Synthesis of pH- and thermoresponsive poly(2-n-propyl-2-oxazoline) based copolymers

Article abstract of DOI:10.1002/pola.28011

Polymers that possess lower critical solution temperature behavior such as poly(2-alkyl-2-oxazoline)s (PAOx) are interesting for their application as stimulus-responsive materials, for example in the biomedical field. In this work, we discuss the scalable and controlled synthesis of a library of pH- and temperature-sensitive 2-n-propyl-2-oxazoline P(nPropOx) based copolymers containing amine and carboxylic acid functionalized side chains by cationic ring opening polymerization and postpolymerization functionalization strategies. Using turbidimetry, we found that the cloud point temperature (CP) is strongly dependent on both the polymer concentration and the polymer charge (as a function of pH). Furthermore, we observed that the CP decreased with increasing salt concentration, whereas the CP increased linearly with increasing amount of carboxylic acid groups. Finally, turbidimetry studies in PBS-buffer indicate that CPs of these polymers are close to body temperature at biologically relevant polymer concentrations, which demonstrates the potential of P(nPropOx) as stimulus-responsive polymeric systems in, for example, drug delivery applications.

Full text of DOI:10.1002/pola.28011

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Synthesis of pH- and Thermoresponsive Poly(2-n-propyl-2-Oxazoline)
Based Copolymers
Marcel A. Boerman,1,2,3 Harry L. Van der Laan,1 Johan C. M. E. Bender,3
Richard Hoogenboom,4 John A. Jansen,2 Sander C. Leeuwenburgh,2 Jan C. M. Van Hest1
1Radboud University Nijmegen, Institute for Molecules and Materials (IMM), Heyendaalseweg 135, 6525 AJ Nijmegen,
The Netherlands
2Radboudumc, Department of Biomaterials, 6500 HB Nijmegen, The Netherlands
3Bender Analytical Holding B.V, Parksesteeg 8, 6611 KH Overasselt, The Netherlands
4Supramolecular Chemistry Group, Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4,
9000 Ghent, Belgium
Correspondence to: J. C. M. Van Hest (E-mail: j.vanhest@science.ru.nl)
Received 18 October 2015; accepted 25 November 2015; published online 5 January 2016
DOI: 10.1002/pola.28011
ABSTRACT: Polymers that possess lower critical solution tem-
perature behavior such as poly(2-alkyl-2-oxazoline)s (PAOx) are
interesting for their application as stimulus-responsive materi-
als, for example in the biomedical field. In this work, we dis-
cuss the scalable and controlled synthesis of a library of pH-
and temperature-sensitive 2-n-propyl-2-oxazoline P(nPropOx)
based copolymers containing amine and carboxylic acid func-
tionalized side chains by cationic ring opening polymerization
and postpolymerization functionalization strategies. Using tur-
bidimetry, we found that the cloud point temperature (CP) is
strongly dependent on both the polymer concentration and the
observed that the CP decreased with increasing salt concentra-
tion, whereas the CP increased linearly with increasing amount
of carboxylic acid groups. Finally, turbidimetry studies in PBS-
buffer indicate that CPs of these polymers are close to body
temperature at biologically relevant polymer concentrations,
which demonstrates the potential of P(nPropOx) as stimulus-
responsive polymeric systems in, for example, drug delivery
C
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applications. 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part
A: Polym. Chem. 2016, 54, 1573–1582
KEYWORDS: stimuli-sensitive polymers; poly-2-oxazolines;
biocompatibility
polymer charge (as
a function of pH). Furthermore, we
occasionally challenging synthesis procedure, the occurrence of
hysteresis between heating and cooling, as well as poor tunabil-
ity of the transition temperature have, however, stimulated the
search for polymer alternatives.11 New candidates have been
identified recently, such as the poly(oligo ethylene glycol) meth-
acrylate family.12–14 Another very promising type of polymers
are the poly(2-alkyl-2-oxazoline)s15–18 or PAOx, which are a
class of pseudo-polypeptides that have gained increasing
research interest because of their ease of synthesis, structural
versatility, limited hysteresis compared with P(NIPAM) and
potential for application as biomaterials because of their promis-
ing biocompatibility profile.19–21 The wide range of studies on
the thermoresponsive properties of PAOx that have been per-
formed to date include the LCST behavior of 2-ethyl-2-oxazoline
(EtOx, CP 5 65 8C), 2-isopropyl-2-oxazoline (iPropOx, CP 5 37
8C) and 2-n-propyl-2-oxazoline (nPropOx, CP 5 24 8C) homopol-
ymers, as well as a great number of PAOx based copoly-
mers.22–24 For application in the human body, a transition
INTRODUCTION Stimuli-responsive polymeric systems are of
great interest due to their wide potential in a range of appli-
cations (for e.g., drug delivery1–3 and coating applications4).
Polymers that possess lower critical solution temperature
(or LCST) behavior are particularly interesting in that
respect, since these thermosensitive polymers are water
soluble below their so-called cloud point (CP), but insoluble
above this critical temperature. This behavior is driven by an
unfavorable entropy of mixing above the CP, which causes
precipitation of the polymer from aqueous solutions. This CP
depends on a large number of parameters including mono-
mer composition, polymer concentration, overall hydropho-
bic/hydrophilic nature, polymer length, as well as external
factors like the type of solvent, pH, salt concentration and
other additives.5–10
Poly(N-isopropyl acrylamide) (P(NIPAM)) is one of the most fre-
quently studied thermoresponsive polymers (LCST5 32 8C). Its
Additional Supporting Information may be found in the online version of this article.
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temperature around body temperature is essential. As a conse-
quence, iPropOx based copolymers have been studied most
extensively. The lower transition temperature of nPropOx based
copolymers appears to make them the less obvious choice for
biomedical purposes. Nevertheless, these more hydrophobic
nPropOx based polymers can be of interest because of their sol-
ubility in a wide range of organic solvents, which renders them
particularly suitable in, for example, coating applications and
which facilitates the preparation of polymer-drug conjugates.
Moreover, in comparison to P(iPropOx), P(nPropOx) has the
additional advantage to be amorphous and does not show irre-
versible crystallization upon annealing above the CP.25–27 Finally,
taken into account that cationic ring opening polymerization
(CROP) allows for the introduction of a wide range of (pro-
tected) hydrophilic moieties, for example carboxylic acid or
amine functional groups, these polymers can be made both tem-
perature and pH responsive around body temperature.
in the polymerizations. Acetonitrile was dried and dispensed
under nitrogen atmosphere by using an MBraun MB SPS-800
solvent dispersing system. For turbidimetry measurements,
ultrapure Milli-Q water (MQ) was obtained from a WaterPro
PS polisher (Labconco, Kansas City, MO) set to 18.2 MX/cm.
Characterization
1H-NMR and 13C-NMR spectra were recorded on a Bruker
Avance III 500MHz spectrometer using the solvents D2O,
MeOD, CDCl3 or DMSO-d6. FT-IR measurements were per-
formed on
a Bruker Tensor 27 IR ATR spectrometer.
Microwave-assisted polymerizations were performed in a
Biotage Initiator1, equipped with an autosampler. Size exclu-
sion chromatography (SEC) was performed on an Agilent
1260 - series HPLC system equipped with a 1260 online
degasser, a 1260 ISO-pump, a 1260 automatic liquid sampler,
a thermostatted column compartment, a 1260 diode array
detector and a 1260 refractive index detector. Analyses were
performed on two Mixed-D and a guard column in series at
50 8C. As an eluent, N,N-dimethylacetamide (DMA), contain-
ing LiCl (concentration 50 mM), was used at a flow rate of
0.593 mL min21. The SEC traces were analyzed using the
Agilent Chemstation software with the GPC add on. Number
average molecular weights (Mn), weight average molecular
weights (Mw), and dispersity (Ð) values were calculated
against poly(methyl methacrylate) (PMMA) standards.
To date however, no polymeric systems have been designed
that combine both the beneficial properties of nPropOx and
the structural versatility of PAOx in forming both pH and
thermoresponsive polymers. Moreover, the solution proper-
ties of both pH and temperature responsive polymeric sys-
tems consisting entirely of PAOx are only scarcely reported
and have not systematically been investigated in the field.28
Since the direct incorporation of both amine and carboxylic
acid moieties would interfere with the CROP reaction of 2-
oxazolines, both functionalities have to be introduced in a
protected manner. To this end, the methyl ester-
functionalized MestOx 3 is highly suitable.18 It allows, after
polymerization, the introduction of both functional groups
via a one-step modification reaction. The polymerization
kinetics of this monomer with various 2-alkyl-2-oxazoline
comonomers has been reported by Bouten et al.,29 and this
monomer has also been copolymerized with P(NIPAM) and
postmodified forming temperature- and pH-responsive poly-
mers.28,30 In this respect, MestOx seems to be an ideal
monomer for the synthesis of more polar and stimuli-
responsive nPropOx-based copolymers.
Monomer Distillations
Monomers were distilled using a KDL-1 Wiped film evaporation
setup (UIC GmbH) containing both an oil pump and an oil diffu-
sion pump. Details can be found in the supporting information.
Turbidimetry
UV-VIS spectra were recorded on a Jasco V650 containing a
temperature controller at a wavelength of 500 nm. Measure-
ments were conducted at various polymer concentrations
using different aqueous solutions, both specified in the main
text. For these measurements, two subsequent cycles of heat-
ing and cooling were conducted. The CPs were recorded as
the temperature (8C) at which 50% transmittance was
observed during the second heating cycle without stirring.
Herein, we present the preparation and stimulus-responsive
properties of P(nPropOx)-based copolymers containing car-
boxylic acid and amine functional side chains. We demon-
Synthesis
Monomers
nPropOx (1) was synthesized using a modified literature
procedure31 - Butyronitrile (500 mL, 5.75 mol, 1 eq.), 2-
amino ethanol (520 mL, 8.61 mol, 1.5 eq.), and
Zn(OAc)2Á2 H2O (24.7 g; 0.11 mol, 0.02 eq.) as a catalyst
were mixed and heated to reflux (ꢀ130 8C) overnight. The
resulting yellow-orange reaction mixture was cooled down
to room temperature. Afterwards DCM (1000 mL) was added
and the organic layer was washed with water (2 3 500 mL)
and brine (500 mL). The organic phase was dried over anhy-
drous Na2SO4 and subsequently filtered. The solvent was
evaporated under reduced pressure, yielding a yellow oil
(256.8 g). After two distillations (1.5Á1022 mbar) using the
KDL-1, nPropOx (1) was obtained as a colorless liquid
(167.74 g, 26% yield). 1H NMR (500 MHz, CDCl3) d 4.13 (t,
J 5 9.5 Hz, 2H, CH2AO), 3.74 (t, J 5 10 Hz, 2H, CH2N), 2.16
strate
a versatile scalable synthesis strategy of these
polymers based on the copolymerization of nPropOx and
MestOx followed by a modification reaction, yielding both
pH- and thermoresponsive copolymers. Finally, using tur-
bidimetry, a detailed study in various aqueous solutions is
performed to determine the behavior of these polymers at
various biologically relevant conditions.
EXPERIMENTAL
Materials
All reagents (synthesis grade) for the synthesis of the mono-
mers were purchased at Sigma Aldrich and used without fur-
ther purification, unless stated otherwise. All reagents for
the synthesis of the polymers were distilled twice before use
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(m, 2H, CH2ACH2ACH3), 1.58 (m, 2H, CH2ACH2ACH3), 0.89
(t, J 5 7.4 Hz, 3H, CH2ACH2ACH3) 13C NMR (500 MHz,
CDCl3) d 167.96, d 66.66, d 53.94, d 29.43, d 18.98, d 13.30
TLC Rf 5 0.82, (MeOH/DCM, v/v, 9:1)
P(nPropOx) (P1) was obtained as a white foam (1.9 g, 76%
yield). 1H NMR (500 MHz, CDCl3) d 3.48 (b, nÁ4H, NCH2CH2N),
3.05 (b, 3H, CH3ANCHCHA), 2.35–2.20 (b, nÁ2H,
COACH2ACH2ACH3), 1.60–1.40 (b, nÁ2H, COACH2ACH2ACH3),
0.92–1.01 (b, nÁ3H, COACH2ACH2ACH3) FT-IR: 1636 cm21
MestOx-precursor (2) was synthesized following a modified
literature procedure32 - Methyl succinyl chloride (271.8 g,
1.805 mol, 1 eq.) was dissolved in DCM (2.00 L) and 2-
chloroethylamine HCl (209.2 g, 1.804 mol, 1 eq.) was added.
The reaction mixture was cooled to 0 8C and triethylamine
(535 mL, 3.84 mol, 2.13 eq.) was added drop wise over 2 h.
The reaction mixture was stirred for 4 days. The reaction
mixture turned orange and a precipitate (Et3N-HCl) was
formed. Subsequently, the reaction mixture was filtered and
divided into three equal parts. Each part was washed twice
with water (2 3 80 mL) and brine (1 3 80 mL). The organic
layers were combined and dried over anhydrous Na2SO4 and
subsequently filtered. The mixture was evaporated under
reduced pressure yielding a brown oil (323.7 g). After two
distillations (4.4Á1023 mbar) using the KDL-1 the MestOx
precursor (2) was obtained as a colorless oil which solidified
in time (186.5 g, 53% yield). 1H NMR (500 MHz, CDCl3) d
6.32 (s, 1H, NHCO), 3.70 (s, 1H, CH3AOACO), 3.66 (t, J 5 7.4
Hz, 2H, CH2ACl), 3.54 (t, J 5 7.4 Hz, 2H, CH2ANHACO), 2.69
(t, J 5 7.4 Hz, 2H, NHACOACH2), 2.59 (t, J 5 7.4 Hz, 2H,
CH2ACOAO)
-
(CO amide) SEC (PMMA) Mn 5 20.2 kg/mol, D 5 1.20.
P2-P(nPropOx-c-MestOx) 90-10 - Methyl tosylate (0.131 mL,
0.870 mmol), nPropOx (4.83 mL, 43.5 mmol), MestOx (2.99 mL,
21.8 mmol), and acetonitrile (7.60 mL) were mixed in a Schlenk
flask under inert atmosphere (Ar). After the addition, the flask
was put in a preheated oil bath (80 8C) and stirred during 10 h.
Next, dry piperidine (0.430 mL, 4.35 mmol) was added to the
reaction mixture, which was stirred for three hours. The poly-
mer was dissolved in an appropriate amount of DCM and pre-
cipitated in diethyl ether (DCM/Et2O, v/v, 1:20). This procedure
was performed two times. The resulting suspension was fil-
tered and the residue dissolved in DCM (100 mL). The solvent
was evaporated under reduced pressure. P(nPropOx)-c-Mes-
tOx) 90-10 (P2) was obtained as a white foam (53.1 g, 76%
yield). 1H NMR (500 MHz, CDCl3)
d
3.66 (b, mÁ3H,
COAOACH3), d 3.48 (b, (m 1 n)Á4H, NCH2CH2N), 3.03 (b, 3H,
CH3ANCHCHA), 2.70–2.60 (b, mÁ4H, COCH2CH2CO), 2.35–2.20
(b, nÁ2H, COACH2ACH2ACH3), 1.70–1.60 (b, nÁ2H,
COACH2ACH2ACH3), 0.91–1.02 (b, nÁ3H, COACH2ACH2ACH3)
-
SEC (PMMA) Mn 5 16.7 kg/mol, D 5 1.23
P3- P(nPropOx-c-MestOx) 70-30 - The reaction was per-
formed similar to P2 using methyl tosylate (0.131 mL, 0.870
mmol), nPropOx (4.83 mL, 43.5 mmol), MestOx (2.99 mL, 21.8
mmol) and acetonitrile (7.60 mL). P(nPropOx)-c-MestOx) 90-10
(P3) was obtained as a white foam (23.4 g, 86% yield). 1H
NMR (500 MHz, CDCl3) d 3.66 (b, mÁ3H, COAOACH3), d 3.48
(b, (m 1 n)Á4H, NCH2CH2N), 3.03 (b, 3H, CH3ANCHCHA), 2.70–
2.60 (b, mÁ4H, COCH2CH2CO), 2.35–2.20 (b, nÁ2H,
COACH2ACH2ACH3), 1.70–1.60 (b, nÁ2H, COACH2ACH2ACH3),
0.93–1.01 (b, nÁ3H, COACH2ACH2ACH3) SEC (PMMA)
MestOx (3) was synthesized following a modified literature
procedure32 - MestOx-precursor 2 (132.3 g, 0.68 mol, 1 eq.)
was heated to 45 8C to melt the solid. Subsequently, sodium
carbonate (70.62 g, 0.67 mol, 1 eq.) was added gently, which
caused the formation of CO2. The reaction mixture was
heated and stirred overnight (60 8C, 20 mbar) using a rotary
evaporator. After overnight reaction the absence of CO2 for-
mation indicated full conversion. The reaction mixture was
diluted with Et2O (500 mL) and the resulting viscous sus-
pension was filtered. The solvent was removed under
reduced pressure yielding the crude material (104.7 g). After
two distillations (2.3Á1022 mbar) using the KDL-1, MestOx
(3) was obtained a colorless oil which solidified over time.
(86.3 g, 80% yield). 1H NMR (500 MHz, CDCl3) d 4.20 (t,
J 5 9.5 Hz, 1H, CH2O), 3.78 (t, J 5 9.4 Hz, 1H, CH2N), 3.66 (s,
3H, CH3AOCO), 2.64 (t, J 5 9.5 Hz, 2H, CH2CH2COO), 2.55 (t,
J 5 7.5 Hz, 2H, CH2CH2COO). 13C NMR (500 MHz, CDCl3) d
172.67, d 166.97, d 67.45, d 54.35, d 51.73, d 30.01, d 23.04
-
Mn 5 20.1 kg/mol, D 5 1.17.
P4- P(nPropOx-c-MestOx) 50-50 - The reaction was per-
formed similar to P2 using methyl tosylate (0.131 mL, 0.870
mmol), nPropOx (4.83 mL, 43.5 mmol), MestOx (2.99 mL, 21.8
mmol), and acetonitrile (7.60 mL). P(nPropOx)-c-MestOx) 50-
1
50 (P4) was obtained as a white foam (14.2 g, 81% yield). H
NMR (500 MHz, CDCl3) d 3.66 (b, nÁ3H, COAOACH3), d 3.48 (b,
(m1 n)Á4H, NCH2CH2N), 3.03 (b, 3H, CH3ANCHCHA), 2.70–
2.60 (b, mÁ4H, COCH2CH2CO), 2.35–2.20 (b, nÁ2H,
COACH2ACH2ACH3), 1.70–1.60 (b, nÁ2H, COACH2ACH2ACH3),
0.90–1.01 (b, nÁ3H, COACH2ACH2ACH3) SEC (PMMA)
Polymers
P1- P(nPropOx) - Methyl tosylate (0.131 mL, 0.870 mmol),
nPropOx (4.83 mL, 43.5 mmol) and acetonitrile (7.60 mL) were
mixed in a dry microwave vial under inert atmosphere (Ar).
The polymerization was heated for 30 min under microwave
irradiation after which dry piperidine (0.430 mL, 4.35 mmol)
was added to the reaction mixture, which was stirred for three
hours. The polymer was dissolved in an appropriate amount of
DCM and precipitated in diethylether (DCM/Et2O, v/v, 1:20).
This procedure was performed two times. The resulting sus-
pension was filtered and the residue dissolved in DCM
(100 mL). The solvent was evaporated under reduced pressure.
-
Mn 5 20.3 kg/mol, D 5 1.12.
P5- P(nPropOx-c-MestOx) 30-70 - The reaction was per-
formed similar to P1 using methyl tosylate (0.032 mL, 0.214
mmol), nPropOx (0.713 mL, 6.4 mmol), MestOx (2.06 mL, 21.8
mmol), and acetonitrile (2.54 mL). P(nPropOx)-c-MestOx) 30-
1
70 (P5) was obtained as a white foam (2.7 g, quant. yield). H
NMR (500 MHz, CDCl3) d 3.60 (b, mÁ3H, COAOACH3), d 3.45
(b, (m1 n)Á4H, NCH2CH2N), 2.70–2.50 (b, mÁ4H, COCH2CH2CO),
2.35–2.15 (b, nÁ2H, COACH2ACH2ACH3), 1.50 (b, nÁ2H,
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COACH2ACH2ACH3), 0.91–1.01 (b, nÁ3H, COACH2ACH2ACH3)
acid), 3363 cm21 (OH carboxylic acid) SEC (PMMA)
-
-
SEC (PMMA) Mn 5 20.7 kg/mol, D 5 1.32.
Mn 5 19.2 kg/mol, D 5 1.25.
Protocol 1: General Procedure Ester Hydrolysis (P6-P9)
P(nPropOx-c-MestOx) (P2-P5) was dissolved in a 0.1 M-solution
of NaOH (100 mL) and stirred overnight. The reaction mixture
was acidified to pH 4 by dropwise addition of a 0.1 M-solution of
HCl. The reaction mixture was stirred in a heated water bath (45
8C), which caused precipitation of the polymer as a sticky white
precipitate. The precipitate was dissolved in cold water (100 mL)
and the reaction mixture was stirred again in the heated water
bath, which caused the polymer to precipitate again. The pH of
the solution was 4. The sticky white precipitate was either (1) dis-
solved again in DCM (100 mL) and concentrated under reduced
pressure or (2) precipitated from DMF (20 mL) into diethyl ether
(2000 mL) and subsequently filtered and dried under high vac-
uum, yielding the final polymer as a white fluffy powder.
Amidation
P10-P(nPropOx-c-NH2) 90-10 was synthesized following a modi-
fied literature procedure33 - P2 (4.21 g, 4 mmol functional groups)
was dissolved in ethylene diamine (25 mL, 22.5 g, 0.37 mol, 10
eq.). The reaction mixture was allowed to stir overnight. Excess
ethylene diamine was removed under reduced pressure. The poly-
mer was dissolved in methanol (20 mL) and precipitated in diethyl
ether (250 mL). A sticky precipitate was formed. This was
repeated twice. In order to remove residual ethylene diamine, the
polymer was subsequently dissolved in cold water (50 mL), after
which brine (10 mL) was added and the mixture was stirred in a
water bath (50 8C), causing the polymer to precipitate. After this,
the polymer was dissolved in methanol (30 mL). After concentra-
tion of the solvent under reduced pressure, P(nPropOx-c-NH2)
1
(P10) was obtained as a white solid (1,4 g, 30% yield) H NMR
P6-P(nPropOx-c-COOH) 90-10 was synthesized from P2
(10.2 g, 9 mmol functional groups) according to protocol 1,
yielding P(nPropOx-c-COOH) 90-10 (P6) as a white solid (6.8 g,
68% yield). 1H NMR (500 MHz, D2O) d 3.71–3.30 (b,
(m1 n)Á4H, NCH2CH2N), 2.60–2.50 (b, mÁ4H, COCH2CH2CO),
2.35–2.20 (b, nÁ2H, COACH2ACH2ACH3), 1.50–1.40 (b, nÁ2H,
COACH2ACH2ACH3), 0.90–1.00 (b, nÁ3H, COACH2ACH2ACH3)
(500 MHz, DMSO-d6) d 12.01 (b, mÁ1H, COOH) FT-IR:
1639 cm21 (CO amide), 1729 cm21 (CO carboxylic acid),
3503 cm21 (OH carboxylic acid) SEC (PMMA) Mn 5 20.6 kg/
(500 MHz, D2O) d 3.55–3.35 (b, nÁ4H, NCH2CH2N), 3.35–3.30 (b,
mÁ2H,
CONHACH2CH2ANH2),
2.95–2.90
(b,
mÁ2H,
CONHACH2CH2ANH2), 2.70–2.55 (b, mÁ2H, COCH2CH2CO), 2.55–
2.40 (b, mÁ2H, COCH2CH2CO), 2.35–2.20 (b, nÁ2H,
COACH2ACH2ACH3), 1.55–1.40 (b, nÁ2H, COACH2ACH2ACH3),
0.90–0.80 (b, 3H, nÁCOACH2ACH2ACH3) FT-IR: 1670 cm21 (CO
amide), 3470 cm21 (NH stretch) SEC (PMMA) Mn 5 21.4 kg/mol,
-
D 5 1.20.
RESULTS AND DISCUSSION
-
mol, D 5 1.16.
Synthesis
Monomers
P7-P(nPropOx-c-COOH) 70-30 was synthesized from P3
(10.0 g, 24 mmol functional groups) according to protocol 1,
yielding P(nPropOx-c-COOH) 70-30 (P7) as a white solid (2.4 g,
25% yield). 1H NMR (500 MHz, D2O) d 3.72–3.32 (b,
(m1 n)Á4H, NCH2CH2N), 2.60–2.50 (b, mÁ4H, COCH2CH2CO),
2.35–2.20 (b, nÁ2H, COACH2ACH2ACH3), 1.50–1.40 (b, nÁ2H,
COACH2ACH2ACH3), 0.90–1.00 (b, nÁ3H, COACH2ACH2ACH3)
The monomers were synthesized via procedures described
in literature.34,35 In short, nPropOx (1) was formed by
refluxing butyronitrile and ethanolamine with Zn(OAc)2 as
a catalyst (Scheme 1, i). For MestOx, a two-step approach
was followed because of the labile character of the methyl
ester side chain. First, a monomer precursor (2) was
formed, after which ring closure was facilitated by reaction
with Na2CO3, which resulted in the formation of MestOx
(3). This reaction was performed on the rotary evaporator
to drive the reaction to completion by removal of the
formed water and CO2. Both monomers 1 and 3 were puri-
fied twice by wiped film evaporation (for details see ESI)
and were kept under argon atmosphere to ensure dry con-
ditions for polymerization. Both monomers were obtained
in large amounts, 168 g and 86 g for (1) and (3), respec-
tively, demonstrating the scalability of both the synthesis
and distillation methods.
-
SEC (PMMA) Mn 5 24.8 kg/mol, D 5 1.16.
P8-P(nPropOx-c-COOH) 50-50 was synthesized from P4
(10.0 g, 40 mmol functional groups) according to protocol 1,
yielding P(nPropOx-COOH) 50-50 (P8) as a white solid (6.8 g,
68% yield). 1H NMR (500 MHz, D2O) d 3.70–3.30 (b,
(m1 n)Á4H, NCH2CH2N), 2.60–2.50 (b, mÁ4H, COCH2CH2CO),
2.35–2.20 (b, nÁ2H, COACH2ACH2ACH3), 1.50–1.40 (b, nÁ2H,
COACH2ACH2ACH3), 0.90–1.00 (b, nÁ3H, COACH2ACH2ACH3)
(500 MHz, DMSO-d6) d 11,68 (b, nÁ1H, COOH) FT-IR:
1619 cm21 (CO amide), 1721 cm21 (CO carboxylic acid),
3413 cm21 (OH carboxylic acid) SEC (PMMA) Mn 5 21.2 kg/
-
mol, D 5 1.11.
Polymers
The polymers were synthesized by CROP, using microwave
reaction conditions36 (Scheme 2). For the copolymerization
of MestOx and nPropOx, the monomers were mixed in the
desired ratios to form statistical copolymers with close to
random monomer distribution as recently reported32 (Table
1). In these experiments, methyl tosylate was used as an ini-
tiator and piperidine as a terminating agent which prevents
saponification of the methyl ester side chains of MestOx.
P9- P(nPropOx-c-COOH) 30-70 was synthesized from P4
(2.21 g, 10 mmol functional groups) according to protocol 1,
yielding P(nPropOx-COOH) 30-70 (P9) as a white solid
(1.4 g, 66% yield). 1H NMR (500 MHz, D2O) d 3.71–3.42 (b,
(m 1 n)Á4H, NCH2CH2N), 2.71–2.51 (b, mÁ4H, COCH2CH2CO),
2.42–2.12 (b, nÁ2H, COACH2ACH2ACH3), 1.60–1.41 (b, nÁ2H,
COACH2ACH2ACH3), 0.91–0.89 (b, nÁ3H, CO–CH2–CH2–CH3)
FT-IR: 1627 cm21 (CO amide), 1714 cm21 (CO carboxylic
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SCHEME 1 Overview of monomer synthesis, (i) Zn(OAc)2 (cat.), 120 8C, overnight, (ii) Et3N, CH2Cl2, 0 8C, (iii) Na2CO3 (0.9 eq.), 60 8C,
20 mbar, overnight.
After threefold precipitation in ether, P1-P5 were obtained
in multigram scale in overall high yields (76%). The poly-
mers containing MestOx-side chains P2-P5 were post-
functionalized after polymerization. This was achieved either
by (1) hydrolysis of the methyl ester side chains to carbox-
ylic acid moieties using 1M NaOH, (Scheme 2, ii) or by (2)
an amidation reaction in bulk using ethylene diamine as a
solvent, yielding the polymer with amine-functionalized side
chains (Scheme 2, iii)
nary experiments on the direct amidation demonstrated
the feasibility of this approach (results not shown). How-
ever, for this study, we chose to continue with the poly-
mer that was successfully synthesized (P10) for proof-of-
concept for the pH-responsivity and have not synthesized
copolymers with other amine contents. Since the work-up
of this polymer (evaporation of solvent, followed by
threefold precipitation in ether) proved insufficient to
remove all ethylene diamine, a similar workup approach
as for P6-P9 was performed. The crude polymer was dis-
solved in water and brine was added, which caused pre-
cipitation of the polymer. After drying the polymer, P10
was obtained in a pure state (30% yield). We suspect
that in this case, intermolecular crosslinking is absent
because in this case soluble polymers were obtained.
Moreover, intramolecular crosslinking is minimal because
this would result in both a lower NH2 percentage com-
pared with MestOx and additional signals by 1H-NMR,
which were both not observed. Moreover, the dispersity
of the obtained copolymer is very similar to the starting
MestOx copolymer further confirming that no significant
chain coupling occurred.
The workup of P6-P9 was performed by decreasing the
pH of the solution to 4 to protonate the side chains, and
subsequent heating, which caused the polymers to precipi-
tate. After multiple cycles of dissolving the polymers and
precipitation by heating, the polymers P6-P9 were
obtained as pure white powders in multigram quantities.
This demonstrates that the thermoresponsive behavior of
these copolymers can be used for the workup of these
polymers.
For the amidation reaction, we aimed to synthesize poly-
mers containing various molar percentages of amine
functionalities. Unfortunately, we observed intermolecular
crosslinking for polymers with higher degrees (>20%) of
MestOx-functionalization by gelation of the reaction mix-
ture, which resulted in insoluble polymers. In future
experiments, this could be circumvented by using, for
example, mono-Boc-protected ethylene diamine, which
can be coupled via the free amine group and subse-
quently be deprotected under acidic conditions. Prelimi-
The analytical data of the synthesized polymers are dis-
played in Table 1 and the 1H-NMR spectra of P1, P2, P6,
and P10 are shown in Figure 1. The amount of MestOx
that was incorporated into the polymers was well con-
trolled as evidenced by 1H-NMR spectroscopy and close to
the theoretical value. This was also the case for the postpo-
lymerization modifications (COOH and NH2), indicating
SCHEME 2 Synthesis of polymers (P1-P10), conditions: (i) 140 8C, mW, 30 min, (ii) NaOH (1M), rt, overnight, (iii) ethylene diamine,
rt, overnight.
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TABLE 1 Analytical Data of Synthesized Polymers (P1-P10)
Theor.
1H-NMR
Yield
Mn (kg/mol)
mol %
mol %
mol %
COOH
mol %
NH2
SECa
D
-
Polymer
P(nPropOx)
MestOx
MestOx
%
g
Theor.
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
–
–
–
–
–
–
–
–
–
–
–
–
9
76
86
87
81
q.
1.9
11.4
11.9
12.7
13.6
14.5
11.5
12.3
12.9
13.5
12.1
20.2
16.7
20.1
20.3
24.1
20.6
24.8
21.2
19.2
21.4
1.20
1.23
1.17
1.12
1.32
1.16
1.16
1.11
1.25
1.20
P(nPropOx-c-MestOx)
P(nPropOx-c-MestOx)
P(nPropOx-c-MestOx)
P(nPropOx-c-MestOx)
P(nPropOx-c-COOH)
P(nPropOx-c-COOH)
P(nPropOx-c-COOH)
P(nPropOx-c-COOH)
P(nPropOx-c-NH2)
10
30
50
70
10
30
50
70
10
10
30
49
72
–
–
51.3
23.4
14.2
2.7
–
–
–
8
69
25
60
66
30
6.8
–
30
49
65
–
2.4
–
5.7
–
1.4
–
1.4
a
SEC was calibrated against PMMA standards, eluent: 0.1% LiCl in DMA.
near-quantitative conversions. The deviations between the-
oretical and experimental Mn values as observed for all the
synthesized polymers can be attributed to the differences
in hydrodynamic volume of the synthesized PAOx com-
pared with the PMMA standards which were used for cali-
bration. Integration of the end groups of the synthesized
polymers proved to be too inaccurate for proper determi-
1
-
nation of the Mn by H-NMR. The D of the polymers ranged
from 1.1 to 1.3, indicating control over the distribution of
polymer chains.
FIGURE 1 Overview of 1H-NMR spectra of selected polymers. A: P2 recorded in CDCl3, (B) P10 recorded in D2O, (C) P1 recorded in
CDCl3, and (D) P6 recorded in D2O 1 DMSO-d6 (COOH 12 ppm). The integrals of the end groups [Me (3 ppm) and piperidine] were
observed only in case of P1 and P2, but these were too small for accurate integration.
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polymer concentrations hydrogen bonds are more easily
formed with surrounding water molecules, while at higher
polymer concentration competition with interchain aggrega-
tion of hydrophobic regions in the polymer chain will occur,
which results in CP-induction. At higher polymer concentra-
tions (20 to 5 mg/mL) the CPs of P6-P8 were similar to the
CPs of P1 (ꢀ25 8C), indicating that at these concentrations
interchain aggregation causes these polymers to precipitate
out of solution which results in CP formation, despite the
hydrophilic side chains. At lower polymer concentrations
(1 mg/mL) however, the ability of P6-P8 to form hydrogen
bonds with the surrounding water molecules was more pre-
dominant. This effect became more pronounced with increas-
ing hydrophilicity of the side chains, as can be observed for
polymers containing 10, 30, and 50% of carboxylic acid
groups (from P6 to P8), which showed CPs of 38, 47, and 55
8C, respectively. This is in contrast with P1, which was not
easily hydrated due to the lack of hydrophilic side chains. As
a result, the CPs recorded for this polymer were around 25 8C
for all polymer concentrations.
Amount of COOH Groups
Next, the influence of the molar percentage of carboxylic
acid groups on the polymer’s transition temperature was
investigated.
It was expected that incorporation of hydrophilic carboxylic
acid groups would increase the water solubility, resulting into
a higher CP. These studies were performed using polymers
with 0 to 70 mol % of COOH. It was observed, as depicted in
Figure 2(B) that a linear relationship existed between the
amount of carboxylic acid groups on the polymer chain and
the CP of the polymer solution in MQ (5 mg/mL).
These results are in agreement with the findings of Hoogen-
boom et al.25 and Park et al.37 in which statistical copoly-
mers of nPropOx with non-ionic hydrophilic comonomers
showed similar trends. Overall, increasing the amount of
hydrophilic groups results in better polymer hydration and
consequently a shift in CP from 24 8C (0% COOH) to 39 8C
(70% COOH), as can be observed from P1 to P9.
Influence of NaCl
To study the Hofmeister-related salting out effect38 on the
CP of the synthesized polymers, a turbidimetric study was
performed for P6 and P10 at various salt concentrations, as
depicted in Figure 2(C,D). As expected, at increasing salt con-
centrations, the CPs of the polymers dropped since the
increased ionic strength of the solution as well as a salting
out effect of NaCl facilitated precipitation of both polymers.
This phenomenon was exploited in the synthesis of P6 and
P10, as was discussed before. This effect was more pro-
nounced for the carboxylic acid-functionalized polymer as
compared to the amine-functionalized polymer. It should be
emphasized that P6 [Fig. 2(C)] seems to be at the limit of
solubility for the measurement in a 100 mg/mL NaCl solu-
tion, since 100% transmittance was not reached in the con-
secutive heating and cooling cycles.
FIGURE 2 Turbidimetry study. A: Overview of CPs of P1, P6-P8
at various polymer concentrations in MQ water, (B) Overview
of CPs of P1, P6-P9 in MQ-water (polymer concentration: 5 mg/
mL), (C) P6 at various concentrations of NaCl, and (D) P10 at
various concentrations of NaCl. Polymer concentration: 5 mg/
mL (C and D). [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
Turbidimetry
Polymer Concentration
Since polymer concentration is a key parameter in the solubil-
ity of polymers and as a result the CP, this was investigated
for P1 and P6-P8 [Fig. 2(A)]. It was expected that at lower
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FIGURE 3 Turbidimetry plots of P1, P6, and P10 in phosphate buffers pH 2 (A) and pH 12 (C). The ionization states are given,
respectively, in (B; pH 5 2) and (D; pH 5 12). Polymer concentration: 10 mg/mL. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
Effect of pH
charge and polarity of the side chains. The resulting poly-
mers were less hydrophilic and as a consequence CP forma-
tion was observed. Another possible explanation for this
phenomenon could be related to the formation of intramo-
lecular hydrogen bonds with the amide groups present in
the backbone of PAOx resulting from protonation of the car-
boxylic acid groups, thereby reducing the interaction with
surrounding water molecules. This is in agreement with the
findings of Weber et al. on copolymers based on P(EtOx) and
methacrylic acid.39 In contrast, P10 was positively charged
and consequently soluble over the entire temperature range.
The CPs of polymers P1, acid-functional P6 and amine-
functional P10 were determined using turbidimetry measure-
ments in buffers of different pH. This study was performed to
investigate the influence of the ionization state/charge of the
side chains of the polymers on their CPs. In view of the con-
secutive pKa values of carboxylic acids (ꢀ4–5) and amines
(ꢀ8–9), this experiment was conducted at pH 2 and pH 12 to
ensure that the majority of the functional groups were in the
protonated or deprotonated state. P1 was measured as well
to study the effect on the CP of a polymer without functional
side chains in these solvent systems.
At pH 12, a similar relationship between the charge of the
side chains and the transition temperature was observed
[Fig. 3(C)]. In this case, however, P6 was fully deprotonated
[Fig. 3(D)] and did not show any transition temperature
between 10 and 50 8C due to the hydrophilic side chains.
At pH 2 only P1 and P6 showed a CP (around 24 8C), while
P10 did not in the temperature range of 15–50 8C [Fig.
3(A)]. A CP was observed for P6 since the carboxylic acid
groups were protonated [Fig. 3(B)], thereby reducing the
FIGURE 4 Overview of CPs of P1, P6, and P10 at various polymer concentrations in PBS buffer (0.1 M). [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
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ACKNOWLEDGMENTS
Both P1 and P10 polymers, however, showed a transition
temperature of 19 and 27 8C respectively, which can be
explained by the charge of the side chains, which plays a
role in the hydration of the polymer chains. The lower CP of
P1 at pH 12 compared with pH 2 might be explained by the
partial deprotonation of the piperidine end groups, thereby
making the polymer less charged (less hydrophilic), which
induced a small decrease in the CP compared with the mea-
surement at pH 2. In addition, the minor change in ionic
strength may also have a small influence on the CP.
This work was supported by the Netherlands Institute for
Regenerative Medicine (NIRM, grant No. FES0908), NWO
(KIEM 731.013.107), Europees Fonds voor Regionale Ontwikk-
eling (EFRO 2011-014237), and GATT Technologies bv.
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Various P(nPropOx-c-MestOx) copolymers were successfully
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