Synthesis and thermoresponsive solution properties of poly[oligo(ethylene glycol) (meth)acrylamide]s: Biocompatible PEG analogues

Article abstract of DOI:10.1021/ma202700y

A library of (meth)acrylamido (co)polymers was prepared by reacting poly(pentafluorophenyl (meth)acrylate) with α-amino, ω-methoxy functionalized di(ethylene glycol), tri(ethylene glycol), and poly(ethylene glycol) (PEG)-350, PEG-750, and PEG-5k, in combination with hexylamine or thyroxine. The resulting copolymers showed an improved solubility in water (higher or absent LCST values) and in alcohols (lower or absent UCST values) than the analogous common series of poly[oligo(ethylene glycol) methyl ether (meth)acrylates]. The polyacrylamido species showed a better solubility than the corresponding polymethacrylamido derivatives of similar molecular weight with all polyacrylamides investigated being water-soluble at temperatures exceeding 90.0 °C. Tunable thermosensitive behavior could be effected by the incorporation of the hydrophobic hexylamide comonomer. Similarly, an acrylamido backbone with grafted oligo(propylene glycol 600) amides exhibited a sharp LCST-type transition around 22.0 °C. The UCST-type transitions of the (meth)acrylamido homopolymers were evaluated in 2-propanol and 1-octanol and were found to increase with an increasing ethylene glycol side chain length, but were essentially independent of the alcohol chain length with polymers exhibiting higher UCST transitions in 2-propanol vs 1-octanol. Cytotoxicity tests on MRC5 fibroblast cells of the di- and tri(ethylene glycol) methyl ether acrylamido homopolymers revealed no toxicity up to concentrations of 10.0 g/L. By employing mixtures of di(ethylene glycol) methyl ether amine and the prohormone thyroxine (T₄), water-soluble copolymers containing varying amounts of T₄ could be easily synthesized. Because of enhanced solubility, low toxicity, and higher hydrolytic stability of amides versus ester linkages, activated ester polymers in combination with amino-functionalized ethylene glycol based side chains are presented as a versatile platform for highly soluble, biocompatible, bioconjugated materials.

Full text of DOI:10.1021/ma202700y

Article
pubs.acs.org/Macromolecules
Synthesis and Thermoresponsive Solution Properties of
Poly[oligo(ethylene glycol) (meth)acrylamide]s: Biocompatible PEG
Analogues
Giles B. H. Chua, Peter J. Roth,* Hien T. T. Duong, Thomas P. Davis, and Andrew B. Lowe*
Centre for Advanced Macromolecular Design (CAMD), School of Chemical Engineering, University of New South Wales,
Kensington, Sydney, New South Wales 2052, Australia
S
* Supporting Information
ABSTRACT: A library of (meth)acrylamido (co)polymers
was prepared by reacting poly(pentafluorophenyl (meth)-
acrylate) with α-amino, ω-methoxy functionalized di(ethylene
glycol), tri(ethylene glycol), and poly(ethylene glycol) (PEG)-
350, PEG-750, and PEG-5k, in combination with hexylamine
or thyroxine. The resulting copolymers showed an improved
solubility in water (higher or absent LCST values) and in
alcohols (lower or absent UCST values) than the analogous
common series of poly[oligo(ethylene glycol) methyl ether
(meth)acrylates]. The polyacrylamido species showed a better
solubility than the corresponding polymethacrylamido derivatives of similar molecular weight with all polyacrylamides
investigated being water-soluble at temperatures exceeding 90.0 °C. Tunable thermosensitive behavior could be effected by the
incorporation of the hydrophobic hexylamide comonomer. Similarly, an acrylamido backbone with grafted oligo(propylene glycol
600) amides exhibited a sharp LCST-type transition around 22.0 °C. The UCST-type transitions of the (meth)acrylamido
homopolymers were evaluated in 2-propanol and 1-octanol and were found to increase with an increasing ethylene glycol side
chain length, but were essentially independent of the alcohol chain length with polymers exhibiting higher UCST transitions in
2-propanol vs 1-octanol. Cytotoxicity tests on MRC5 fibroblast cells of the di- and tri(ethylene glycol) methyl ether acrylamido
homopolymers revealed no toxicity up to concentrations of 10.0 g/L. By employing mixtures of di(ethylene glycol) methyl ether
amine and the prohormone thyroxine (T4), water-soluble copolymers containing varying amounts of T4 could be easily
synthesized. Because of enhanced solubility, low toxicity, and higher hydrolytic stability of amides versus ester linkages, activated
ester polymers in combination with amino-functionalized ethylene glycol based side chains are presented as a versatile platform
for highly soluble, biocompatible, bioconjugated materials.
LCSTH2O ∼ 50 °C), poly/oligo(ethylene glycol) (meth)-
acrylates (OEGMA, LCSTH2O varies depending on the degree
of polymerization of the ethylene oxide side chain), methyl
vinyl ether (MVE, LCSTH2O ∼ 36 °C),7 pyrrolidone containing
polymethacrylates (LCSTH2O∼29−34 °C),8 oligo(ethylene
glycol) methyl ether methacrylate (OEGMA, UCSTROH varies
from ca. 75−20 °C depending on the alcohol) and examples of
polysulfopropylbetaines that can exhibit both UCST and LCST
behavior.9,10
Of these thermoresponsive materials the PEG analogues, and
especially the acrylic and methacrylic species, have attracted
significant attention due in part to their LCST and UCST
behaviors but also because of their well-established biocompat-
ibility.11−20 For example, Bebis et al.20 recently described the
effect of polymer concentration and added solutes on the
LCST behavior of a series of well-defined, RAFT-synthesized,
methacrylic-based oligo(ethylene glycol) species. The authors
INTRODUCTION
■
The ability of certain (co)polymers in solution (aqueous or
nonaqueous) to undergo conformational or phase transitions in
response to a change in solution temperature, i.e., thermore-
sponsive polymers, are of interest as so-called “smart” materials.
The response to a change in temperature can be exploited to
induce self-directed assembly (micellization or vesiculation in
block copolymers for example) or to promote the uptake or
release of a particular molecular species and as such thermal
‘triggers’ have attracted significant attention.1,2 The primary
transition temperatures of interest to researchers are the lower
critical solution temperature (LCST) in which a (co)polymer
undergoes a coil-to-globule transition upon heating and is
commonly associated with nonionic water-soluble species,
and the upper critical solution temperature (UCST) whereby
phase separation is induced by cooling. Examples of
(co)polymers that exhibit LCST or UCST behavior are those
containing, either wholly or partially, N-isopropylacryl-
amide (NIPAM, LCSTH2O ∼ 32 °C), N,N-diethylacrylamide
(DEAM, LCSTH2O ∼ 32 °C),3−5 N-vinylcaprolactam (LCSTH2O
∼ 32.5 °C),6 2-(dimethylamino)ethyl methacrylate (DMAEMA,
Received: December 13, 2011
Revised: January 17, 2012
Published: February 1, 2012
© 2012 American Chemical Society
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reported that the cloud point of such materials can increase by
as much as 6 °C when polymer concentrations are decreased
from 5.0 to 0.5 g/L and that the same measurements performed
in bovine blood plasma indicated an additional further decrease
of the cloud point (compared to water or PBS). The results,
collectively, indicate that researchers should apply appropriate
testing protocols for such materials when being examined for
potential biomedical applications such as drug delivery. Roth,
Jochum, and Theato recently detailed the behavior of poly-
OEGMA (POEGMA) in a wide range of alcohols.12 POEGMA
solutions showed sharp, reversible transitions with low
hystereses. The cloud points increased with an increasing alcohol
chain length and were further tunable through end group struc-
ture and molecular weight. This UCST behavior in alcohols is
promising because it occurs in both hydrophilic solvents such as
ethanol as well as in the hydrophobic longer chain alcohols.
Reversible addition−fragmentation chain transfer (RAFT)
radical polymerization is an example of a reversible deactivation
radical polymerization (RDRP) process that is mediated by
thiocarbonylthio compounds.7,21−26 Since its discovery, RAFT
has evolved into arguably the most robust and versatile of the
RDRP systems and is now employed routinely in research
laboratories worldwide. In recent years there has been
significant interest in combining RDRP with a range of click
and other highly efficient coupling chemistries. These include
Cu-catalyzed alkyne−azide,27−30 Diels−Alder,31−34 and a range
of thiol-based chemistries35−41 with the aim of developing
routes for the preparation of complex macromolecular
architectures or facilitating the efficient postpolymerization
modification of preformed (co)polymers. Indeed, RAFT has
lent itself perfectly to such click and click-like chemistries in
part due to the availability of a protected thiol, in the form of
the thiocarbonylthio group, at the polymer chain end(s) and
also due to its inherently high functional group tolerance
allowing the synthesis of parent (co)polymers containing an
impressive array of functionality.42,43 Of the efficient coupling
chemistries, the use of pentafluorophenyl (PFP) activated
(meth)acrylic esters has proven to be particularly useful for the
introduction of amine-containing species via the formation of
or alcohols (UCST). We highlight how these (meth)-
acrylamido-based materials represent an important addition to
the family of PEG-analogues possessing fundamentally different
solution properties from the corresponding (meth)acrylic
species and as such may find application in areas not accessible
to such (meth)acrylic derivatives.
EXPERIMENTAL PART
■
Instrumentation. Nuclear magnetic resonance (NMR) spectro-
scopic measurements were performed in CDCl3 solutions on a Bruker
DPX 300 instrument and evaluated using MestReC 4.7.0.0 software.
The internal solvent signal was used as the reference signal (7.26 ppm).
Size exclusion chromatography (SEC) was performed on a Shimadzu
system with four phenogel columns in dimethylacetamide (DMAc)
as eluent at a flow rate of 1.0 mL/min. Chromatograms were ana-
lyzed with Cirrus SEC software (version 3.0). The system was
calibrated with a series of narrow molecular weight distribution
polystyrene (PS) standards. Turbidity measurements were performed
on a Varian Cary 300 Scan spectrophotometer equipped with a Cary
temperature controller and a Peltier heating element in quartz cuvettes
of 10 mm path length at a wavelength of 520 nm. Heating rates were
1.0 °C/min for all measurements. For clear solutions (cold water;
warm alcohols), the baseline was set to zero absorbance, A.
Transmittance, T = 10−A, was plotted against temperature and cloud
points were determined at 50% transmittance. The maximum
temperature evaluated for aqueous solutions was 93.0 °C. Fourier
transform infrared spectroscopy (FTIR) was performed on a Bruker
IFS 66/S instrument under attenuated total reflectance (ATR) and
was analyzed on OPUS software version 4.0. Differential scanning
calorimetry measurements were performed with a Perkin-Elmer DSC7
instrument at a heating rate of 2.0 °C/min on 40 μL of 20 g/L
solutions and were analyzed with PE Pyris software.
Synthesis. All reagents were purchased from the Aldrich Chemical
Co. and used as received unless noted otherwise. 4-Cyano-4-
((phenylcarbonothioyl)thio)pentanoic acid (CPADB) was prepared
according to the literature.22 2,2′-Azobis(2-methylpropionitrile)
(AIBN) was recrystallized from methanol.
Synthesis of Pentafluorophenyl (Meth)acrylate Monomers.
Pentafluorophenyl acrylate and pentafluorophenyl methacrylate were
synthesized according to the general procedure described by Eberhardt
et al.50
Synthesis of Ethylene Glycol Methyl Ether Amines. Di- and
triethylene glycol methyl ether amine were prepared according to the
method of Clerc et al.51 Diethylene glycol methyl ether amine: 65%
amides.11,44−49 For example, Gibson, Frohlich, and Klok
̈
detailed the RAFT synthesis of three poly(pentafluorophenyl
methacrylate)s with measured average molecular weights in the
range 13 750−36 800 g/mol.49 These precursor activated
polymers were reacted with a range of hydrophilic primary
amines including glycine, glucosamine, 2-hydroxypropylamine,
and taurine yielding the corresponding functional, hydrophilic
amides in generally high yields under facile conditions. The
authors also demonstrated that the degree of modification was
readily controlled by functional group stoichiometry (penta-
fluorophenyl ester:primary amine). In the case of precursor
polymers modified with hydroxypropylamine cytotoxicity tests
indicated no difference in the inherent toxicity of the materials
via the postpolymerization route vs, for example, polymers
obtained by the direct polymerization of 2-hydroxypropyl
methacrylamide.
1
overall yield; H NMR, 300 MHz, CDCl3, δ (ppm) = 3.55 (m, 2 H),
3.50−3.40 (m, 4 H), 3.32 (m, 3 H, −OCH3), 2.80 (m, 2 H,
−CH2NH2), 1.73 (bs, −NH2). Triethylene glycol methyl ether amine:
64% overall yield; 1H NMR, 300 MHz, CDCl3, δ (ppm) = 3.52 (m, 6 H,
PEG), 3.43/3.38 (m, 4 H, −CH2−), 3.24 (m, 3 H, −OCH3), 2.73
(m, 2 H, −CH2NH2), 1.50 (bs, −NH2).
Poly(ethylene glycol) methyl ether amines with average molecular
weights of 350, 750 and 5000 were prepared following the procedure
of Mongondry and co-workers.52 PEG-350 amine: 50% overall yield;
1H NMR, 300 MHz, CDCl3, δ (ppm) = 3.53 (bs, ∼ 29 H, PEG),
3.44−3.38 (m, 4 H, −CH2−), 3.36 (s, 3 H, −OCH3), 2.75 (bt, 2 H,
−CH2NH2), 2.12 (bs, −NH2). PEG-750 amine: 86% overall yield; 1H
NMR, 300 MHz, CDCl3, δ (ppm) = 3.71−3.44 (bs, ∼ 83 H, PEG),
3.31 (bs, 3 H, −OCH3), 2.81 (m, 2 H, −CH2NH2), 2.49 (bs, −NH2).
1
PEG-5000 amine: 73% overall yield; H NMR, 300 MHz, CDCl3,
δ/ppm =3.75−3.40 (bs, PEG), 3.33 (bs, −OCH3), 2.80 (m,−CH2NH2).
All PEG amines were of sufficient purity for the conversions of
polymeric activated esters without further purification.
RAFT Homopolymerization of PFP Ester Monomers. Below is
given a typical procedure for the RAFT homopolymerization of
pentafluorophenyl acrylate (PFPA). The same general procedure was
employed for the homopolymerization of pentafluorophenyl meth-
acrylate (PFPMA).
Employing a combination of RAFT radical polymerization
with PFP activated ester chemistry herein we report our
findings regarding the synthesis and thermal solution properties
of a library of poly[oligo(ethylene glycol) (meth)acrylamide]s
which serve as PEG analogues. While a few examples of such
materials have been previously prepared there has not, to the
best of our knowledge, been a systematic detailed investigation
of their thermoresponsive phase behavior in water (LCST)
PFPA (5.0 g, 21.0 mmol), CPADB (78 mg, 0.28 mmol), AIBN
(9.2 mg, 0.056 mmol), and 1,4-dioxane (8.0 mL) were combined in a
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round bottomed flask equipped with a magnetic stir bar. The flask was
sealed with a rubber septum and nitrogen was bubbled through the
solution for 20 min. The reaction vessel was subsequently immersed in
a preheated oil bath set to 70 °C. Polymerization was allowed to
proceed for 12 h. The resulting polyPFPA was isolated via two
precipitations into methanol and subsequently dried under vacuum.
PolyPFPA: SEC 26.6 kg/mol, PDI 1.19. 1H NMR, 300 MHz, CDCl3,
δ (ppm) = 3.40, 2.52, 2.15 (m, backbone). 19F NMR, 282 MHz,
CDCl3, δ (ppm) = −153.1 (bs, 2 F, ortho), −153.7 (bs, 1 F, para),
−162.1 (2 F, meta). IR, ν (cm−1) = 2931 (w), 1780 (m), 1513 (s),
1218 (w), 1076 (m), 985 (s), 860 (w), 852 (w). PolyPFPMA: SEC
(v/v) foetal bovine serum (FBS) in a ventilated tissue culture flask
T-75 and passaged every 2−3 days when the monolayer reached around
80% confluence. The cells were used only when stable cell growth was
obtained (approximately 3−4 passages). The cells were incubated at
37 °C in a 5% CO2 humidified atmosphere. The cell density was
determined by counting the number of viable cells using a trypan blue
dye exclusion test. The cells were detached using 0.05% trypsin-
EDTA (Invitrogen), stained using trypan blue dye, and loaded on a
hemocytometer. One day prior to the treatment, the cells were seeded
at required cell densities on a 96-well plate. Cell viability: The
cytotoxicity of the (meth)acrylamido-based polymers was tested in
vitro by a standard Alamar Blue assay which provides a homogeneous,
fluorescent method for monitoring cell viability. The assay is based on
the ability of living cells to convert a redox dye (blue resazurin) into a
fluorescent end product (red resorufin). Nonviable cells rapidly lose
metabolic capacity and thus do not generate a fluorescent signal. The
cells were seeded in a tissue culture treated 96-well plate in 100 μL
medium per well at a density of 5000 cells/well and incubated for
24 h. The medium was then replaced with fresh medium containing
the polymer samples and incubated for 72 h. The final concentration
of polymer in the wells was adjusted to the desired concentrations
ranging from 0.005 to 10.0 g/L in DMEM media containing 10% v/v
FBS. Alamar Blue dye (20 μL) was added to each well and the cells
were incubated for 6 h. After the incubation step, data was recorded
using a fluorescence plate reader (λex = 540 nm; λem = 595 nm).
1
15.4 kg/mol, PDI 1.21. H NMR, 300 MHz, CDCl3, δ (ppm) = 2.44,
2.19, 1.46, 1.40 (m, backbone). 19F NMR, 282 MHz, CDCl3, δ (ppm)
= −150.9, −151.9 (2 m, 2 F, ortho), −157.4 (bs, 1 F, para), −162.5
(2 F, meta). IR, ν (cm−1) = 2967 (w), 1758 (m), 1515 (s), 1240 (w),
1043 (s), 989 (s), 854 (w).
Reaction of Pentafluorophenyl (Meth)acrylate Polymers with
Primary Amines in the Presence of Methyl Methanethiosulfonate.
Below is a typical example for the reaction of a pentafluorophenyl
(meth)acrylate (PFPMA) polymer with an oligo(ethylene glycol)
methyl ether amine.
To a glass vial equipped with a magnetic stir bar was added
polyPFPA (214 mg, 0.9 mmol of pentafluorophenyl esters, 1 equiv)
and DMF (2.0 mL). To a separate flask was added diethylene glycol
methyl ether amine (273.2 mg, 2.25 mmol, 2.5 equiv), DBU (202 μL,
1.35 mmol), methyl methanethiosulfonate (22 μL, 10 equiv. with
respect to polymer end groups) and DMF (0.9 mL). After complete
dissolution the solutions were combined and the reaction allowed to
proceed overnight at room temperature. For poly(pentafluorophenyl
methacrylate) the excess of amines was increased to 5 equiv. and THF
and triethylamine were used as solvent and auxiliary base, respectively.
IR, ν (cm−1) = 3457 (w), 3300 (m), 2865 (m), 1644 (s), 1539 (s),
1452 (m), 1093 (s), 1022 (m), 844 (w). Reaction of polyPFPMA
with diethylene glycol methyl ether amine: IR, ν (cm−1) = 3505 (w),
3352 (m), 2868 (m), 1637 (s), 1518 (s), 1452 (m), 1198 (m), 1095 (s),
845 (w).
Copolymer Synthesis. Reaction of PFPA with two different primary
amines in the presence of methyl methanethiosulfonate. Below is a
typical example for the reaction of a polyPFPA with an oligo(ethylene
glycol) methyl ether amine (85 mol %) and hexylamine (15 mol %).
To a glass vial equipped with a magnetic stir bar was added poly-
PFPA (119 mg, 0.5 mmol of PFP esters, 1 equiv) and DMF (1.5 mL).
To a separate flask was added di(ethylene glycol) methyl ether amine
(60.8 mg, 0.51 mmol, 1.02 equiv, 20% excess), hexylamine (9.1 mg,
0.09 mmol, 0.18 equiv, 20% excess), methyl methanethiosulfonate
(10 μL) and DMF (1.0 mL). The amine solution was quickly added to
the polymer solution under vigorous stirring and allowed to react at
room temperature overnight. IR, ν (cm−1) = 3469 (w), 3284 (m),
2918 (m), 1641 (s), 1540 (s), 1440 (m), 1095 (s), 845 (m).
The same protocol was employed for the preparation of thyroxine
(T4) containing copolymers where ratios of 9:1 and 8:2 of di(ethylene
glycol) methyl ether amine to thyroxine with an excess of 20 mol % of
amines toward PFP esters were used.
RESULTS AND DISCUSSION
■
Synthesis. The desired oligo(ethylene glycol) methyl ether
amines (OEGMEAs) were obtained via two different routes the
choice of which was dictated by the molecular weight of the
precursor oligo(ethylene glycol) methyl ether (OEGME). The
di- and tri(ethylene glycol) methyl ether amines (MEO2 and
MEO3) were prepared from the corresponding OEGME via a
three step procedure involving tosylation, nucleophilic
substitution with azide followed by Staudinger reduction. The
higher molecular weight target OEGMEAs with average
precursor molecular weights of 350, 750, and 5000 g/mol
were prepared via a Mitsunobu reaction with phthalimide
followed by a Gabriel synthesis-like reaction of the intermediate
functional phthalimide via hydrazinolysis, Scheme 1. In all
instances the target OEGMEAs were obtained in moderate to
high overall yield and purity.
With the OEGMEAs in hand two different pentafluoro-
phenyl- (PFP-) containing homopolymers (one derived from
PFP-acrylate (PFPA) and the other from PFP-methacrylate
(PFPMA)) were prepared under standard RAFT conditions
employing 4-cyano-4-((phenylcarbonothioyl)thio)pentanoic
acid (CPADB) as the mediating agent and AIBN as the source
of primary radicals, Scheme 2. The RAFT (co)polymerization
of PFPA and PFPMA is well-documented46,50 and the
polymerizations were allowed to proceed for a predetermined
period of time yielding two homopolymers with measured
number-average molecular weights (Mn) of 26 600 g/mol
(polyPFPA) and 15 400 g/mol (polyPFPMA) and relatively
low polydispersity indices (Mw/Mn = 1.19 and 1.21
respectively). With the polyPFPA and polyPFPMA parent
homopolymers in hand a library of oligo(ethylene glycol)-based
(meth)acrylamido (co)polymers were prepared via the reaction
of the OEGMEAs prepared above (as well as with some
commercially available primary amine species, vide infra) with
the PFPA and PFPMA homopolymers, Scheme 2. The use of
two simple precursor homopolymers as activated substrates
thus allowed the preparation of a library of amide-based PEG
analogues with identical average main-chain degrees of
polymerization. It should be noted that the acyl substitution
General Purification Protocols. After stirring overnight, 0.2 mL of
reaction mixture was withdrawn, 0.45 mL of CDCl3 was added and a
19F NMR measurement was made (example poly[di(ethylene glycol)
methyl ether acrylamide]: 19F NMR, 282 MHz, CDCl3, δ (ppm) =
−170.7 (m, 2 F, ortho), −171.7 (m, 2 F, meta), −189.5 (m, 1 F, para),
pentafluorophenol). The NMR sample was combined with the
remaining reaction mixture which was then placed into a dialysis
membrane (regenerated cellulose, 3500 g/mol MWCO for di- and
triethylene glycol amine and PEG-350 amine; MWCO 6−8 kg/mol for
PEG-750 amine; MWCO 12−14 k for PEG-5000 amine) and dialyzed
against methanol for 3 days with solvent changes twice per day. The
solvent was removed by blowing in air; the residue was then dissolved
in a small amount of water and lyophilized to obtain the final products.
Cytotoxicity Testing. The toxicity of the prepared polymers was
tested on a MRC5 fibroblast cell line. Cell culture: MRC5 fibroblast
cells were cultured in growth media consisting of Dulbecco’s modified
Eagle’s medium: Nutrient Mix F-12 (DMEM) supplemented with 10%
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Scheme 1. Synthetic Approaches for the Preparation of Oligo(ethylene glycol) Methyl Ether Amines
Scheme 2. Outline for the RAFT Homopolymerization of Pentafluorophenyl (Meth)acrylate Monomers and Procedures for the
Subsequent Post-Polymerization Acyl Substitution Reactions with OEGMEAs
reactions were performed in the presence of S-methyl
methanethiosulfonate (MMTS). Thiocarbonylthio functional
groups (found at the ω-chain end of the RAFT-prepared PFPA
and PFPMA homopolymers in the form of dithiobenzoate
species) are extremely reactive towards primary and secondary
amines with reactions resulting in the cleavage of the
thiocarbonylthio end groups yielding the corresponding
macromolecular thiol. Indeed, this represents the most
commonly employed route for the removal of such end-groups
in RAFT-synthesized (co)polymers with the liberated thiol
being able to undergo a range of further chemical
reactions.36,43,53 However, one common problem with the
formation of macromolecular thiols is their propensity to
undergo oxidative coupling forming polymeric disulfides. Such
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Figure 1. Monitoring the conversion of PFP esters with OEGMEAs. (A) 1H NMR spectra of parent polyPFPA (bottom), polyMEO2AM (middle),
and polyMEO3AM (top) showing quantitative formation (within NMR accuracy) of the PEG-amides. (B) FTIR spectra of the activated polymers
polyPFPA and PFPMA and after conversion of each with di(ethylene glycol) amine, with the inset highlighting the shift of the carbonyl band.
reactions, while reversible, consume the thiol, which is clearly
problematic if further modification is the goal but also results in
an effective doubling of the average molecular weight or degree
of polymerization. Since molecular weight is an important
parameter affecting the solution phase behavior of thermore-
sponsive (co)polymers this precautionary step of capturing the
macromolecular thiol was adopted. Indeed, it has previously
been shown that MMTS, and functional derivatives thereof, are
extremely effective reagents for ‘trapping’ liberated thiols pro-
duced during the aminolysis of end-groups in RAFT-prepared
(co)polymers12,54−57 thus negating any undesirable polymer−
polymer disulfide formation.
formation of methacrylic acid anhydrides, as opposed to the
desired activated esters, between neighboring −CO2H groups,
which subsequently formed one amide and one acid group
upon exposure with amines. On the other hand, poly(acrylic
acid) gave a high amide yield. While different to the current
research, where the activation step took place prior to poly-
merization, the low amidation of methacrylic PFP derivatives
and the fundamental difference compared to the polyacrylate
should be noted. Here, the ratio of amine with respect to
methacrylic PFP esters was increased to 5 to minimize the
formation of hydrolysis byproducts. For the acrylate derivative
on the other hand, as little as 1.2 equiv of amines (as used in
the production of copolymers) was sufficient to achieve a
quantitative conversion to the target amides as evidenced by
1H, 19F NMR and FTIR spectroscopies.
Initially, both polyPFPMA and polyPFPA were reacted with
four OEGMEAs: di(ethylene glycol) methyl ether amine,
tri(ethylene glycol) methyl ether amine and two OEGMEAs
with average Mn’s of 350 and 750 g/mol, Scheme 2A. The
resulting acrylamido (AM) and methacrylamido (MAM)
analogues are denoted polyMEOnAM and polyMEOnMAM
for the dimer and trimer ethylene glycol species (n = 2 and 3)
and polyOEGyAM and polyOEGyMAM for those derived from
the OEGMEAs with y = 350 and 750 g/mol. Conversion of the
parent polyPFPA and polyPFPMA homopolymers to the
corresponding PEG-amides was quantified employing a
combination of 1H and 19F NMR spectroscopy, SEC and
FTIR spectroscopy. For the acrylic activated esters quantitative
conversion was achieved with 2.5 equiv of amine with regards
to PFP esters. This procedure did not, however, give a full
conversion of the analogous methacrylic esters to the de-
1
For example, Figure 1A shows the H NMR spectra, plotted
between 1.0 and 4.0 ppm, for the polyPFPA homopolymer
(bottom) and the corresponding amides obtained after reaction
with di(ethylene glycol) methyl ether amine (polyMEO2AM)
(middle) and tri(ethylene glycol) methyl ether amine
(polyMEO3AM) (top). The spectrum associated with
polyPFPA is relatively simple with only backbone hydrogens
visible which span the range of ∼3.2−2.0 ppm. After
modification with the di- and tri(ethylene glycol) amino
species peaks appear in the range ∼3.8−2.8 ppm which
correspond to the side-chain hydrogens associated with the
ethylene glycol methyl ether segments. Analysis of the integrals
associated with these new peaks indicated quantitative for-
mation of the PEG-acrylamides when compared to the integrals
associated with the backbone hydrogens. Figure 1B shows the
FTIR spectra for polyPFPA, polyPFPMA, polyMEO2MAM,
and polyMEO2AM. The most distinctive feature when
comparing the parent (meth)acrylic homopolymers and the
resulting PEG-(meth)acrylamides, and qualitatively verifying
complete reaction, relates to the CO peak associated with
the parent esters vs the product amides. In the parent
homopolymers the ester CO peak appears at ca. 1780
cm−1 while in the product (meth)acrylamides the amide CO
1
sired amides. In case of polyPFPMA, H NMR spectroscopy
showed less than quantitative formation of the PEG amides and
FTIR spectroscopy revealed the presence of acid groups
(∼1710 cm−1). The nonquantitative conversion of methacrylic
PFP esters with 2 equiv of amine, followed by hydrolysis of
unreacted esters during work-up, has been reported.49 Similarly,
nonquantitative amidation of surface tethered poly(methacrylic
acid) during the activation step with EDC/NHS (N-ethyl-N′-
(3-(dimethylamino)-propyl)carbodiimide/N-hydroxysuccini-
mide) was recently described.58 The authors detailed the
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Figure 2. SEC traces of (A) the acrylamido series including the precursor polyPFPA and (B) the methacrylamide series with its parent polymer
polyPFPMA.
Table 1. Summary of Prepared OEG Amide Homopolymers and Statistical Copolymers, SEC-Measured Molecular Weights,
Polydispersities, and Cloud Points in Water
(meth)acrylamido (co)polymers
b
cloud point (5 g/L)
heating (°C)
reference (meth)acrylates
a
a
b
entry
polymer
Mn (kg/mol)
PDI
cooling (°C)
polymer
cloud point (°C)
reference
c
1
2
polyMEO2AM
polyMEO3AM
22.5
24.7
31.1
47.0
177.4
19.7
20.7
21.3
20.7
42.5
49.7
18.0
21.3
28.2
27.8
42.5
1.22
1.25
1.2
s
s
s
polyMEO2A
polyMEO3A
polyOEG370
38
59
59
60
s
58
e
3
polyOEG350AM
s
s
s
A
s
4
polyOEG750AM
1.25
1.74
1.26
1.24
1.24
1.25
1.21
1.18
1.26
1.32
1.38
1.26
1.29
s
5
polyOEG5kAM
s
s
d
6
poly[(MEO2)49-co-Hex51]AM
poly[(MEO2)76-co-Hex24]AM
poly[(MEO2)85-co-Hex15]AM
poly[(MEO2)93-co-Hex7]AM
poly[(MEO2)91-co-T49]AM
poly[(MEO2)87-co-T413]AM
polyOPG600AM
ins
ins
21.1
43.0
75.9
s
7
26.0
46.7
77.7
s
8
9
10
11
12
13
14
15
16
s
s
22.1
55.8
69.3
s
16.7
52.7
63.2
s
polyMEO2MAM
polyMEO2MA
polyMEO3MA
polyOEG375MA
26
52
90
19
19
17
polyMEO3MAM
f
polyOEG350MAM
polyOEG750MAM
s
s
a
b
c
Data obtained by SEC. Cloud points depend on concentration, molecular weight and polymer end groups. Soluble between 0 and 93 °C.
d
e
f
Insoluble between 0 and 93 °C. [Poly(ethylene glycol) monomethyl ether acrylate], Mmonomer = 454 g/mol. [Poly(ethylene glycol) monomethyl
ether methacrylate], Mmonomer = 475 g/mol.
peak appears at ca.1640 cm−1 with no evidence of residual ester
CO species. Additionally, we note the appearance of bands at
ca. 2850 cm−1 that may be assigned to the C−H stretches in the
CH2 and CH3 groups associated with the newly introduced
ethylene glycol side chains and the broad bands between 3500
and 3100 cm−1 due to the N−H stretch of the newly introduced
secondary amide functionality. Representative 19F NMR spectra
demonstrating the complete cleavage of the activated esters are
given in Figure S1 of the Supporting Information.
Figure 2 shows the measured molecular weight distributions
for the parent polyPFPA (Figure 2A black line) and
polyPFPMA (Figure 2B black line) as well as the resulting
(meth)acrylamides derived from reaction with the OEGMEAs.
The experimentally determined molecular weight and poly-
dispersity data is summarized in Table 1 (entries 1−4 for the
acrylamides and 13−16 for the methacrylamides). In the case of
the acrylamido derivatives we observe an increase in the Mn as
the length of the oligo(ethylene glycol) side chain increases, as
expected. In all instances the measured polydispersity indices
increase slightly compared to the parent polyPFPA but are all
between 1.20 and 1.25 with the molecular weight distributions
being unimodal and essentially symmetrical. This also indicates
that the macromolecular thiols were successfully trapped with
MMTS since there is no evidence of high molecular weight
(coupled) species in the chromatograms. The same general
trends are observed in the case of the methacrylamide deriva-
tives, with an increase in the measured Mn as side-chain degree
of polymerization increases and polydispersity indices in the
range 1.26−1.38.
Aqueous Solution Properties. With a library of
acrylamido and methacrylamido-based PEG analogues prepared
the aqueous phase behavior of the polymers was investigated at
a concentration of 5.0 g/L (0.5 wt %). Consider first the range
of acrylamido derivatives (entries 1−4 Table 1). In all cases, at
the concentration evaluated, the PEG-based acrylamido
homopolymers did not exhibit any LCST behavior in pure
water and were completely soluble between 0 and 93 °C. This
is in sharp contrast to acrylate derivatives in which polymers
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with di- and tri(ethylene glycol) side chains have reported
LCSTs of 38 and 58 °C respectively.59 This difference in
solubility is, presumably, due to the enhanced hydrophilicity of
the amide-based materials due to the H-bond donor and
acceptor properties of the amide functional group and
highlights how subtle functional group changes can have a
marked effect on the aqueous solution properties of such PEG-
containing polymers. The absence of LCST behavior for the
acrylamido-based PEGs with the longer side chains is consistent
with the enhanced aqueous solubility reported for analogous
acrylic derivatives.60
is completely soluble and does not exhibit a phase transition
upon heating (solid blue line). With the introduction of as little
as 7 mol % hexylamide residues the amphiphilic copolymer
undergoes a phase transition upon heating with a measured
cloud point of 77.7 °C (taken at 50% transmittance, solid red
line) although the transition is not particularly sharp. Increasing
the hexylamide content to 15 and 24 mol % results in a
systematic lowering of the cloud point to 46.7 °C (solid black
line) and 26.0 °C (solid green line) respectively and also
appears to result in sharper, more distinct transitions.
Increasing the hexylamide content to just above equimolar
(51 mol %) yields a copolymer that is completely insoluble in
water even when cooled to temperatures approaching 0 °C.
Also shown in Figure 3 are the corresponding cooling curves
for the three thermoresponsive copolymers, and the corre-
sponding phase transition temperatures are listed in Table 1.
Little hysteresis is observed for all three samples and in all
instances the redissolution temperatures are only several
degrees lower than the cloud points found for heating. This
is consistent with the well-known kinetic effect and the process
of rehydration of the highly compact globules and aggre-
gates thereof. Shown inset in Figure 3 is a plot of the cloud
point vs mol % hexylamide residues. It can be seen that this plot
is essentially linear and indicates that the synthetic route
described herein offers a simple and convenient approach to
thermoresponsive copolymers with tunable, and essentially
predetermined, LCST behavior simply by varying the molar
content of hexylamide residues.
Another strategy was also investigated to induce LCST
behavior into the acrylamido polymers, this time by a
“hydrophobic modification” of the ethylene glycol side chains.
Poly(propylene glycol) (PPG) is a well documented thermo-
responsive polymer that exhibits a marked dependence of
its LCST on the polymer molecular weight. For example,
increasing the MW from 1000 to 2000 to 3000 results in a
decrease in the LCST from 42.0 to 23.0 to 15.5 °C.64 PPG,
while not perhaps as currently widely evaluated or employed in
polymer synthesis as PEG, due in part to such low thermal
transitions, finds wide use as the hydrophobic middle section of
the commercially important Pluronic series of surfactants.65 To
evaluate the effect of relative hydrophobicity of the oligoether
side chains the polyPFPA homopolymer was also reacted with
a commercially available oligo(propylene glycol) amine (Jeff-
amine) with an average molecular weight of ca. 600 g/mol.
Consistent with the previous postpolymerization conjugations
the reaction of the Jeffamine proceeded smoothly, quantita-
tively yielding a well-defined oligo(propylene glycol) methyl
ether acrylamido homopolymer, polyOPG600AM, entry 12 in
Table 1, Figure S4 in the Supporting Information. Evaluation of
the cloud point behavior of this PPG-acrylamido derivative,
Figure 4, indicates a sharp phase transition upon heating at
22.1 °C with the cooling curve also indicating moderate hysteresis.
Thus, the combination of PPG side-chains linked to a backbone
via a hydrophilic acrylamido functional group allows for the
preparation of PPG-containing polymers that exhibit distinctly
different aqueous phase behavior compared to simple linear
PPG materials and facilitates the preparation of water-soluble
PPG-containing substrates with much higher effective molec-
ular weights than the linear species.
It is well documented that the LCST behavior of
thermoresponsive polymers may be tuned via the incorporation
of a hydrophobic comonomer61,62 or, in the case of suitably low
molecular weight species, via variation of the hydrophilicity/
hydrophobicity of polymer end-groups.5,63 In an effort to
induce LCST behavior the polyPFPA parent homopolymer
was reacted with varying ratios of di(ethylene glycol) methyl
ether amine and hexylamine, Scheme 2 route B, yielding,
presumably, random amphiphilic copolymers containing 51,
24, 15, and 7 mol % hydrophobic hexylamide residues. Com-
1
positions were determined by H NMR spectroscopy by a
comparison of the hexyl −CH3 group with the ethylene glycol
−OCH3 group. Both amines exhibited a similar reactivity
toward the activated esters yielding polymers with essentially
the same composition as the feed ratio of amines (see Figure S2,
and Table S1 in Supporting Information). Molar com-
positions, molecular weights and polydispersity indices for
these copolymers are listed in Table 1 (entries 6−9). SEC
traces are shown in Figure S3. It should be noted that this
yields statistical copolymers with the same degree of polymer-
ization whose individual components are either wholly soluble
in water (di(ethylene glycol) methyl ether amide residues) or
completely insoluble, i.e. hydrophobic (hexylamide residues)
and thus should allow for a tuning of the LCST behavior.61,62
The LCST characteristics of these amphiphilic copolymers
were evaluated under the same conditions as noted above for
the acrylamide homopolymers. Two additional amphiphilic
statistical copolymers were also prepared by substituting
hexylamine for the prohormone thyroxine (Table 1 entries 10
and 11) and these will be discussed in the final part of this
contribution.
Figure 3 shows the cloud-point curves for the poly-
[(MEO2)1‑x-co-Hexx]AM statistical copolymers as well as the
polyMEO2AM homopolymer. As noted above, polyMEO2AM
The thermal phase transitions of two samples, polyOP-
G
600AM and poly[(MEO2)85-co-Hex15]AM, were also briefly
Figure 3. Turbidity curves of the poly[(MEO2)1‑x-co-Hexx]AM series
demonstrating tunable LCST behavior: heating curves (solid lines);
cooling curves (dashed lines).
examined by differential scanning calorimetry (DSC). Experi-
ments were performed at 20.0 g/L, four times higher than the
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Next, the thermal behavior of the methacrylamido polymers,
Table 1 entries 13−16, was investigated. In contrast to the
polyMEO2AM and polyMEO3AM acrylamido homopolymers
both the polyMEO2MAM and polyMEO3MAM species exhibit
LCST behavior in water with measured cloud points of 55.8
and 69.3 °C respectively, Figure 6. Both of these values are
Figure 4. Turbidity measurement (dashed line cooling curve) of the
Jeffamide derivative showing a sharp LCST transition.
concentration examined above in cloud point measurements.
The measured transition temperatures were in agreement
with turbidity measurements performed at the same concen-
tration (Supporting Information, Figure S5). The endotherms
of both polymers do however show distinct differences.
PolyOPG600AM shows a very broad transition occurring over
a range ∼10 °C with a heat of 22.94 kJ/mol monomer units
calculated from the peak area, Figure 5. On the other hand, the
Figure 6. Turbidity curves (dashed curve cooling) for the
methacrylamido polymers with 2 and 3 ethylene glycol units showing
sharp transmittance decreases and low to moderate hystereses.
higher than identical methacrylic derivatives whose reported
cloud points are 26.0 and 52.0 °C19 again highlighting the
enhanced hydrophilicity associated with these (meth)-
acrylamido derivatives vs the corresponding (meth)acrylates.
Interestingly, the observed phase transition behavior of the
methacrylamido vs acrylamido species is opposite to that which
has been reported for poly(N-isopropylacrylamide) (PNIPAM)
vs poly(N-isopropylmethacrylamide) species (PNIPMAM).66,67
In the case of the latter two polymers, PNIPAM has a well-
documented LCST of ca. 32.0 °C whereas the methacrylamido
species, PNIPMAM, has a reported higher LCST of ∼47.0 °C
even though a cursory examination of the structure, with the
extra hydrophobic methyl group, would suggest that
PNIPMAM should undergo a temperature induced phase
transition at a temperature below that of PNIPAM. This
interesting behavior has been attributed to the methyl group
hindering the coil-to-globule transition and subsequent
aggregation and phase transition. Such an effect is not,
apparently, at play in the case of the ethylene glycol-based
methacrylamido polymers described here. In agreement with
the enhanced water solubility of amides vs esters and with an
increase of OEG side chain length, polyOEG350MAM and
polyOEG750MAM (Table 1, entries 15, 16) did not show a
phase transition between 0 and 93 °C.
The effect of (co)polymer concentration on the cloud point
was determined for two samples: poly[(MEO2)76-co-
Hex24]AM and polyMEO2MAM which have cloud points of
26.0 and 55.8 °C respectively at the concentration of 5.0 g/L
examined previously. The (co)polymers were also examined at
concentrations of 0.5, 1.0, 2.0, and 10.0 g/L, Figure 7. Consider
first the concentration effect on poly[(MEO2)76-co-Hex24]AM.
At the lowest concentration evaluated, 0.5 g/L, this amphi-
philic copolymer exhibits a measured cloud point of 37.2 °C.
Doubling this concentration results in a decrease of the cloud
point of nearly seven degrees to 30.4 °C while increasing the
concentration further to 2.0 g/L results in a further, but less
pronounced decrease to 26.7 °C. Beyond this concentration
Figure 5. Heating endotherms of two samples obtained by differential
scanning calorimetry showing the significant difference of the
transitions of a copolymer with few hydrophobic comonomer units
(blue curve, top) and a polymer with thermally responsive side chains
(red curve, bottom).
poly[(MEO2)85-co-Hex15]AM copolymer produced only a very
small narrow peak spanning ∼2 °C and an area of 0.073 kJ/mol
average monomer units. The endotherms represent molecular
events occurring during the phase transition and account for
the loss of solvation (e.g., through hydrogen bonding) when
the polymer undergoes a coil-to-globule transition. The large
difference between the two observed polymers thus suggests
a much larger number of hydrogen bonds are breaking dur-
ing the phase transition of the OPG derivative. This is not
surprising as the OPG graft homopolymer carries stronger
hydrophobic side chains that are in fact thermally responsive
themselvesin contrast to the poly[(MEO2)85-co-Hex15]AM
copolymer.
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Figure 7. Influence of (co)polymer concentration on the cloud point for an acrylamido copolymer (blue curves) and a methacrylamido polymer (red
curves). (A) Turbidity curves. (B) Plot of cloud point versus concentration showing the stronger increase of phase separation temperature with
decreasing temperature for the methacrylamido derivative.
there appears to be little, if any, concentration dependence on
the LCST with phase transitions occurring at 26.0 and 26.2 °C
at concentrations of 5.0 and 10.0 g/L. The concentration
dependence of the cloud point for polyMEO2MAM is
significantly more pronounced. Recall that the cloud point at
a concentration of 5.0 g/L for this homopolymer was
determined to be 55.8 °C. A similar value of 55.6 °C was
found for a 10.0 g/L solution. At 2.0 g/L, however, the cloud
point jumps by 13 to 68.8 °C, while at the a concentration of
1.0 g/L the cloud point jumps an additional ∼30 to 95.7 °C
(determined by extrapolation of the measured cloud point
curve to 50% transmittance). Below this concentration the
polymer was soluble over the entire observed temperature
range. These results indicate that the acrylamido copolymer
would be the better choice for applications where a larger range
of, essentially, concentration independent responsiveness is
desired.
Urea is a well-documented H-bond breaking molecule that
can have a dramatic effect upon the solution behavior of
(co)polymers in aqueous media.68−70 For example, Sagle
et al.68 noted that the LCST of PNIPAM gradually decreases from
∼32.0 to ca. 23.0 °C upon increasing the urea concentration to
7.0 M; an observation rationalized in terms of urea facilitating
the hydrophobic collapse via intramolecular H-bonding to the
PNIPAM amide residues. Similarly, the addition of urea can
induce insoluble-to-soluble transitions upon heating (UCST-
type behavior) whereby urea disrupts the inter- or intra-
molecular H-bonding interactions responsible for initial
insolubility.70 The effect of added urea upon the aqueous
phase transitions of polyMEO2MAM and poly[(MEO2)85-co-
Hex15] was examined with the expectation that these
(meth)acrylamido-PEG species would behave in a fashion
similar to that reported for PNIPAM, i.e., urea addition would
result in a decrease in the LCST. As described above,
polyMEO2MAM has a measured cloud point, in pure water,
of 55.8 °C at 5.0 g/L. At a urea concentration of 26.7 mM,
corresponding to one molecule of urea per repeat unit, a small
increase in the LCST is observed with the phase transition
occurring at 56.5 °C. Increasing the concentration to 0.5 M
urea results in a more significant increase in the LCST to 63.1 °C
and finally at concentrations ≥2.0 M the LCST behavior
completely vanishes and the polymer remains soluble up to
temperatures in excess of 93.0 °C. This would suggest that
inter- and intramolecular H-bonding associated with the amide
residues is a very important factor governing the phase behavior
of these materials and has a pronounced effect on the LCST.
Disruption of these H-bonding associations raises the LCST by
eliminating an important physical pathway by which such
polymers can self-aggregate and undergo the coil-to-globule
phase transition. A similar increase in the LCST was also
observed for poly[(MEO2)85-co-Hex15] upon addition of 0.5 M
urea (46.7 to 48.8 °C), Figure S6 in Supporting Information.
Solution Properties in Alcohols. In addition to an
evaluation of the LCST behavior in aqueous media of the
(meth)acrylamido PEG derivatives their UCST behavior in
2-propanol and 1-octanol was also investigated at a concentration
of 5.0 g/L. The experimentally determined UCST values are
given in Table 2 and are plotted in Figure 8A. Turbidity curves
Table 2. Cloud Points (50% Transmittance) Found for
(Meth)acrylamido Homopolymers in 2-Propanol and
1-Octanol
cloud point 2-
propanol (5.0 g/L)
cloud point 1-octanol
a
a
(5.0 g/L)
cooling
(°C)
heating
(°C)
cooling
(°C)
heating
(°C)
entry
polymer
b
1
2
polyMEO2AM
polyMEO3AM
polyOEG350AM
polyOEG750AM
pOEG5kAM
s
s
s
s
s
s
s
s
s
s
3
s
s
4
9.3
30.2
s
16.0
38.5
s
7.5
25.5
s
18.9
45.5
s
5
6
polyMEO2MAM
polyMEO3MAM
polyOEG350MAM
polyOEG750MAM
PEG 6k
c
c
7
n.d.
4.4
13.9
15.8
−3
8.1
n.d.
4.0
9.0
16.5
2
8
8.8
9
17.4
41.5
18.6
49.2
10
11
d
e
polyMEO4/5MA
18.7
a
Cloud points depend on concentration, molecular weight and
b
polymer end groups. Measurements in alcohols were carried out
c
between 0 and 70.0 °C. Determined visually with a thermometer.
d
[Poly(ethylene glycol) monomethyl ether methacrylate], Mmonomer
=
e
300 g/mol. Found for a 10.3 kg/mol sample at 5.0 g/L, value taken
from reference12
measured in 2-propanol are shown in Figure 8B, curves
measured in 1-octanol may be found the Supporting
Information, Figure S7.
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Figure 8. UCST-type behavior of OEG amide homopolymers in alcohols. (A) Cloud point values of polyacrylamides (blue), polymethacrylamides
(purple), and reference PEG 6k in 2-propanol (squares) and 1-octanol (circles) in dependence of OEG side chain length (dotted lines are added to
guide the eye). (B) Cooling curves of homopolymers in 2-propanol.
Several important observations were made. First, both di-
ethylene glycol (meth)acrylamido polymers polyMEO2AM
and polyMEO2MAM exhibit high solubility in both 2-propanol
and 1-octanol with no clouding occurring when kept at
−25.0 °C for several days (Table 2, entries 1 and 6). For longer
EG side chains, the acrylamido derivatives showed a better
solubility in both tested alcohols compared to the methacry-
lamido versions, very similar to the observations made on
aqueous solutions. Whereas polyMEO3MAM and poly-
OEG350MAM (Table 2, entries 7 and 8) were found to cloud
in 2-propanol at low temperatures (below −3.0 °C and at 4.4 °C,
respectively), the solutions of the corresponding acrylamido
polymers polyMEO3AM and polyOEG350AM (Table 2, entries
2 and 3) in both alcohols remained transparent down to
−25.0 °C. In the same fashion, but significantly less pronounced,
the cloud points of polyOEG750MAM (Table 2, entry 9, purple
symbols in Figure 8A) were found to be slightly higher (4.6 °C
higher in 2-propanol, 1.5 °C higher in 1-octanol) than those of
polyOEG750AM (Table 2, entry 4, blue symbols in Figure 8A)
indicating a decreased solubility. Second, with an increasing
length of the pendant EG chains the UCST-type cloud point
increases, as indicated by the dotted lines in Figure 8A. For
example, in 2-propanol, the phase separation of poly-
OEG750MAM (13.9 °C) occurred at a temperature 9.5 °C
higher than that of polyOEG350MAM (4.4 °C) (dotted purple
lines in Figure 8A). In order to evaluate whether this was also
true for acrylamido polymers and to determine the properties
of a graft-copolymer with much longer PEG side chains,
polyPFPA was reacted with PEG 5000 amine. As with the
lower molecular weight amines, 19F NMR of the reaction
mixture indicated a complete cleavage of the activated esters.
However, SEC analysis revealed a bimodal distribution with a
(PS standard) molecular weight of 177.4 kg/mol and a PDI of
1.74 (Table 1, entry 5, Figure S4 in Supporting Information).
This very broad distribution may be due to an incomplete
conversion involving hydrolysis reactions and suggesting that
this sterically challenging graft reaction poses a limitation to the
PFP ester approach. It is also plausible that the PEG 5000
amine contained some diamine impurities that caused cross-
linking. In spite of its broad molecular weight distribution,
polyOEG5KAM represented a significantly larger PEG amide
analogue. As anticipated, it was found to have increased cloud
points (i.e., decreased solubility) in both alcohols compared to
the acrylamido polymer with shorter OEG side chains, with
transitions occurring at 30.2 °C (2-propanol) and 25.5 °C
(1-octanol), respectively (Table 2, entry 5, dotted blue lines in
Figure 8A). It was, however, noted that the increase of
transition temperature with increasing OEG side chain length
also caused an increase in the hysteresis. Heating and cool-
ing transition temperatures highlighting the hysteresis are
plotted in Figure S8A, B in the Supporting Information. This ef-
fect was especially pronounced in 1-octanol, where the
temperature difference between cooling and heating cycles
was found to increase from 4.8 to 9.6 °C when going from
polyOEG350MAM to polyOEG750MAM and from 11.4 to
20.0 °C when increasing the PEG chain length from
polyOEG750AM to polyOEG5KAM. For comparison, a stand-
ard PEG 6K, with OH end groups, was measured in 2-propanol
and 1-octanol giving cloud points of 15.8 and 16.5 °C,
respectively, values in between those of the acrylamido OEG750
and OEG5K graft polymers (Table 2, entry 10). The hystereses
of PEG 6K in 2-propanol and 1-octanol were found to be very
large with 25.7 and 32.7 °C, respectively, suggesting that in
terms of retarded redissolution characteristics, the OEG amide
polymers approach the behavior of pure PEG with an
increasing OEG side chain length. Similar to the case in
aqueous solutions, the amide species exhibited a better solubil-
ity than the corresponding ester analogues. As a reference,
the cloud point of the polymethacrylate polyMEO4/5MA in
2-propanol was found to be 18.7 °C12 which is higher than the
measured transition temperatures even for the methacrylamido
polymers with longer OEG side chains, polyOEG350MAM (4.4 °C)
and polyOEG750MAM (13.9 °C). A surprising observation
was the very little difference between 2-propanol and 1-octanol,
with all transition temperatures (except PEG 6K) in 2-propanol
being somewhat higher than those in 1-octanol. This is in
complete contrast to the behavior of the methacrylate deriva-
tives for which a transition temperature increase of roughly
5.0 °C per additional methylene group in n-alcohols was
found.12 It therefore seems that the ester group plays an im-
portant role in the unfavorable interactions between an OEG-
based polymer and the alcoholic solvent, that cause the polymer
to lose solvation during the chain collapse. The amide group on
the other hand seems to be capable of forming hydrogen bonds
of similar thermal stability independent of the chain length of
the alcohol. Upon exchanging the ester for an amide group,
there is no decreased solubility with an increased alcohol chain
length. The amide analogues therefore present an interesting
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class of materials with predictable thermal properties
independent of alcohol polarity that can range from hydrophilic
(2-propanol, miscible with water in all proportions) to
hydrophobic (1-octanol, essentially insoluble in water).
demonstrate the potential of the route described herein for
preparing biocompatible copolymers containing biorelevant
molecules, the precursor polyPFPA homopolymer was treated
with a mixture of di(ethylene glycol) methyl ether amine and
thyroxine (T4), Table 1 entries 10 and 11. T4 is a prohormone
serving as a precursor to triiodothyronine (T3) secreted by
the thyroid gland and is involved in controlling the rate of
metabolic processes and also influences physical develop-
ment.71,72 The relevant region of T4, where recognition and
metabolic conversion into T3 takes place, is the outer
diiodophenyl ring. T4 has been shown to selectively recognize
and bind its transport protein transthyretin even when the
amino group at the opposite end has been functionalized.11,73
The synthesis was accomplished by employing a mixture of
di(ethylene glycol) methyl ether amine and T4, the
Biocompatibility and Bioconjugation. As noted earlier,
besides the interesting phase behavior exhibited by (meth)-
acrylic PEG analogues, and also demonstrated herein for
(meth)acrylamido species, PEG and its derivatives have
attracted significant attention in the biomaterials arena due to
the nontoxic, antifouling and so-called ‘stealth’ properties
exhibited by such materials. To demonstrate the potential of
these (meth)acrylamido (co)polymers in biomedical applica-
tions the cytotoxicity of two samples, polyMEO2AM and
polyMEO3AM, were tested on MRC5 fibroblast cells at
concentrations ranging from 0.005 to 10.0 g/L. These two
samples were chosen since both are completely soluble in
aqueous media up to temperatures in excess of 93.0 °C, in
contrast to the acrylic analogues, and highlights how the
(meth)acrylamido species may offer advantages in biomedical
applications over the (meth)acrylic species by virtue of both
enhanced solubility (higher or absent LCSTs) and significantly
improved hydrolytic stability of the amide vs ester functional
groups. Cell viability was determined via a fluorescence method
by which living, metabolically active, MRC5 cells convert a
redox dye (blue resazurin) into a fluorescent species (red
resorufin). Thus, measuring the fluorescence affords a
straightforward method for determining cell concentration
and thus, indirectly, the cytotoxicity of the acrylamido-PEG
polymers. Figure 9 shows a plot of cell viability vs polymer
1
incorporation ratio was verified and quantified by H NMR
spectroscopy, which showed the distinctive broad aromatic
signals at 7.81 and 7.11 ppm originating from the incorporated
T4 groups. The activated esters were quantitatively cleaved, as
1
determined by 19F NMR spectroscopy, and H NMR of the
product indicated a complete presence of MEO2 + T4 groups in
both cases (Figure S9 in Supporting Information). SEC
analyses gave unimodal narrow distributions with the apparent
molecular weight increasing with increasing T4 contents which
can be attributed to a conformational influence of the bulky T4
side groups (Figure S10 in Supporting Information). Two
copolymers, poly[(MEO2)91-co-T49]AM and poly[(MEO2)87-
co-T413]AM with 9 and 13 mol % of T4 units respectively were
prepared. This demonstrates that the PFP precursors provide a
versatile platform for the preparation of biofunctionalized PEG
analogues. In particular, in light of specific binding of proteins
to main chain bound targets, the short di(ethylene glycol)
amide side chains would be at an advantage over longer,
sterically more challenging, ester-bound OEG chains that would
be necessary to provide a similar thermally independent water
solubility. Both copolymers with 9 and 13% of T4 were found
to be totally soluble in water up to 93 °C, again highlighting the
enhanced solubility provided by the OEG amides also in the
presence of the biological hydrophobic comonomer.
CONCLUSIONS
■
Acrylamido and methacrylamido (co)polymers with ethylene
glycol (EG) side chains of varying lengths were prepared from
pentafluorophenyl activated ester precursor polymers with the
respective ethylene glycol amines. Quantitative formation of
EG amides, as well as of amide copolymers was found for the
acrylic derivatives with as low as 1.2 equiv of amines per
activated ester. For the methacrylate polymers a larger excess of
amines was necessary to minimize the formation of acid groups.
The PEG amide analogues showed an enhanced solubility in
water and alcohols compared to the more commonly used ester
derivatives which is attributed to the H-bond donor/acceptor
properties of the amide linkage. All acrylamido species showed
a better solubility (higher LCST transitions in water, lower
UCST transitions in alcohols) than the corresponding
methacrylamido polymers, and in contrast to the PNIPAM/
PNIPMAM pair in water. Of the examined homopolymers,
only the polymethacrylamides with MEO2 or MEO3 side
groups showed LCST behavior in water, however with an
unusually strong concentration dependence of the cloud point,
as determined for polyMEO2MAM. The acrylamido polymers
can be rendered thermosensitive by the incorporation of
hydrophobic comonomers such as n-hexylamide via the
Figure 9. Cell viability after incubation of MRC5 cells with varying
concentrations of acrylamido PEG polymers for 72 h.
concentration after incubation of the MRC5 cells in the
presence of polyMEO2AM and polyMEO3AM for 72 h. In
both instances these acrylamido-based PEG analogues were
found to be noncytotoxic with measured cell viability at ≥85%
after 3 days even at high concentrations. These results confirm
that these acrylamido-PEG derivatives exhibit desirable
“biocompatible” properties which when taken in conjunction
with the enhanced solubility and hydrolytic stability (vs the
(meth)acrylic species) suggests that these materials may find
potential application in areas not accessible to the ester-
analogues.
As discussed above, the aqueous phase behavior of the
acrylamido-based PEG analogues can be tuned via the
incorporation of a hydrophobic comonomer. In addition to
statistical copolymers containing hexylamide residues, and to
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Macromolecules
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ASSOCIATED CONTENT
* Supporting Information
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■
S
Representative 19F NMR spectra, 1H NMR spectra of
hexylamide copolymers, list of feed ratios and obtained ratio
for hexylamide copolymers, SEC traces of hexylamide
copolymers and of polyOPG600AM and polyOEG5kAM,
turbidity measurements of DSC samples, turbidity measure-
ments of samples containing urea, turbidity curves of
(meth)acrylamido homopolymers in 1-octanol, plot of UCST
cloud points found in 2-propanol and 1-octanol showing
hysteresis values, 1H NMR spectra and SEC chromatograms of
copolymers containing thyroxine. This material is available free
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AUTHOR INFORMATION
Corresponding Author
*E-mail: (A.B.L.) a.lowe@unsw.edu.au; (P.J.R.) p.roth@unsw.
edu.au.
■
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
Dr. Anthony Granville is kindly thanked for help with the DSC
measurements.
■
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