Stimuli-responsive polymers based on L-phenylalanine residues: Protonation thermodynamics of free polymers and cross-linked hydrogels

Article abstract of DOI:10.1021/ma047652i

Vinyl polymers carrying L-phenylalanine residues were synthesized in the free and in the cross-linked hydrogel form, as homo- and copolymers with N-isopropylacrylamide (NIPAAm). The protonation reaction thermodynamics of the COO^- group were stu

Full text of DOI:10.1021/ma047652i

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2460 Macromolecules 2005, 38, 2460-2468
Stimuli-Responsive Polymers Based on L-Phenylalanine Residues:
Protonation Thermodynamics of Free Polymers and Cross-Linked
Hydrogels
Mario Casolaro,*,† Eugenio Paccagnini,‡ Raniero Mendichi,§ and Yoshihiro Ito¥
Dipartimento di Scienze e Tecnologie Chimiche e dei Biosistemi, and Dipartimento di Biologia
Evolutiva, Via Aldo Moro, Universita` degli Studi di Siena, I-53100 Siena, Italy, Istituto per lo Studio
delle Macromolecole (CNR), via E.Bassini 15, I-20133 Milano, Italy, and Kanagawa Academy of
Science and Technology, KSP East 309, Sakado 3-2-1, Takatsu-ku, Kawasaki, Kanagawa 213-0012
and RIKEN, (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako,
Saitama 351-0198, Japan
Received November 15, 2004; Revised Manuscript Received January 5, 2005
ABSTRACT: Vinyl polymers carrying L-phenylalanine residues were synthesized in the free and in the
cross-linked hydrogel form, as homo- and copolymers with N-isopropylacrylamide (NIPAAm). The
protonation reaction thermodynamics of the COO- group were studied in aqueous media, at different
temperatures and at increased concentrations of sodium chloride, mainly by potentiometry and solution
calorimetry. The soluble polymer, namely poly(N-acryloyl-L-phenylalanine), and its copolymers with low
NIPAAm content displayed characteristic features. Their basicity constants (log K), as well as the enthalpy
(-∆H°) changes in relation to R (degree of protonation) showed an abrupt drop at R ) 0.5. This was
ascribed to the formation of hydrogen bonds between the protonated and the ionized neighboring COO-
groups. The process was driven by the side-chain aromatic rings that improved hydrophobic interactions.
The entropy (∆S°) changes sharply increased as a result of the increased macromolecular conformational
freedom and the release of water molecules surrounding the hydrophilic groups of the polymer. The
corresponding cross-linked polymers formed hydrogels that were responsive to pH, temperature, and
ionic strength. The two hydrogels, P9 (homopolymer with 9 mol% cross-links) and CP2 (copolymer with
90 mol% of NIPAAm and 2 mol% cross-links), were characterized for their pH- and temperature-responsive
behavior by equilibrium and oscillatory swelling studies. They demonstrated a strong pH-dependent
volume phase-transition and an unusual sodium chloride phase-transition phenomenon. Moreover, the
hydrogel CP2 exhibited a temperature-dependent volume phase-transition (LCST, lower critical solution
temperature) behavior in aqueous solution, where the LCST decreased by lowering the pH. It was nontoxic
against the RAW264 cell line.
Introduction
Previously, we studied the protonation thermodynam-
ics of a series of (meth-) acrylate polymers containing
R-amino acid residues (L-valine and L-leucine) that
showed a close structure to PNIPAAm and had a
carboxyl acid group in the monomer unit.1-4,27,28 The
polymers were studied in aqueous media as homo- and
copolymers with NIPAAm, either in the soluble and
hydrogel forms.29 Recently, a poly(ampholyte) polymer,
containing L-histidine residues was also reported.30 The
latter polymer was studied because its LCST may be
changed by tuning pH at low or high values, i.e., below
and above the isoelectric point.31,32
The purpose of this work is to synthesize a novel vinyl
monomer containing L-phenylalanine residues. From
this, a series of acrylate polymers (Chart 1) and copoly-
mers with NIPAAm were obtained in the free and in
the cross-linked hydrogel form.
L-Phenylalanine is an essential amino acid and ty-
rosine precursor that works on the central nervous
system as an anti-depressant and mood elevator.33 The
phenyl group of the phenylalanine residue should
improve a rather large hydrophobic domain in which
the cohesive forces play a major role during the confor-
mational transition of the macromolecules. Morcellet et
al. reported that the methacrylate analogue, poly(N-
methacryloyl-L-phenylalanine), showed a pH-induced
conformational transition due to the hydrophobic inter-
actions between the aromatic side chains.34
In previous papers of recent years, several stimuli-
responsive polymers were synthesized and studied from
a thermodynamic point of view.1-7 The polymers con-
tained, besides the carboxyl group, amido and isopropyl
groups structurally related to the poly(N-isopropylacryl-
amide) (PNIPAAm).8 The latter is a well-known non-
ionic polymer that exhibits a sharp, thermoreversible
phase-transition (lower critical solution temperature,
LCST) behavior at 32°C in aqueous solution.9 The
possibility of tuning the LCST of PNIPAAm by incor-
porating comonomers with different hydrophilic char-
acters is an interesting feature to obtain “intelligent”
polymer systems for many biomedical and pharmaceuti-
cal applications,10 including controlled delivery of
drugs,11-14 molecular separation,15 tissue culture sub-
strates,16 and materials for improved biocompatibility.17
Among these “intelligent” materials, the pH- and tem-
perature-responsive light-cross-linked polymers (hydro-
gels) are the most widely investigated.8,18-26
* Author to whom correspondence should be addressed. Phone:
+39 0577 234388. Fax +39 0577 234177. E-mail: casolaro@unisi.it.
† Dipartimento di Scienze e Tecnologie Chimiche e dei Bio-
sistemi, Universita` degli Studi di Siena.
‡ Dipartimento di Biologia Evolutiva, Universita` degli Studi di
Siena.
§ Istituto per lo Studio delle Macromolecole (CNR).
¥ Kanagawa Academy of Science and Technology.
10.1021/ma047652i CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/12/2005

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Macromolecules, Vol. 38, No. 6, 2005
Stimuli-Responsive Polymers Based on L-Phenylalanine 2461
Chart 1. Structure of the Monomer Unit of Poly(Phe)
product was separated; it was filtered with suction and purified
by recrystallization from water. The yield of the dry product
was 38 g (56%). The compound was characterized by spectros-
copy (proton NMR and infrared spectra), and elemental
analysis.
Polymers. Poly(N-acryloyl-L-phenylalanine) [poly(Phe)] and
poly(N-acryloyl-L-phenylalanine-co-N-isopropylacrylamide) [poly-
(Phe-co-NIPAAm)] at different Phe/NIPAAm molar ratios were
prepared by a radical polymerization of the corresponding
monomers.3,4,29,30 The poly(Phe) was prepared in two different
solvents, in methanol and in ethanol/benzene (1/1) mixture,
dissolving 2.00 g of Phe in 20 mL of a well-degassed and
nitrogen-purged solution. To each solution, 30 mg of recrystal-
lized (from methanol) AIBN was added. The mixture was
allowed to react in a thermostated water bath at 65 °C for 24
h. In the methanol solution, the polymer started to precipitate
after 30 min. It was washed with fresh methanol and treated
with ethyl ether three times. The polymer from the ethanol/
benzene mixture was precipitated in ethyl ether (200 mL),
under magnetic stirring. The voluminous white polymer was
repeatedly washed with fresh ethyl ether and dried in vacuo.
The yield was 1.75 g (87%) and 1.10 g (55%) for the poly(Phe)s,
respectively, from methanol and an ethanol/benzene mixture.
Both compounds were characterized by proton NMR and
infrared spectra, along with elemental analysis. The poly(Phe-
co-NIPAAm)s were obtained with a similar procedure at three
different Phe/NIPAAm molar ratios (Table 1). In all cases, the
Phe and the NIPAAm monomers were dissolved in ethanol/
benzene (1:1) solution. The mixture was purged with nitrogen
and was allowed to react at 60 °C in a thermostated water
bath for 24 h. The copolymer was precipitated in ethyl ether
(300 mL), washed with fresh ether, and dried in vacuo. The
amount of COOH groups incorporated into the compounds was
determined by potentiometric titrations.
Cross-Linked Polymers (Hydrogels). Two polymers were
obtained with different amounts of cross-linking N,N′-ethylene-
bis-acrylamide (EBA).30 The first one (P9) was a poly(Phe)
containing 9 mol% of EBA, and the second one (CP2) was a
poly(Phe-co-NIPAAm) with a NIPAAm/Phe molar ratio of 10
and cross-linked with 2 mol% of EBA. The synthesis was
carried out at room temperature in a glass tube and under
nitrogen atmosphere by the following procedure. Hydrogel P9
(14.5 wt% monomer concentration): the monomer Phe (2.00
g, 9.12 mmol) was dissolved in twice-distilled water (15 mL)
containing TEA (93 mg) and EBA (156 mg, 0.91 mmol).
Hydrogel CP2 (14 wt% monomer concentration): the monomer
Phe (0.57 g, 2.58 mmol) was dissolved in twice-distilled water
(27 mL) containing TEA (275 mg) and EBA (97 mg, 0.56
mmol). Both mixtures were repeatedly flushed with nitrogen,
and then APS (20 mg) was added. The reaction mixtures were
kept at room temperature (r.t.) for 24 h even if the gelation
was observed within 15 min. Afterward, the hydrogels were
removed, thoroughly washed with twice-distilled water for 1
week, and then slowly dried at r.t. up to constant weight in a
desiccating cabinet. Unlike the hydrogel CP2, which was cut
in small disks, the hydrogel P9 was treated with acetone to
give a fine powder for potentiometric analysis.
The solution properties of this polymer, as well as of
the polymers with L-valine and L-leucine residues previ-
ously reported, are those of polyelectrolytes with a
compact conformation in water due to hydrophobic
forces between phenyl or isopropyl groups.1,3,4,27,28 These
attractive forces outweigh the repulsive electrostatic
interactions between the negatively charged COO-
groups when the polymer reaches a critical degree of
protonation, R. This critical R value strongly depends
on the nature of the R-amino acid residues and on the
kind of acrylate or methacrylate polymer.1,3,27 The
magnitude of the hydrophobic character may be re-
vealed also by calorimetric data. Thus, the thermody-
namic study is of special interest because it enables a
correlation to be established between the increasing
hydrophobicity and the appearance of compact struc-
ture.34,35 Moreover, the results of this preliminary
investigation should be of interest in designing slightly
cross-linked polymer networks (hydrogels) that should
be responsive to different external stimuli. We evaluated
the thermodynamic functions of poly(Phe) and described
the protonation mechanism of the polymer in the free
and in the cross-linked form and the corresponding
copolymers with NIPAAm by using mainly potentio-
metric and solution calorimetric techniques. Moreover,
the swelling behavior of the hydrogels with respect to
ionic strength, pH, and temperature was investigated.
A copolymer hydrogel, which is responsive to both pH
and temperature, would be able to respond to conditions
where both variables are coupled.
Experimental Section
Materials. L-Phenylalanine (99%), acryloyl chloride (97%),
R,R′-azobisisobutironitrile (AIBN), 2,6-di-tert-butyl-p-cresol,
ammonium peroxodisulfate (APS, 98%), triethylamine (TEA,
99%), and N,N′-ethylene-bis-acrylamide (EBA, 98%) were
purchased from Fluka Co. N-Isopropylacrylamide (NIPAAm,
97%) was purchased from Aldrich Co.
Spectroscopic Measurements. Proton NMR spectra of
the monomer and the polymers dissolved in DMSO-d6 were
recorded on a Bruker AC200 spectrometer using tetrameth-
ylsilane as internal reference. The FT-IR spectra of the
samples were recorded on a FTS 6000 Biorad spectrophotom-
eter.
Syntheses. Monomer. N-Acryloyl-L-phenylalanine (Phe)
was obtained by the usual synthetic route to introduce the
vinyl group in the monomers.1,3,4,28,30,36 To a well-stirred
aqueous solution of L-phenylalanine (54.5 g, 0.33 mol), sodium
hydroxide (26.7 g, 0.67 mol) and 2,6-di-tert-butyl-p-cresol (0.10
g) in twice-distilled water (100 mL) was added dropwise
acryloyl chloride (29.3 g, 0.31 mol) over a 30 min period while
the reaction mixture was kept below 0 °C by external ice-bath
cooling. After the addition was completed, the stirring was
continued for additional 1 h with the temperature raising to
room temperature. The mixture was acidified to pH 2 with
concentrated hydrochloric acid (28 mL). A voluminous white
Molecular Characterization. The molecular character-
ization of the poly(Phe) homopolymer and of the poly(Phe-co-
NIPAAm) copolymer (co-2) was performed by a multi-angle
laser light scattering (MALS) Dawn DSP-F photometer from
Wyatt (Santa Barbara, CA). As the co-2 did not elute at all
from the usual aqueous size exclusion chromatography (SEC)
columns, both the homopolymer and the copolymer were
characterized by means of a hydrodynamic chromatography
(HC) method. In such a system, the classical SEC column was
replaced with a long capillary tube. Obviously, the separation
performance of this HC-MALS system was not adeguate in
obtaining the whole molar mass distribution but sufficient in

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2462 Casolaro et al.
Macromolecules, Vol. 38, No. 6, 2005
Table 1. Feed Composition for the Copolymerization of NIPAAm with Phe
NIPAAm
COOH group content (wt%)b
Phe
compd
g
mmol
g
mmol
AIBN mg
vol.a mL
yield g
theor
exp
co-10
co-2
co-0.5
2.04
2.04
2.14
9.30
9.30
9.76
0.11
0.55
2.06
0.94
4.71
17.4
37
43
53
25
25
40
1.34
2.07
2.89
94.9
78.8
51.3
83.4
71.8
46.5
a Ethanol/benzene (1:1) solution; b From the acid-base titration.
obtaining a reliable weight-average molar mass, Mw, value of
the polymers. The HC-MALS experimental conditions were the
following: 0.2 M NaCl + 0.1 M Tris buffer pH 8.0 as mobile
phase, 35 °C temperature, 0.4 mL/min flow rate, and 50 µL
injection volume. The wavelength of the MALS laser was 632.8
nm. The light scattering signal was simultaneously detected
at 15 scattering angles ranging in the solvent from 14.5° to
151.3°. The calibration constant was calculated using toluene
a weighed amount of compound (monomer, 0.20-0.25 mmol;
polymer, 0.18-0.21 mmol; copolymer 0.17-0.22 mmol) and a
measured volume of standard sodium hydroxide, was titrated
with standard 0.1 M HCl solution with a constant BDR (buret
delivery rate) of 0.0837 mL/min through a Gilmont buret. A
chemical calibration (Tris/HCl) of the instrument and correc-
tions for the heats of the titrant dilution were made before
each titration experiment. All of the experiments were auto-
matically controlled by the Thermal program from Tronac, Inc.,
which was renewed to operate through a NI-DAQ driver
software in Windows, from National Instruments. The graphi-
cal programming language LabVIEW was used to create the
application. The enthalpy changes were evaluated with the
Fith program40 by taking into account the dependence of the
log K’s on the degree of protonation, R, for the polymer and
the copolymers. The entropy change values were calculated
by the relation ∆S° ) (∆H° - ∆G°)/T with -∆G° ) RT ln K.
The results of at least two replicates were averaged.
as standard assuming a Rayleigh Factor of 1.406 × 10-5 cm-1
.
The angular normalization was performed by measuring the
scattering intensity of a concentrated solution of a BSA
globular protein in the mobile phase assumed to act as an
isotropic scatterer. The MALS photometer was described in
detail elsewhere.37,38 The refractive index increment, dn/dc, of
the poly(Phe) and of the co-2, with respect to the used solvent,
was measured by a KMX-16 differential refractometer from
LDC Milton Roy (Riviera Beach, FL). The dn/dc values for poly-
(Phe) and for co-2 were, respectively, 0.215 and 0.202 mL/g.
Scanning Electron Microscopy (SEM). The morphology
of the hydrogels P9 and CP2 was examined by the XL20
Philips scanning electron microscopy. Samples were mounted
on SEM stubs with Leit-C Conductive Carbon Cement and
sputtered with 20 nm gold by a Balzers Med 01 sputter-coater.
The morphology of the hydrogels was analyzed at 20 kV and
at different magnifications.
Swelling Measurements. The equilibrium and oscillatory
swelling studies were carried out on the two hydrogels P9 and
CP2 as functions of environmental pH, temperature, and ionic
strength of the bathing medium. The hydrogel CP2 had a dried
thin-disk form. In the equilibrium swelling experiments, a
dried CP2 sample was weighed (Wdry
) and placed in a Stainer
cell (100 µm pore size); then, the cell was immersed in a buffer
solution of known pH and was allowed to swell to equilibrium
at the desired pH value, temperature, or ionic strength. A
similar procedure was followed for the hydrogel P9, even if in
the form of a fine powder. The degree of swelling (DS) was
monitored at intervals, until a plateau of DS/time plot was
reached. Afterward, for swelling studies in buffer solutions
(0.01 M Tris, 0.01 M phosphate, 0.01 M acetate, and 0.01 M
hydrochloric acid) contained in a thermostated glass cell (100
mL of 0.15 M NaCl) and having pH values ranging from 3 to
9, the gel sample was placed in the proper medium and allowed
to equilibrate for a further 24 h under stirring. The temper-
ature changes were monitored by the Haake D8 thermostat.
To study the effect of sodium chloride concentration, both the
gels P9 and CP2 were swollen at high pHs in Tris buffer (0.01
M). Thus, weighed portions of NaCl were consecutively added
to the solution to have the desired final concentration of the
simple salt. The degree of swelling was taken after 24 h. In
the oscillatory swelling experiments of CP2, the pH was
kept constant at 4.80 by a buffer solution (0.01 M acetate
in 0.15 M NaCl) and the temperature was varied between
25 and 35 °C. In all cases, the sample was removed from the
bath at intervals, quickly blotted with tissue paper to remove
any surface water, and weighed (wet weigh, Wwet). The
procedure was repeated to a constant weight. The equilibrium
Potentiometric Measurements. Potentiometric titrations
were performed according to a previously described proce-
dure.30 The measurements were carried out in aqueous solu-
tion at 25 °C by using a TitraLab 90 titration system from
Radiometer Analytical. TitraLab 90 consists of three compo-
nents: the powerful Titration Manager (TIM900), the high-
precision autoburet, and a convenient sample stand (SAM7).
The TimTalk 9, Windows-based software, was used in con-
nection with the TIM900 Titration Manager for remote control.
The titrations of the compounds were performed in the
thermostated glass cell filled with 100 mL of 0.15 M NaCl in
which a weighed amount of solid material (monomer, 0.18-
0.35 mmol; polymer, 0.17-0.21 mmol; copolymers, 0.20-0.34
mmol; hydrogel, 0.19-0.26 mmol) and a measured amount of
standard sodium hydroxide solution were dispersed by mag-
netic stirring. A presaturated nitrogen stream was maintained
over the surface of the solution to avoid CO2 contamination.
Unlike the monomer, which was soluble over the whole range
of pH, the forward titrations of the equilibrated alkaline
solution containing the polymer, the copolymer, or the hydrogel
were performed with standard 0.1 M hydrochloric acid solution
at the equilibration time of 300 s for each titration step (0.04
mL). The backward titrations with standard 0.1 M NaOH
solution showed rielable results. The endpoint of the poten-
tiometric titration curve was taken to calculate the excess of
sodium hydroxide equivalents. The difference between the
initial amount and the equivalents of hydroxide ions was
attributed to the COOH proton equivalent of the polymer and
the copolymers. The basicity constants (log K’s) of the monomer
and the polymers were evaluated with the Superquad39 and
the ApparK40 programs, respectively. The results of three
replicates were averaged.
degree of swelling (EDS) was calculated as: EDS ) (Wwet
-
Wdry)/Wdry
.
Evaluation of Cytotoxicity. The evaluation of cytotoxicity
was performed as previously reported.30 The RAW264 cells,
derived from murine leukemoic monocyte and having a mac-
rophage-like morphology, were provided by Riken (Tsukuba,
Japan). They were cultured in a minimum essential medium
(Sigma) with 10% fetal bovine serum (FBS) and 1% nones-
sential amino acids (Invitrogen Life Technologies). The cells
were harvested with a 0.25% trypsin solution containing 0.5
mM EDTA. The recovered cells were washed with the culture
medium and suspended in the medium. The cell suspension
was added to the well of the 24-well plate in the presence of
the gel CP2 and was allowed to stand for 3 days at 37 °C. After
the incubation, the cell number was counted by microscopy.
The results were expressed as viability (%) relative to a control
Calorimetric Measurements. Calorimetric titrations were
performed following a previously reported procedure.30 The
measurements were carried out in aqueous solution at 25 °C
with a Tronac 1250 titration calorimeter, operating in the
isothermal mode by using a stainless steel reaction vessel of
25 mL capacity. The water bath was thermostated by means
of a PTC 40 (precision temperature controller, from Tronac,
Inc.). The aqueous solution (25 mL of 0.15 M NaCl), containing

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Macromolecules, Vol. 38, No. 6, 2005
Stimuli-Responsive Polymers Based on L-Phenylalanine 2463
Scheme 1. Synthetic Route to Phe
bone and side-chain resonances, consistent with the
presence of a slowly tumbling macromolecular species
in solution. The used HC-MALS method was able to
recover a rielable weight-average molar mass, Mw, value
of 47.6 kDa.
A series of copolymers with NIPAAm were prepared
at different Phe/NIPAAm comonomer compositions. The
composition was determined by potentiometric titrations
of the carboxyl acid groups. Compared with the feed
composition, a slight decreasing of the Phe unit content
was revealed in all cases (Table 1). These results suggest
that the comonomer feed ratios almost reflected the
relative comonomer incorporation levels. The radical
polymerization probably obeyed Bernouillian statistics,
forming copolymers with a random distribution of
COOH groups along the chain. This was confirmed by
the thermodynamic data, as the increasing amounts of
uncharged comonomer led to a decrease of the polyelec-
trolyte effect because the NIPAAm units shielded the
charges.41 The disappearance of the 1596 and 990 cm-1
very strong bands of the IR spectrum, in conjunction
with the NMR results, indicated a total conversion of
the monomers into the corresponding polymers and
copolymers. In all cases, the copolymers of acidic
monomers with NIPAAm showed a greater Mw value;28
with the HC-MALS method, it was possible to recover
a rielable weigh-average molar mass, Mw, of 146.1 kDa
for the co-2 copolymer.
Two hydrogels were prepared to study the effect of
the pH and the pH/temperature sensitivity on the
swelling properties of P9 and CP2, respectively. The gel
P9 was a homopolymer of Phe cross-linked with 9 mol%
of EBA, while the gel CP2 was a copolymer of Phe and
NIPAAm containing 10 mol% of Phe and cross-linked
with 2 mol% of EBA. The water solution containing the
monomers gelled at r.t. within 15 min, giving a soft and
clear product. The two cross-linked polymers were
treated in different ways. Unlike CP2, which was cut
in small disks, the hydrogel P9 was treated with acetone
to give a dispersed powder for the potentiometric study.
In both cases, the samples were slowly dried at r.t. for
15 days and then under vacuum to a constant weight.
Protonation Study. The protonation behavior of the
polymers in the free form and in the hydrogel form was
studied at 25 °C in aqueous media (0.15 M NaCl) by
potentiometric and solution calorimetric techniques.
Basicity Constants. The potentiometric titrations al-
lowed for the evaluation of the carboxylic acid groups
amount and of the basicity constants (log K). The values
of the latter, evaluated at the degree of protonation R
) 0.5, are reported in Table 2, along with all the
thermodynamic functions.
The peculiar polyelectrolyte behavior of the polymeric
compounds are depicted in Figures 2 and 3. It is evident
that the log K for the protonation of the Phe ionized
carboxyl group is greater than that of the corresponding
simple L-phenylalanine42 but more than 2 orders of
magnitude lower with respect to the poly(Phe) (Table
2). Moreover, the basicity of the acrylate monomer is
lower compared to the methacrylate analogue,34 while
the corresponding polymers revealed the greatest basic-
ity in the poly(acrylate) compound. This is the usual
trend already observed in vinyl polymers containing
R-amino acid residues.1,3,4,30 In the present case, the log
K of the monomer was lower than that of the other
monomers previously studied and the basicity of the
corresponding polymers was higher. The different in-
containing no polymer. The means ((SD) of four experiments,
each one containing three replicates, are reported.
Results and Discussion
Synthesis and Characterization. Monomer and
Polymers. The vinyl monomer N-acryloyl-L-phenylala-
nine (Phe) was obtained following the usual acylation
reaction of acryloyl chloride with the L-phenylalanine,
dissolved in aqueous solution and containing 2 equiv of
sodium hydroxide (Scheme 1).1,3,4,28,30,36When the solu-
tion was neutralized with concentrated hydrochloric
acid, a white solid formed. The crystallization from
water gave rise to white crystals of analytical grade, as
revealed by acid-base titrations, elemental, and spec-
troscopic analysis. It was freely soluble in water and in
most organic solvents. The proton NMR (Figure 1) and
the FT-IR spectra were consistent with the proposed
structure. The main IR frequencies observed (COOH,
1711 cm-1; amide I, 1650 cm-1; -CdC-, 1596 cm-1;
amide II, 1530 cm-1) were lower than those found in
the previously synthesized N-acryloyl derivatives con-
taining L-valine, L-leucine, or L-histidine residues,1,3,4,30
so the benzene ring provided a great inductive effect.
The elemental analysis was found to be in satis-
factory agreement: found (%) C, 65.2; N, 6.1; H, 6.2;
C12H13NO3; required C, 65.7; N, 6.4; H, 6.0. The
monomer could be cold-stored for a long time and
showed no sensitivity to the atmospheric moisture.
Unlike the monomer, which was soluble over the wide
pH range investigated, the corresponding polymer and
copolymers dissolved in aqueous solution only in the
ionized form. They formed a cloudy solution when a
stoichiometric amount of hydrochloric acid was added
to neutralize the COO- groups. The poly(N-acryloyl-L-
phenylalanine), the corresponding copolymers with
NIPAAm, and the hydrogels were synthesized by the
free-radical polymerization of the corresponding mono-
mers. The poly(Phe) was prepared in methanol and in
an ethanol/benzene (1:1) mixture using AIBN as a
radical initiator. Unlike the polymer obtained by the
ethanol/benzene mixture, which was recovered from
precipitation in ethyl ether, the compound from metha-
nol precipitated out during the polymerization. In both
cases, the purified compound showed a similar struc-
ture, as confirmed by spectroscopy (1H NMR and
FT-IR) and thermodynamic (potentiometry and solution
calorimetry) data. The elemental analyses were in
substantial agreement: found (%) C, 62.2; N, 5.9; H,
6.1; (C12H13NO3‚0.75H2O)x; required C, 61.9; N, 6.0; H,
6.3. Moreover, the FT-IR spectra revealed the disap-
pearance of the vinyl double bond at 1596 and 990
cm-1 and a slight wavenumber shift of the COOH to
1725 cm-1, while the other IR frequencies remained
almost the same as those in the monomer. The 1H NMR
spectra also showed the complete absence of the vinyl
double bond and the presence of broad lines for back-