 mM, 37 °C, 24 h.
oxidation in water, the original copolymers (PF3a−PF5a)
completely disappeared, and only the low molecular weight
mPEG5K was observed in the GPC traces. After the
degradation in PB, a similar GPC trace was obtained, indicating
the complete degradation of PF4a.
pH-Dependent Degradation of Copolymer Nano-
particles of the PF Series As Studied by NR Fluorescence
Probe. NR is a solvatochromic dye with strong fluorescence in
a hydrophobic microdomain, but its quantum yield is drastically
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Figure 8. Time-dependent change in the normalized intensity of NR fluorescence in PF4a nanoparticles (a) at different H2O2 concentrations and
(b) at different pHs without (empty) or with (solid) H2O2 (2.0 mM), and (c) in PF3a, PF4a, and PF4b nanoparticles without (empty) or with
(solid) H2O2 (2.0 mM). NR concentration: 5 × 10−6 mol/L; 37 °C; pH 7.4.
amines but slows down the phenylboronic ester oxidation and
the subsequent self-immolative elimination.
*Phone +86-10-62755543; Fax +86-10-62751708; e-mail zcli@
pku.edu.cn (Z.L.).
Figure 8c shows the degradation kinetics of PF3a, PF4a, and
PF4b nanoparticles in PB. PF3a and PF4a nanoparticles
exhibited similar degradation profiles, while PF4b nanoparticles
degraded at a much slower rate. These results might
demonstrate that the degree of PEGylation, not the molecular
weight of the polymer precursors (F3 vs F4), mainly influenced
the degradation kinetics of the copolymer nanoparticles.
Compared with PF3a or PF4a nanoparticles, the oxidation-
responsive segments in PF4b nanoparticles are located in the
more hydrophobic microdomains that have a lower perme-
ability to H2O2 as well as water molecules.21 H2O2-induced
oxidative degradation of the PF4b nanoparticles was further
confirmed by the light scattering and TEM measurements
(Figure S25).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Natural
Science Foundation of China (21174002 and 21090351) and
the National Science Fund for Distinguished Young Scholars of
China (21225416).
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ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed synthetic procedures, H and 13C NMR spectra and
1
FT-IR spectrum of A1, A2, and A3; NMR spectra of the model
molecules, GPC curves of polymers, more H NMR spectra of
the oxidation products. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
AUTHOR INFORMATION
■
Corresponding Authors
*Phone +86-10-62757155; Fax +86-10-62751708; e-mail
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