Tuning the sugar-response of boronic acid block copolymers

Article abstract of DOI:10.1002/pola.26125

A detailed study of the pH- and sugar-responsive behavior of poly(3-acrylamidophenylboronic acid pinacol ester)-b-poly(N,N- dimethylacrylamide) (PAPBAE-b-PDMA) block copolymers is presented. Reversible addition-fragmentation chain transfer (RAFT) polymerization of the pinacol ester of 3-acrylamidophenylboronic acid resulted in homopolymers with molecular weights between 12,000 and 37,000 g/mol. The resulting homopolymers were employed as macro-chain transfer agents during the polymerization of N,N-dimethylacrylamide (DMA). Successful chain extension and removal of the pinacol protecting groups to yield poly(3-acrylamidophenylboronic acid)-b-PDMA (PAPBA-b-PDMA) with free boronic acid moieties resulted in pH- and sugar-responsive block copolymers that were subsequently investigated for their behavior in aqueous solution. The PAPBA-b-PDMA block copolymers were capable of solution self-assembly due to the PAPBA block being water-insoluble below its pK_a. The resulting aggregates were demonstrated to solubilize and release model hydrophobic compounds, as demonstrated by fluorescence studies. Dissociation of the aggregates was induced by raising the pH above the pK _a of the boronic acid residues or by adding sugars capable of forming boronate esters. Aggregate size, dissociation kinetics, and the effect of various sugars were considered. The critical sugar concentration needed to induce aggregate dissociation was tuned by incorporation of hydrophilic DMA units within the PAPBA responsive segment to yield PDMA-b-poly(3- acrylamidophenylboronic acid-co-DMA) block copolymers.

Full text of DOI:10.1002/pola.26125

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Tuning the Sugar-Response of Boronic Acid Block Copolymers
Jennifer N. Cambre, Debashish Roy, Brent S. Sumerlin
Department of Chemistry and Center for Drug Discovery, Design, & Delivery, Southern Methodist University,
3215 Daniel Avenue, Dallas, Texas 75275-0314
Correspondence to: B. S. Sumerlin (E-mail: bsumerlin@smu.edu)
Received 28 February 2012; accepted 21 April 2012; published online
DOI: 10.1002/pola.26125
ABSTRACT: A detailed study of the pH- and sugar-responsive
behavior of poly(3-acrylamidophenylboronic acid pinacol
ester)-b-poly(N,N-dimethylacrylamide) (PAPBAE-b-PDMA) block
copolymers is presented. Reversible addition-fragmentation
being water-insoluble below its pK
a
. The resulting aggregates
were demonstrated to solubilize and release model hydropho-
bic compounds, as demonstrated by fluorescence studies. Dis-
sociation of the aggregates was induced by raising the pH
chain transfer (RAFT) polymerization of the pinacol ester of 3-
acrylamidophenylboronic acid resulted in homopolymers with
molecular weights between 12,000 and 37,000 g/mol. The
resulting homopolymers were employed as macro-chain trans-
fer agents during the polymerization of N,N-dimethylacryla-
mide (DMA). Successful chain extension and removal of the
pinacol protecting groups to yield poly(3-acrylamidophenylbor-
onic acid)-b-PDMA (PAPBA-b-PDMA) with free boronic acid
moieties resulted in pH- and sugar-responsive block copoly-
mers that were subsequently investigated for their behavior in
aqueous solution. The PAPBA-b-PDMA block copolymers were
capable of solution self-assembly due to the PAPBA block
a
above the pK of the boronic acid residues or by adding sugars
capable of forming boronate esters. Aggregate size, dissocia-
tion kinetics, and the effect of various sugars were considered.
The critical sugar concentration needed to induce aggregate
dissociation was tuned by incorporation of hydrophilic DMA
units within the PAPBA responsive segment to yield PDMA-b-
poly(3-acrylamidophenylboronic acid-co-DMA) block copoly-
mers. VC 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym
Chem 000: 000–000, 2012
KEYWORDS: inorganic polymers; reversible addition fragmenta-
tion chain transfer (RAFT); solution properties
INTRODUCTION Stimuli-responsive polymers respond to var-
examples of sugar-responsive polymers have been reported,
with the most common systems being glucose-responsive
polymers that undergo changes in solubility upon the glu-
cose oxidase-catalyzed reaction of glucose with oxygen or
iations in the local environment with dramatic changes in
1
their properties. A variety of applications have been envi-
sioned for such polymers, with the most commonly studied
stimuli-responsive systems being either temperature- or pH-
competitive binding of glucose with glycopolymer–concanav-
2
–7
4,12,13
responsive.
Temperature-responsive polymers undergo
alin A complexes.
These glucose-responsive systems are
8
,9
volume phase transitions on heating or cooling.
pH-re-
versatile and highly specific, though the reliance on protein-
based components may limit applications under non-biologi-
cal conditions or over long times that might promote
denaturation.
sponsive polymers are generally polyacids or polybases that
undergo solubility changes in aqueous media on acid/base-
8
,9
induced ionization events.
Recently, there has been increased interest in polymers that
respond to a variety of other stimuli that have been less fre-
An alternative mechanism of glucose-response is exhibited
by polymers endowed with purely synthetic components. In
particular, the ability of boronic acids to bind sugars with
high affinity has led to polymeric boronic acids finding use
as glucose sensors and as ligand moieties during chromato-
graphy. Similar to other examples of stimuli-responsive
polymers, the water solubility of polymeric boronic acids can
be tuned by changes in the surrounding solution (Scheme 1).
In aqueous systems, boronic acids exist as either a neutral
trigonal form (1) or an anionic tetrahedral form (2), with
4
,10
quently considered.
Specifically, to enable access to stim-
uli-responsive materials for many biomedical applications, it
is important to prepare polymers that respond to specific
chemical stimuli encountered in vivo. For instance, polymers
that respond to enzymes, antigens, and naturally occurring
reducing/oxidizing agents may enable new therapeutic deliv-
1
4
4
,11
ery routes during the treatment of disease.
We are partic-
ularly interested in polymers that respond to changes in the
concentration of sugars in the surrounding medium. Several
the direction of the equilibrium depending on the pK of the
a
Additional Supporting Information may be found in the online version of this article.
2012 Wiley Periodicals, Inc.
V
C
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SCHEME 1 Ionization equilibria of polymeric boronic acids in aqueous media.
boronic acid and the solution pH. Upon the addition of 1,2-
or 1,3-diols, cyclic boronic esters formed from the neutral
trigonal boronic acid and diols are generally considered
groups can lead to polymers that are highly hygroscopic and
difficult to handle and characterize.
In this report, we describe the RAFT polymerization of the
pinacol ester of 3-acrylamidophenylboronic acid. The result-
ing boronic ester polymers were employed as macro chain
transfer agents (macroCTAs) for the polymerization of a
hydrophilic monomer, with the resulting block copolymers
being deprotected to afford free boronic acid functionality.
The solution behavior of these responsive polymers was
investigated, with particular attention being paid to the abil-
ity of the block copolymers to reversibly self-assemble and
dissociate in response to changes in pH and sugar concentra-
tion. The block copolymer aggregates were capable of solubi-
lizing and releasing model hydrophobic compounds, suggest-
ing they may hold promise for controlled release
applications. Additionally, we found it was possible to tune
the glucose sensitivity of the block copolymers by incorpo-
rating hydrophilic comonomers within the responsive bor-
onic acid-containing block. This approach may prove useful
for increasing the versatility of boronic acid polymeric mate-
1
5
hydrolytically unstable. On the other hand, the tetrahedral
anionic form (2) is able to reversibly bind with diols to form
a boronate ester (3), shifting the equilibria to the anionic
forms (2 and 3). In the absence of hydrophilic comonomers,
polymers containing neutral boronic acid groups are typi-
cally hydrophobic, whereas the polymers with anionic boro-
nate moieties are water-soluble. Therefore, as the concentra-
tion of sugar is increased, the contribution of the anionic
species increases, along with the hydrophilicity of the
system. This behavior results in the solubility of boronic
acid-containing polymers being dependent on both pH and
the concentration of compatible diols in the surrounding
medium. Thus, by exploiting the reaction of boronic acids
with glucose (i.e., a model diol), the possibility for glucose-
responsive materials exists.
A variety of sugar-responsive materials based on boronic
1
6
acid polymers have been reported,
including random
and other crosslinked materi-
The majority of these materials were composed of
1
7–24
25–29
16
copolymers,
gels,
rials for biological applications.
3
0,31
als.
polymers prepared by conventional radical polymerization.
Controlled radical polymerization (CRP) techniques facilitate
the preparation of well-defined (co)polymers with a high
degree of chain-end functionalization, an important require-
ment for the efficient preparation of complex macromolecu-
lar architectures (e.g., block copolymers) that lead to self-as-
sembly and other advanced materials applications. Of the
various CRP techniques, stable free radical polymerization
EXPERIMENTAL
Materials
2-Dodecylsulfanylthiocarbonylsulfanyl-2-methyl-propionic acid
(DMP) was prepared as previously reported. N,N-Dimethy-
lacrylamide (DMA, TCI, 98%) was passed through a small
column of basic alumina prior to polymerization. 2,2 -Azobis-
isobutyronitrile (AIBN, Sigma, 98%) was recrystallized from
2 2
ethanol. Dichloromethane (CH Cl , Fisher) was dried over-
5
3
0
3
2
33,34
(
SFRP), atom transfer radical polymerization (ATRP),
and reversible addition-fragmentation chain transfer (RAFT)
night over 4 Å molecular sieves. 3-Aminophenylboronic acid
(Boron Molecular), acryloyl chloride (Alfa Aesar, 96%), 1,3,5-
trioxane (Acros Organics, 99.5%), pinacol (Acros Organics,
99%), sodium bicarbonate (Acros Organics, 99.5%), sodium
hydroxide (Alfa Aesar, 97%), triethylamine (Mallinckrodt,
99.5%), ethyl acetate (Fisher), sodium chloride (EMD,
99.0%), anhydrous sodium sulfate (EMD, 99.0%), toluene
(Aldrich, 99.5%), acetonitrile (Aldrich, 99.9%), trifluoroacetic
acid (Acros Organics, 99%), D-glucose (Mallinckrodt), D-fruc-
tose (Acros Organics, 98%), N,N-dimethylformamide (DMF,
Acros Organics,> 99%), hexanes (Fisher, 98.5%), dimethyl-
sulfoxide-d (DMSO-d , Cambridge Isotope, 99.9% D), chloro-
3
5–40
polymerization
have been utilized to synthesize well-
defined boronic acid (co)polymers through postpolymeriza-
4
1–45
tion modification
ing monomers.
or by polymerization of boron-contain-
One route of polymerizing organoboron
4
6–52
monomers to obtain boronic acid polymers can involve the
polymerization of monomers with boronic ester groups that
are subsequently hydrolyzed after polymerization to yield
4
8,50
boronic acid functionality.
We employed this route to
prepare block copolymers by the polymerization of the pina-
4
8,52
col esters of boronic acid monomers.
Alternatively,
monomers that contain boronic acid groups can be directly
polymerized by RAFT. We have demonstrated that this latter
route leads to controlled polymerizations and well-defined
6
6
form-d (CDCl
3
, Cambridge Isotope, 99.8% D), methanol-d
4
(Cambridge Isotope, 99.8% D), deuterium oxide (D O, Cam-
2
4
6,47
sugar-/pH-responsive polymers.
However, as opposed to
bridge Isotope, 99.9% D), sodium deuteroxide (NaOD, 40%
the presence of boronic esters, a high density of boronic acid
in D O, Cambridge Isotope, 99.5% D), molecular sieves (4 Å,
2
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Mallinckrodt), Nile Red (Sigma Aldrich), and polystyrene-
supported boronic acid resin (Lancaster) were used as
received. Dialysis was conducted with Fisherbrand regener-
ated-cellulose membranes of 10,000 Da molecular weight
cut-off (MWCO).
crude solid product was suspended into ethyl acetate (200
mL) and stirred for 30 min followed by filtration to remove
any solid particles. The organic layer was extracted with
water (100 mL ꢁ2), saturated aqueous sodium bicarbonate
(100 mL ꢁ2), water (100 mL ꢁ2), and brine (100 mL ꢁ2).
The organic layer was dried over anhydrous sodium sulfate.
After filtration, the solvent was removed under reduced
pressure (13 g of APBAE, 70% yield). The yellowish product
was recrystallized twice from a mixture of toluene and hex-
Analyses
Size exclusion chromatography (SEC) was conducted in DMF
ꢀ
(
(
with 0.05 M LiBr) at 55 C with a flow rate of 1.0 mL/min
Viscotek VE 2001 GPCmax; Columns: guard þ two Polymer
1
ane (1:1 v/v) to afford a white powder. H NMR (500 MHz,
Laboratories PolarGel-M mixed bed columns: molecular
DMSO-d ) (d, ppm): 10.12 (s, 1H, NH), 7.95, 7.83–7.81, 7.34–
6
3
3
weight range 0–60 ꢁ 10 and 0–2000 ꢁ 10 g/mol, respec-
tively). Detection consisted of a Viscotek VE 350 refractive
index detector operating at k ¼ 660 nm and a Viscotek
Model 270 Series Platform, consisting of a laser light scatter-
ing detector (operating at 3 mW, k ¼ 670 nm with detection
7
.29, 7.31–7.29 (s, d, m, 1H, 1H, 2H, ArH), 6.41–6.36, 6.24–
6.21 (2d, dd, 1H each, vinyl CH ), 5.73–5.71 (dd, 1H, vinyl
2
CH), 1.26 (s, 12H, CH3).
RAFT Polymerization of APBAE
ꢀ
ꢀ
angles of 7 and 90 ) and a four capillary viscometer. Molec-
ular weights were determined by the triple detection
method. For the poly(3-acrylamidophenylboronic acid pina-
col ester) (PAPBAE) homopolymers, a dn/dc ¼ 0.121 mL/g
was employed. Block copolymer molecular weights were
determined by in situ calculation of dn/dc, assuming 100%
APBAE (0.200 g, 0.732 mmol), DMP (2.70 mg, 0.00740
mmol), AIBN (0.240 mg, 0.00146 mmol), and trioxane (9.80
mg) were dissolved in DMF (1.00 mL) in a sealed 20-mL
vial. The vial was purged with nitrogen for 30 min and
placed in a preheated reaction block at 70 C. Samples were
removed periodically by syringe to determine molecular
weight, polydispersity index (PDI), and monomer conversion
by SEC and H NMR spectroscopy. DMSO-d was used as the
solvent for NMR spectroscopy. Homopolymers employed as
macroCTAs were purified by precipitating into cold hexane
four times and drying the sample under vacuum.
ꢀ
1
11
mass recovery. H- and B NMR spectroscopy was con-
ducted in CDCl , DMSO-d , methanol-d , or D O/NaOD with a
Bruker Avance 400 spectrometer or a JEOL Delta 500. For
1
3
6
4
2
6
1
1
B NMR spectroscopy, 5-mm thin-walled quartz NMR tubes
were used. Dynamic light scattering (DLS) was conducted with
a Malvern Zetasizer Nano-ZS equipped with a 4 mW, 633 nm
He–Ne laser, and an Avalanche photodiode detector at an angle
RAFT Block Copolymerization of DMA with a
PAPBAE MacroCTA
An example RAFT block copolymerization of DMA with PAP-
BAE macroCTA was conducted as follows. DMA (0.200 g,
ꢀ
of 173 . UV–vis spectroscopy measurements were conducted
with a Beckman Coulter DU 800 UV–vis spectrophotometer.
Fluorescence measurements were conducted with a Hitachi
F-7000 fluorescence spectrophotometer.
2.02 mmol), PAPBAE macroCTA (0.550 g, 0.0275 mmol),
AIBN (0.200 mg, 0.00122 mmol), and trioxane (12.0 mg) (in-
ternal standard) were dissolved in DMF (3.00 mL) in a
sealed 20-mL vial. The solution was purged with nitrogen
Synthesis of 3-Aminophenylboronic Acid Pinacol Ester
3
-Aminophenylboronic acid (11.0 g, 0.0803 mol), pinacol
9.0 g, 0.076 mol), and molecular sieves were dissolved in
dry CH Cl (170 mL) under a nitrogen atmosphere in a
(
for 30 min and then placed in a preheated reaction block at
2
2
ꢀ
70
C. The polymerization was quenched after 20 h by
sealed round-bottomed flask. After the reaction was allowed
to proceed for 48 h at room temperature, the flask was
opened, the solids were filtered, and the solvent was
removing the polymerization vial from the heating block and
exposing the reaction solution to air. The resulting PAPBAE-
b-PDMA was isolated by precipitating into cold hexane, filter-
ing, and drying under vacuum.
removed under reduced pressure to yield a pale yellow solid
1
(
15 g, 86% yield). H NMR (400 MHz, CDCl ) (d, ppm):
3
7
.23–7.19 (m, 3 H, ArH), 6.80 (m, 1 H, PhAC H), 3.63 (br-s,
4
Deprotection of Polymeric Pinacol Ester to
Yield Boronic Acid Polymers
2H, NH ), 1.36 (s, 12H, CH ).
2 3
Synthesis of 3-Acrylamidophenylboronic Acid Pinacol
Ester Monomer
An example deprotection of PAPBAE-b-PDMA was conducted
as follows. PAPBAE-b-PDMA (0.218 g, 0.00527 mmol, Mn
¼
3-Acrylamidophenylboronic acid pinacol ester (APBAE) was
41,400 g/mol, M /M
w
n
¼ 1.40) and polystyrene-supported
synthesized by a method similar to that reported by Shinkai
boronic acid (2.92 g, 8.76 mmol, boronic acid loading 2.6–
3.0 mmol/g) were stirred and refluxed for 24 h in a 2 vol %
solution of trifluoroacetic acid in acetonitrile (30.0 mL) in a
50-mL round bottom flask. The solvent was removed under
reduced pressure, and DMF/deionized water (95/5) (30 mL)
was added to the product. The solution was filtered to
recover the polystyrene-supported boronic acid resin, and
the solvent was evaporated. The remaining solid was dis-
solved in 2 wt/vol % aqueous NaOH (5 mL) and dialyzed
against 2 wt/vol % aqueous NaOH, followed by dialysis
5
4
and coworkers. 3-Aminophenylboronic acid ester (14.9 g,
0
.0680 mol) was dissolved in dry CH Cl2 (250 mL) in a
2
ꢀ
round bottom flask. The reaction mixture was cooled to 0 C
with an ice-water bath, triethylamine (12 mL, 0.086 mol)
was added, and the solution was stirred for 30 min. A solu-
tion of acryloyl chloride (6.8 g, 0.075 mol) in CH
2
Cl
2
ꢀ
(
20 mL) was added drop wise for 2 h at 0 C, and the reac-
tion was allowed to continue for 24 h at room temperature.
The solvent was removed under reduced pressure, and the
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against deionized water. The resulting solution was lyophi-
lized to yield a white powder (0.141 g).
0.0200 mmol), AIBN (0.656 mg, 0.00400 mmol), and triox-
ane (21.2 mg) were dissolved in DMF (2.6 mL) in a sealed
2
0-mL vial. The vial was purged with nitrogen for 30 min
ꢀ
Solution Studies of PAPBA-b-PDMA
An example micellization procedure for poly(3-acrylamido-
phenylboronic acid) (PAPBA)-b-PDMA was conducted as fol-
and placed in a preheated reaction block at 70 C. Samples
were removed by syringe to determine the monomer conver-
sion by H NMR spectroscopy. DMSO-d6 was used as a sol-
1
lows. PAPBA-b-PDMA (53.5 mg, 0.00129 mmol, M ¼ 41,400
n
vent for NMR spectroscopy.
g/mol, M /M ¼ 1.40) dissolved in aqueous NaOH (2.80 mL,
w
n
RAFT Polymerization of DMA
DMA (2.99 g, 30.2 mmol), DMP (110 mg, 0.302 mmol), AIBN
2
wt/vol %) was dialyzed against deionized water. The con-
centration of the polymer in the aqueous solution was deter-
mined to be 0.5 wt/vol % from the mass of the polymer
originally dissolved and the volume of the final dialyzed
solution.
(
2.50 mg, 0.0152 mmol), and trioxane (53.8 mg) were dis-
solved in DMF (12.0 mL) in a sealed 20-mL vial. The vial
was purged with nitrogen for 30 min and placed in a pre-
heated reaction block at 70 C. Samples were removed peri-
odically by syringe to determine monomer conversion by H
3
NMR spectroscopy. CDCl was used as the solvent for NMR
spectroscopy. The resulting polymer was precipitated into
cold diethyl ether, dissolved in water, dialyzed against de-
ionized water, and the resulting solution was lyophilized.
ꢀ
To investigate the sugar-responsive behavior of the block co-
polymer aggregates, the dialyzed solutions were sonicated
for 30 min, and the pH was adjusted to 8.7 using a solution
of NaOH (0.1 M). Aggregate dissociation was observed by
turbidity measurements in a UV–vis spectrophotometer. For
sugar-responsive studies, samples were adjusted to 0.5 wt/
vol % by the addition of deionized water. Varying concentra-
tions of glucose or fructose (2 M) were added to the sam-
ples. Throughout the studies, samples were held in a water
1
RAFT Block Copolymerization of APBAE with
a DMA MacroCTA
An example RAFT block copolymerization of APBAE with
PDMA macroCTA was conducted as follows. APBAE (0.436 g,
ꢀ
bath heated to 37 C and were removed every hour for tur-
1.60 mmol), PDMA macroCTA (0.0502 g, 0.0107 mmol),
bidity measurements. Deionized water was used for a blank.
AIBN (0.540 mg, 0.00329 mmol), and trioxane (46 mg) (in-
ternal standard) were dissolved in DMF (2.20 mL) in a
sealed 20-mL vial. The solution was purged with nitrogen
a
To investigate the pK and the pH-responsiveness of the
block copolymers, the originally dialyzed solution of block
copolymer in deionized water was diluted to 0.1 wt/vol %
by the addition of deionized water and sonicated for 30 min.
The resulting solution was titrated to the targeted pH with a
solution of NaOH (0.1 M).
for 30 min and then placed in a preheated reaction block at
ꢀ
70
C. The polymerization was quenched after 21 h by
removing the polymerization vial from the heating block and
exposing the reaction solution to air. The resulting PDMA-b-
PAPBAE was isolated by precipitating into cold hexane, filter-
ing, and drying under vacuum. Samples were deprotected
using the procedure outlined above.
To investigate dye encapsulation and controlled release capa-
ꢂ4
bilities, PAPBA-b-PDMA (7.2 mg, 1.4 ꢁ 10
mmol, Mn
¼
5
3,000 g/mol, M /M
w
n
¼ 1.36) and Nile red (1.5 mg, 0.0047
mmol) were dissolved in methanol (0.7 mL). Deionized
water (3.75 mL) was slowly added to the solution over 25
min while stirring. The resulting solution was dialyzed
against deionized water for 24 h, and the mass of the solu-
tion was recorded. The concentration of the polymer in the
dialyzed solution was calculated from the mass of the poly-
mer originally dissolved and the volume of the final dialyzed
solution. The resulting solution was diluted to 0.1 wt/vol %
by the addition of deionized water. For pH-responsive release
studies, the PAPBA-b-PDMA block copolymer solutions were
adjusted to pH ¼ 12 by the addition of aqueous NaOH (2
wt/vol %). For sugar-responsive release studies, solutions of
fructose or glucose (2 M) were added to yield solutions of
PAPBA-b-PDMA with a targeted concentration of sugar. For
both the pH- and sugar-responsive release studies, the
adjusted and control solutions were allowed to sit at room
temperature for 1.5 h before being centrifuged at 6000 rpm
for 10 min. The supernatant was decanted for subsequent
fluorescence measurements.
RAFT Block Copolymerization of DMA and
APBAE with a DMA MacroCTA
An example RAFT block copolymerization of DMA and
APBAE with a PDMA macroCTA was conducted as follows.
DMA (0.0425 g, 0.429 mmol), APBAE (0.3282 g, 1.20 mmol),
PDMA macroCTA (0.0498 g, 0.011 mmol), AIBN (0.540 mg,
0.00329 mmol), and trioxane (72.4 mg) (internal standard)
were dissolved in DMF (2.20 mL) in a sealed 20-mL vial.
The solution was purged with nitrogen for 30 min and then
ꢀ
placed in a preheated reaction block at 70 C. The polymer-
ization was quenched after 20 h by removing the polymer-
ization vial from the heating block and exposing the reaction
solution to air. The resulting PDMA-b-(PAPBAE-co-PDMA)
was isolated by precipitating into cold hexane, filtering, and
drying under vacuum. Samples were deprotected using the
procedure outlined above.
RESULTS AND DISCUSSION
Block Copolymer Synthesis
Determination of DMA and APBAE Reactivity Ratios
An example RAFT block copolymerization of APBAE and
DMA with DMP was conducted as follows. APBAE (0.218 g,
We have previously reported that unprotected boronic acid
monomers can be directly polymerized by RAFT polymeriza-
4
6,47
tion.
However, polymeric boronic acids are hygroscopic,
0.798 mmol), DMA (0.1184 g, 1.19 mmol), DMP (7.28 mg,
difficult to handle, and often difficult to characterize.
4
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SCHEME 2 Synthesis of boronic ester and boronic acid homopolymers and block copolymers by RAFT polymerization.
Therefore, the polymers for this study were prepared by the
polymerization of a boronate ester monomer and were sub-
sequently deprotected to yield free boronic acid-containing
ment with theoretical molecular weights calculated from
reaction stoichiometry (Fig. 1, Table 1). The increase in mo-
lecular weight versus conversion was fairly linear over the
conversion range considered. SEC plots of the homopolyme-
rization at various stages of conversion demonstrated a
smooth shift to higher molecular weights, indicating the
polymerization proceeded in a controlled manner (Fig. 2).
Collectively, these data indicated the polymerizations of
APBAE were controlled, as expected for the RAFT process
(Table 1).
4
8,52
polymers.
The crude product from the APBAE monomer
synthesis (4) was a lightly yellow colored powder that if
directly polymerized resulted in a long inhibition period or
no polymerization via RAFT. Recrystallization from a mixture
of hexane and toluene resulted in a white, crystalline solid
that was readily polymerized by RAFT with reproducible
rates and minimal inhibition.
APBAE (4) was polymerized by RAFT using DMP (5) as the
CTA and AIBN as the initiator in DMF at 70 C (Scheme 2).
The PAPBAE homopolymers (6) were extended with DMA to
afford PAPBAE-b-PDMA (7) block copolymers (Scheme 2).
Chain extension was observed by SEC. For example, SEC
traces showed a fairly smooth shift from the 20,000 g/mol
PAPBAE macroCTA to a PAPBAE-b-PDMA block copolymer of
25,400 g/mol (Fig. 3). A series of amphiphilic diblock copoly-
mers with varying compositions was prepared (Table 1).
ꢀ
The polymerization stoichiometry was varied to investigate
the effect on reaction kinetics and the molecular weight con-
trol. Pseudo-first-order kinetic plots indicated the radical
concentration remained relatively constant throughout the
polymerizations [Fig. 1(A)]. Mn values were in good agree-
FIGURE 1 A: Pseudo-first-order kinetic plot for RAFT polymerization of APBAE with various ratios of monomer (Mon), CTA, initia-
tor (Init). B: M versus conversion for RAFT polymerization of APBAE with various ratios of [Mon]/[CTA]/[Init].
n
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TABLE 1 Results from the Synthesis of Poly(3-acrylamidophenylboronic acid pinacol ester) (PAPBAE) and PAPBAE-b-poly(N,N-
dimethylacrylamide) (PDMA)
a
b
c
d
d
Polymer
[Mon]/[CTA]/[Init]
Time (min)
Conv (%)
M
n,th (g/mol)
M
n, exp (g/mol)
PDI
PAPBAE
100/1/0.2
100/1/0.2
200/1/0.2
200/1/0.2
73/1/0.05
48/1/0.05
279/1/0.25
60
100
97
43
74
12,100
20,600
22,200
37,500
25,800
40,000
52,700
15,300
21,400
22,600
37,200
25,400
41,400
53,000
1.30
1.30
1.24
1.26
1.36
1.40
1.36
PAPBAE
PAPBAE
40
PAPBAE
150
180
180
180
68
PAPBAE59-b-PDMA67
PAPBAE130-b-PDMA61
PAPBAE75-b-PDMA298
a
80
100
100
d
Stoichiometric ratio of monomer/chain transfer agent/initiator.
Conversion determined by H NMR spectroscopy.
Experimental molecular weight and polydispersity index (M
determined by size exclusion chromatography.
w n
/M )
b
c
1
Theoretical molecular weight calculated using stoichiometry and
conversion.
To afford the desired boronic acid-containing block copoly-
mers, the boronic ester block copolymers were deprotected.
We have previously reported an effective deprotection proce-
dure for polymeric boronic esters utilizing polystyrene-sup-
absent, and the boron signal shifted from 21 ppm to approx-
imately 0.5 ppm, which is consistent with the formation of
negatively charged and sp -hybridized boron.
3
55,56
4
8
ported boronic acid resins. In addition to essentially quan-
titative ester cleavage to yield the desired boronic acid block
copolymers, this method has the benefit of facile purification,
as the resins can be easily removed after reaction via filtra-
tion. Successful deprotection to yield the free boronic acid
Block Copolymer Solution Behavior
Most interest in boronic acid-containing block copolymers
arises from their potential to undergo changes in water solu-
bility upon covalently binding to various sugars in solution.
However, boronic acids also demonstrate interesting pH-re-
sponsive behavior as a result of the Lewis acidic boron cen-
ter that is capable of reacting to yield negatively charged
boronate species (Schemes 1 and 2). A value of pKa ꢃ 10
was determined for the PDMA-b-PAPBA block copolymers by
titration. This value is higher than the pK of 8.8–9.1 previ-
ously reported for poly(N-isopropylacrylamide)-co-poly(3-
acrylamidophenylboronic acid) statistical copolymers,
though this discrepancy is likely the result of a reduction in
the ionization constant of the boronic acid homopolymer as
1
11
block polymers was confirmed by H- and B NMR spec-
troscopy in methanol-d (Supporting Information). The pina-
4
col methyl peaks of the PAPBAE repeat units at 1.2 ppm in
the proton spectrum were absent after the deprotection
2
reaction. Additionally, the sp boronic ester peak at 25 ppm
a
in the boron spectrum shifted to d ¼ 21 ppm, which was
5
5,56
consistent with boronic acid formation.
NMR spectros-
18,27
copy of the final boronic acid block copolymer was also con-
ducted in D O/NaOD. Again the pinacol methyl peaks were
2
5
7–59
a result of a high density of neighboring ionized groups.
FIGURE 2 SEC traces as a function of monomer conversion for
FIGURE 3 SEC traces for a PAPBAE homopolymer and block
the RAFT polymerization of APBAE.
copolymer with PDMA.
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6
0
solutions demonstrated a Tyndall effect, allowing disso-
ciation to be readily observed by a reduction in turbidity
[
Fig. 5(A)].
Micellar solutions with no glucose or fructose added were
used as the control and remained relatively stable through-
out the time range considered [Fig. 5(B)]. The dissociation
behavior was dependent on the identity of the sugar being
added. Fructose is known to bind most phenylboronic acids
1
5,61
with greater affinity than glucose.
Accordingly, at all
ratios of fructose to boronic acid groups, aggregate dissocia-
tion was rapid and efficient. On the other hand, aggregate
dissociation was highly dependent on the ratio of glucose to
boronic acid groups, with higher ratios leading to faster and
more efficient dissociation. When a solution of micelles was
adjusted to 30 mM fructose, only unimers were observed by
DLS. Micelles remained intact at a similar concentration of
FIGURE 4 Relative hydrodynamic diameter versus pH for
PAPBA-b-PDMA block copolymers of varying composition.
The pK of most polyacids indicates the pH below which the
a
polymer will be insoluble. Therefore, a diblock copolymer
with a hydrophilic block and boronic acid block is expected
to form micellar aggregates at pH < pK
a
, as we previously
4
6,47
reported.
Indeed, DLS of a solution of PAPBA131-b-
PDMA138 revealed the presence of aggregates with a hydro-
dynamic diameter (D ) of 35 nm at pH ¼ 8.7.
H
A more direct understanding of the pH-responsive behavior of
the PDMA-b-PAPBA block copolymer was gained by observing
the evolution of solution aggregate size as a function of pH
(
Fig. 4). The initial aggregate size depended on the block co-
polymer composition and copolymer molecular weight. How-
ever, in all cases the sizes of the aggregates increased only
slightly until the pH was near the pK , at which point a more
a
significant increase in size was observed. As the pH was
raised beyond the pK , the aggregates dissociated into unim-
a
ers, as apparent from the dramatic reduction in relative size
observed by DLS. The data suggested that the aggregates
swelled as the hydrophilicity of the core increased due to
gradual ionization of the boronic acid residues until the inte-
rior portion of the aggregates became hydrophilic enough to
result in complete dissociation.
As previously discussed, boronic acid-containing block
copolymers have the ability to modulate their solubility by
the change in ionization that occurs upon binding with sug-
ars in aqueous solution. This behavior has led to boronic
acid polymers being considered for various insulin-release
1
6
applications for the potential treatment of diabetes. How-
ever, in order for the boronic acid block copolymers to fulfill
this potential, a better understanding of the sugar-responsive
aggregation and dissociation phenomena is needed.
FIGURE 5 A: Schematic representation of boronic acid micelle
dissociation in response to increased sugar concentration (blue
block ¼ PDMA; red block ¼ PAPBA). The photographs show
representative solutions of aggregates (turbid) and unimers
We investigated the dissociation of the PAPBA-b-PDMA block
copolymer aggregates as a function of glucose and fructose
concentrations. At pH ¼ 8.7, the block copolymers are
expected to exist as aggregates with hydrophilic PDMA coro-
nas and hydrophobic PAPBA cores [Fig. 5(A)]. The aggregate
(
clear). B: Turbidity as a function of time and concentration of
glucose of fructose. Sugar concentrations are expressed as fac-
tors of molar excess with respect to boronic acid units for solu-
tions of PAPBA59-b-PDMA67 at a concentration of 0.5 wt/vol %
and pH ¼ 8.7.
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of 4 nm was observed after the addition of fructose. Removal
of excess fructose by dialysis against water led to the block
copolymers self-assembling to form aggregates with sizes
nearly identical to the original aggregates (Fig. 6). This re-
versibility is consistent with the dynamic-covalent nature of
6
2
boronic acid–diol complexes.
The PDMA-b-PAPBA block copolymer aggregates were inves-
tigated for their ability to solubilize and release model
hydrophobic small molecules [Fig. 7(A)]. Nile Red is a hydro-
phobic dye that exhibits fluorescence strongly dependent on
the local polarity of its environment. In a hydrophobic micro-
environment, strong fluorescence emission is observed, while
6
3,64
the emission intensity is very weak in aqueous media.
Hydrophobic encapsulation of Nile Red within an aqueous
medium should result in a blue shift and a dramatic increase
6
4,65
in fluorescence intensity.
In the absence of sugar and at
pH 8.7 (i.e., below the pKa of the boronic acid groups), a
strong fluorescence emission at ꢄ 635 nm was observed.
Upon increasing pH or the addition of fructose, a dramatic
reduction in fluorescence was observed [Fig. 7(B)]. This
behavior is consistent with encapsulation and pH- or sugar-
induced release of the model compound. Additionally, the
degree of release, as judged by the reduction in fluorescence
intensity was shown to be dependent on the sugar identity
(fructose>glucose) and concentration [Fig. 7(C)], in agree-
ment with the previously discussed turbidity results.
FIGURE 6 Hydrodynamic diameter distribution of PAPBA75-b-
PDMA298 before fructose addition, after fructose addition, and
after fructose removal by dialysis. [fructose] ¼ 73 mM corre-
sponds to [fructose]/[APBA units] ¼ 52.
glucose (Supporting Information). However, at a concentra-
tion of 90 mM glucose, the micelles were observed to be
completely dissociated.
Interestingly, sugar-induced dissociation of the aggregates
could be reversed by dialysis against water. A solution of
The ultimate utility of boronic acid-based sugar-responsive
polymers depends on their ability to respond at physiologi-
PAPBA -b-PDMA
298
forms aggregates with an average
7
5
5
0,52
hydrodynamic diameter of 70 nm before the addition of
fructose; however only the presence of unimers with a size
cally relevant pH
and glucose concentrations (between
6
6
2.5 and 20 mM). We reasoned that one possible method of
FIGURE 7 A: Proposed encapsulation and release of Nile Red by PAPBA-b-PDMA aggregates. B: Fluorescence emission of solu-
tions of PAPBA75-b-PDMA298 before and after aggregate dissociation by increasing pH. C: Fluorescence emission of solutions of
PAPBA75-b-PDMA298 as a function of sugar concentration.
8
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tuning the critical glucose concentration needed to induce
aggregate dissociation was incorporation of comonomers
within the boronic acid block. For example, dissociation at
lower glucose concentrations could potentially be achieved by
including comonomers that increase the overall hydrophilicity
of the responsive block. With this goal in mind, we investi-
gated the synthesis of PDMA-b-poly(APBA-stat-DMA) block
copolymers, where the DMA content within the responsive
block was tuned to adjust the sugar-responsive behavior.
From a series of RAFT polymerizations, the relative reactivity
ratios for APBAE and DMA were determined by the Kelen–
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2
RAFT of APBAE and subsequent deprotection by a mild
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