Polymeric hydrogels modified with ornithine and lysine: Sorption and release of metal cations and amino acids

Article abstract of DOI:10.1002/pola.25063

A novel, convenient synthesis, using copper ions, is described for the multigram-scale preparation of acryloyl and methacryloyl ornithine and lysine without the need to use protecting groups and chromatographic purifications. Three methods of removing the

Full text of DOI:10.1002/pola.25063

JOURNAL OF
POLYMER SCIENCE
ARTICLE
WWW.POLYMERCHEMISTRY.ORG
Polymeric Hydrogels Modified with Ornithine and Lysine: Sorption and
Release of Metal Cations and Amino Acids
Jan Romanski, Marcin Karbarz, Krystyna Pyrzynska, Janusz Jurczak, Zbigniew Stojek
Department of Chemistry, Warsaw University, Pasteura 1, PL-02-093 Warsaw, Poland
Correspondence to: M. Karbarz (E-mail: karbarz@chem.uw.edu.pl) or J. Romanski (E-mail: jarom@chem.uw.edu.pl
Received 10 August 2011; accepted 14 October 2011; published online 9 November 2011
DOI: 10.1002/pola.25063
ABSTRACT: A novel, convenient synthesis, using copper ions, is
described for the multigram-scale preparation of acryloyl and
methacryloyl ornithine and lysine without the need to use
protecting groups and chromatographic purifications. Three
methods of removing the copper ions from the amino acid
derivatives were examined. The obtained acryloyl and
methacryloyl ornithine and lysine were copolymerized with
N-isopropylacrylamide and N,N0-methylenebisacrylamide as
crosslinking agents, resulting in a series of hydrogels with
varying incorporated amino acid content. The relative content
of a given amino acid was estimated from the 1H NMR data
and compared with its molar fraction used in the polymeriza-
tion process. We investigated the influence of the amount of
amino acid groups incorporated into the polymer network on
the swelling behavior of the gels in the presence of metal ions
of different ability to form complexes (Cu2þ, Co2þ, and Ca2þ
)
with a-amino acid groups and the sorption of copper ions.
Next, the presence of a-amino acid groups attached to the
polymer network was used to bond the compounds which can
cocomplex metal ions. Phenylalanine was selected for exami-
nation of its cocomplexation of Cu2þ with the polymer-network
amino acids and its consecutive release from the gel after
C
V
appropriate change of pH.
2011 Wiley Periodicals, Inc.
J Polym Sci Part A: Polym Chem 50: 542–550, 2012
KEYWORDS: adsorption; amino acids; drug delivery systems;
hydrogels; metal complexes; polyamides; release; sorption;
synthesis
INTRODUCTION Amino acids are biologically important com-
pounds and play an invaluable role as chiral auxiliaries and
building blocks in organic synthesis. Scientists working in
various areas have focused their research on stimuli-respon-
sive amino acid-based polymer networks. Stimuli-responsive
polymers have moreover gained much attention because of
their wide range of potential applications, including in drug
delivery systems, artificial muscles, separation techniques,
enzyme immobilization matrixes, sensor construction, swing
absorbers, and molecular recognition.1–18 The presence of
amino acid moieties may give gels new properties such as
catalytic activity, sorption properties, sensitivity to pH, ionic
strength, and the presence of specific ions. The temperature
of the volume phase transition of thermosensitive gels based
on N-isopropylacrylamide (NIPA) and the corresponding vol-
ume change may increase or decrease, and the volume phase
transition may switch from discontinuous to continuous.19–25
paring functional polymers is that that amino acids are bio-
logically active and should behave similarly in the polymeric
network. However, the amino groups of those a-amino acids
are usually bound due to the way the polymeric chains are
built and, therefore, lose their typical properties.
We have recently reported the synthesis of new polymeric
gels with free a-amino acid groups based on modified L-orni-
thine and L-lysine and optionally NIPA. The presence of a-
amino acid in those polymers makes it possible to control
the charge sign and the excess of charge in the polymeric
network by changing pH. The synthesized gels showed an
interesting swelling behavior in response to changes in tem-
perature, pH, and concentration of divalent metal ions.24,25
Moreover, the gels based on poly(N-d-acryloyl ornithine)
lightly crosslinked with N,N0-methylenebisacrylamide (BIS)
are characterized by relatively high sorption capacity, high
uptake capacity in a wide range of pH, and short time of
reaching equilibrium toward copper ions.19
Most of the polymers designed for biomedical and industrial
use contain amino acid residues in the main chain and are
expected to exhibit biocompatibility and biodegradability
similar to polypeptides. The groups we used for modification
of the polymeric chains were appropriately modified a-amino
acids.21–23,26–30 The advantage of using amino acids in pre-
In this study, we examined a few methods for the prepara-
tion of acryloyl and methacryloyl lysine and ornithine. To
avoid using the protecting group methodology, the copper
complex method was chosen to prepare side-chain deriva-
tives of ornithine and lysine. In this method, a stable
C
V
2011 Wiley Periodicals, Inc.
542
JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY 2012, 50, 542–550

JOURNAL OF
POLYMER SCIENCE
ARTICLE
WWW.POLYMERCHEMISTRY.ORG
copper–amino acid complex is formed. In the next step, the
appropriate acid chloride reacts with the side chain of the
functional group, because the a-amino group and the carbox-
ylic group are bound to the copper ion. Finally, the copper
ion is removed and the desired functionalized amino acid is
obtained. There are several methods for removing copper
from the amino acid–copper complexes, but some of them
fail when an additional functional group is present. In our
investigation, we prepared copper complexes of acryloyl and
methacryloyl L-lysine and L-ornithine and compared the
usability of three methods [ethylenediaminetetraacetic acid
(EDTA), NaBH4, and thioacetamide] for the removal of cop-
per from the amino acids complexes.
SCHEME 1 Synthesis of copper complexes of amino acids.
Next, for producing free monomers, we examined the meth-
ods of removing copper from the synthesized complexes.
There are several procedures based on treatment with EDTA,
thioacetamide, hydrochloric and hydrobromic acids, sodium
borohydride, potassium cyanide, ion exchange resin, and 8-
quinolinol.25,31–38 We selected just three of them for further
examination: EDTA, sodium borohydride, and thioacetamide.
All reactions were carried out in water (see Scheme 2).
We used the L-lysine and L-ornithine derivatives as mono-
mers in the free-radical copolymerization with NIPA and BIS
to prepare a series of hydrogels, obtaining gels with varying
amounts of free a-amino acid groups. We examined the influ-
ence of the amount of amino acid groups incorporated into
the polymer network on the swelling behavior of the gels
The EDTA method is most widely used but seemed inappro-
priate in our case because both copper–EDTA complexes
formed and liberated amino acid derivatives are water solu-
ble, which causes difficulties in the separation and purifica-
tion of the desired products. The separation of the copper–
EDTA complex from the obtained amino acid derivative can
be performed using chromatography.
immersed into pure water and aqueous solutions of Cu2þ
Co2þ, and Ca2þ and the sorption properties toward Cu2þ
,
.
The presence of free amino acid groups attached to the poly-
mer network opens up the possibility of further modification
of the gel using the compounds that contain an amino acid
group capable of cocomplexing the metal ions with the poly-
mer network of the gel and creation of the complexes poly-
mer network–metal ion–compound. The compounds used to
modify the gel could then be released from the gel by alter-
ing, for instance, the pH of the environment. The amount
and mechanism of phenylalanine release as a function of pH
were investigated.
Sodium borohydride, in turn, was successfully applied to the
preparation of mono-protected derivatives of basic amino
acids using copper complexes methodology. The copper ion
in the amino acid derivative complex is reduced to insoluble
copper oxide, so it is easy to separate it. Unfortunately, in
our cases, the acrylic and methacrylic function is reduced by
sodium borohydride in the presence of copper ions.
The last of the selected methods, based on thioacetamide,
proved to be useful. The method is cheap and fast (the reac-
tion usually taking up to 30 min). As a result of this reaction,
copper sulfide precipitates and can be removed by filtration.
The filtrate contains exclusively liberated an amino acid
derivatives 7–10 that can be finally purified by simple
crystallization.
RESULTS AND DISCUSSION
Synthesis of Amino Acid-Based Monomers
We began our study by seeking an appropriate method of
synthesis for both acryloyl and methacryloyl derivatives of
two amino acids lysine (1) and ornithine (2). We decided to
use a method that allowed for the avoidance of the protect-
ing group methodology, chromatographic purification, while
being easy to use in multigram scale.
Swelling Behavior
First, we examined the reaction of hydrochlorides of orni-
thine (1) and lysine (2) with copper sulfate in alkaline solu-
tions, followed by treatment of the reaction mixtures with
acryloyl or methacryloyl chlorides, which leads to the forma-
tion of copper complexes of ornithine (3 and 4) and lysine
(5 and 6) (Scheme 1). The complexes of copper with lysine
and ornithine are water soluble and their a-amino functions
are blocked what allowed selective side-chain functionaliza-
tion, here acylation of d- and e-amino groups of ornithine (1)
and lysine (2), respectively.
The synthesized compounds were then used as comonomers
in the free-radical polymerization reaction with NIPA in the
presence of BIS as the linker to prepare a series of hydrogels
with varying molar compositions of amino acid moieties.
We next characterized the volume change of gels with vary-
ing amounts of incorporated a-amino acid groups in the
presence of a series of metal ions. For this investigation,
NIPA-AcOrn gels of various composition (y ¼ 0, 2, 5, 10, and
20%) were selected. The gels obtained in 7.7-mm-diameter
glass pipes were cut into uniform pieces of ꢀ 4 mm in diam-
eter. Then, the gel samples were immersed into pure water
and 50 mL of Cu2þ, Co2þ, and Ca2þ 5 mM solutions. After a
few days, the gel samples were separated and changes in the
volumes were analyzed (Fig. 1). The volume of gels
immersed into pure water increased as the amino acid group
We found that the modified copper complexes 3–6 are insol-
uble in most common organic solvents as well as in water.
This facilitated the isolation and purification of the deriva-
tives. The precipitated products were filtered and washed
several times. The yield was in the range of 62–84%.
WWW.MATERIALSVIEWS.COM
JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY 2012, 50, 542–550
543

JOURNAL OF
POLYMER SCIENCE
ARTICLE
WWW.POLYMERCHEMISTRY.ORG
SCHEME 2 Procedures for removing copper from amino acid–copper complexes using: (a) EDTA, (b) sodium borohydride, and (c)
thioacetamide.
content increased. This is a simple consequence of introduc-
ing ionizable groups into the polymeric network, increasing
the hydrophilicity of the gels. Upon exposure to solution of
Cu2þ and Co2þ ions, the gels exhibited a reduction in volume
and became colored. Those changes became more visible af-
ter the amount of amino acid groups in the network was
increased. In the case of gel series immersed in Ca2þ solu-
tion, the changes in volume are very similar to those
observed for gel series immersed in pure water. The
observed behavior of the gels could be explained in terms of
the formation of complexes between metal ions and amino
acid groups attached to the polymeric chains of the gel net-
works. It is known that amino acids can form stable com-
plexes with some metal cations. Three complexes of stoichi-
ometry 1:1, 1:2, and 1:3 can be formed:39–41
with unmodified glycine are as follows: log bML ¼ 8.1 and log
bML ¼ 15.3. However, as Figure 1 shows, the strongest influ-
2
ence on the swelling ratio is observed for cobalt ions. In spite
of smaller stability constants (log bML ¼ 5.0 and log bML
¼
2
8.0), cobalt ions show a distinct tendency for the creation of
complexes with stoichiometry 1:3 (log bML ¼ 11.5). It should
3
be noted that copper and cobalt have relatively high overall
stability constants, and, moreover, the complexes with stoichi-
ometry 1:3 are more efficiently crosslinked than complexes
with stoichiometry 1:2 and should lead to a more shrunken
state of the gel. The weak influence of calcium ions on swel-
ling ratio, on the other hand, could be explained in terms of
the formation of very weak complexes only with stoichiome-
try 1:1 between the calcium and a-amino acid groups (log
MemðaqÞ þ RCH2NH3þCOOꢁ ! MeðRCH2NH2COOÞðmꢁ1Þ þ Hþ
(I)
MeðRCH2NH2COOÞðmꢁ1Þ þ RCH2NHþCOOꢁ
3
(II)
! MeðRCH2NH2COOÞðmꢁ2Þ þ Hþ
2
MeðRCH2NH2COOÞ2ðmꢁ2Þ þ RCH2NHþ3 COOꢁ
(III)
! MeðRCH2NH2COOÞðmꢁ3Þ þ Hþ
3
The presence of complexes in the gel may influence its swel-
ling ratio. The first complex (1:1) should expand the polymer
network by introducing an excessive positive charge to the
polymeric chains, which leads to an increase in the osmotic
pressure between the solution and the gel. The second (1:2)
and third (1:3) complexes increase the overall crosslink den-
sity of the gels and lead to the shrinking of the polymer net-
work. Among the metal ions investigated, copper showed the
highest stability constants. The average values for complexes
FIGURE 1 Response of NIPA-AcOrn gels with differing
amounts of incorporated a-amino acid groups to aqueous solu-
tion of metal ions.
544
JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY 2012, 50, 542–550

JOURNAL OF
POLYMER SCIENCE
ARTICLE
WWW.POLYMERCHEMISTRY.ORG
adsorbed and dissolved molecules). The expression for the
Langmuir isotherm is as follows:
KLCe
q ¼ qm
;
(1)
1 þ KLCe
where q is the amount of Cu2þ adsorbed per gram of adsorb-
ent (mg/g); Ce denotes the equilibrium concentration of the
metal ions in the solution (mg/L); qm is the theoretical satura-
tion capacity of the monolayer (mg/g); and KL represents the
Langmuir constant (L/mg) that is related to the affinity of the
binding sites. The values of qm and KL could be calculated from
the intercept and slope of the linear plot of Ce/q versus Ce.
The analyzed data fit the Langmuir isotherm model very
well, as the R2 value for all of Ce/q versus Ce plots was bet-
ter than 0.995. The theoretical saturation capacities (qm)
determined from this model for 5, 10, and 20% NIPA-AcOrn
gels are 6.65, 12.15, and 19.34 mg/g, respectively.
FIGURE 2 Sorption isotherm of Cu2þ by gels with differing
amounts of incorporated AcOrn. Experimental conditions: C0
5–140 mg/L; pH ¼ 5; sorbent dose: 50 mg/10 mL.
¼
Assuming that the relative content of the amino acid incorpo-
rated into the polymeric network of the gels is equal to its mole
ratio in the pregelation solution, and that all a-amino acid
groups are involved in 1:2 complexes with the copper ions, the
calculated maximum sorption capacities for 5, 10, and 20%
NIPA-AcOrn gels are 13.56, 26.30, and 49.61 mg/g, respectively.
bML ¼ 1.4). All average values of stability constants between
metal ions and glycine were taken from ref. 40.
Sorption Process
The obtained values of saturation capacity from the Lang-
muir isotherm model constitute 49.0, 46.2, and 39.0% of the
calculated maximum sorption capacity. Additionally, as has
been reported,20 the gel based on N-d-acryloyl ornithine and
the crosslinking agent BIS could achieve only 11% of theo-
retical maximum sorption capacity. This could be explained
in terms of steric reasons that make many amino acid
groups inaccessible to the metal ions. In other words, the
formation of complexes with copper ions increases the cross-
link density and makes the polymeric gels collapse. The in-
ternal structure of the gel becomes denser, which makes the
penetration of the gel by subsequent ions harder, and limits
Sorption capacity is an important factor because it determines
the amount of sorbent, which is required for the sufficient
enrichment of the analyte (present at trace level) from the
examined solution. NIPA-AcOrn gels with various composition
(y ¼ 0, 5, 10, and 20%) were investigated. Figure 2 shows
the dependence between the metal-ion equilibrium concentra-
tion and the amount of the adsorbed Cu2þ onto the employed
gels. The sorption capacity of the NIPA gel increases slightly
up to an initial concentration of Cu2þ of ꢀ 50 mg/L and then
increases rapidly to the end of the investigated concentration
range, for example, 140 mg/L. The shape of this dependence
curve does not fit the typical adsorption isotherms used to
describe sorption properties polymeric gels, such as the Lang-
muir, Freundlich, Temkin, Reundlich-Peterson, and Sips iso-
therms.42–46 This suggests that the interactions between cop-
per ions and the polymeric network are very weak. For the
gels that contain a-amino acid groups, sorption capacity
increases rapidly with concentration in the low range of cop-
per ion concentration and then, for higher concentrations, the
increase is not that strong.
To better characterize the sorption process on amino acid
groups of the gels, sorption capacities per one mole of NIPA
unit in polymer network were estimated for appropriate Ce,
and then the isotherms for 5, 10, and 20% NIPA-AcOrn were
appropriately corrected. The obtained isotherms for sorption
on amino acid groups, presented in Figure 3, are of similar
shape. The sorption capacities of the polymeric gel used
increase gradually with Cu2þ concentration and reach a pla-
teau, which represents the maximum sorption capacity. Iso-
therms were analyzed in terms of the Langmuir model (the
polymer chains can be covered by only a monolayer of ad-
sorbate, and there is no subsequent interaction between the
FIGURE 3 Sorption isotherm of Cu2þ on amino acid units in gels
with various amount of incorporated AcOrn. Experimental condi-
tions: C0 ¼ 5–140 mg/L; pH ¼ 5; sorbent dose: 50 mg/10 mL.
WWW.MATERIALSVIEWS.COM
JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY 2012, 50, 542–550
545

JOURNAL OF
POLYMER SCIENCE
ARTICLE
WWW.POLYMERCHEMISTRY.ORG
the gel could then be released from the gel by altering the pH
of the environment. The influence of pH on phenylalanine
release from 20% NIPA-AcLys is shown in Figure 4. The
amount of the released amino acid from a moderately acidi-
fied medium is approximately double that for pH range 5.5–
10.4. Additionally, the complete release of phenylalanine is
achieved after 100 min at pH 2.1 and after 500 min for pH in
the range of 5.5–10.4. Photographs of the gel sample taken af-
ter the release processes were completed (Fig. 4), indicating
that the mechanism of release is different in the two cases. At
pH 2.1, the gel samples lose their blue color and become
more swelled; in a more basic medium, gel samples shrunk
and the blue color become darker. The observed results could
be explained in terms of the influence of pH on the complexa-
tion reactions between copper ions and amino acid groups
attached to the polymeric network on one side and to phenyl-
alanine on the other side. The amino acid groups are known
to exist in three forms: as a cation [protonated amino group
(þH2NCHRCOOH)], as
a
neutral form,
a
zwitterion
(þH2NCHRCOOꢁ), and as an anion [dissociated carboxylic
group (H2NCHRCOOꢁ)]. In a more acidic medium, the equilib-
rium of the complex formation reaction is shifted to the left
side (reactions I–III). Free metal ions are formed and uncom-
plexed phenylalanine can be released from the gel. Addition-
ally, at pH 2.1, functional groups of the polymeric network of
the gel are protonated and these ionized groups create an os-
motic pressure in the network and, therefore, prompt the
swelling process. Also, the charges on the ionized groups gen-
erate electrostatic repulsive forces between the polymer
chains, which lead to further swelling of the network. In the
case of a more basic medium, pH 5.5–10.4, the formation of
complexes with amino acid groups is preferred. Shrinking of
gel sample suggests that some copper ions again formed 1:2
complexes with the amino acid groups attached to the poly-
meric network of the gel. The presence of the 1:2 complexes
increases the overall crosslink density of the gels and leads to
the shrinking of the polymer network. The formation of 1:2
complexes with the amino acid groups attached directly to the
polymeric network means that the phenylalanine ligand is
freed and released to the solution.
FIGURE 4 Effect of pH on release of phenylalanine from 20%
NIPA-AcOrn gel. T ¼ 20 ꢅC.
the spatial orientation of the amino acid groups suitable for
the formation of complexes of 1:2 stoichiometry. The sorp-
tion capacities obtained by us are higher than those reported
recently for carbon nanotubes47 as well as for various che-
late-functionalized materials such as iron-based magnetic
nanoparticles with polyacrylic acid48 and imprinted polyme-
thacrylic microbeads with 4-(2-pyridylazo) resorcinol.49 They
are comparable with those reported recently for polymeric
hydrogels containing the chelating groups.50–53
The selectivity of the gels was not investigated. However,
based on the investigation of the swelling behavior of the
gels in the presence of metal ions (Cu2þ, Co2þ, and Ca2þ
)
and the overall stability constants of metal complexes with
a-amino acids,40,54 some conclusions on the selectivity
aspects could be made. The alkaline earth metals have very
low overall stability constants (log b ꢂ 1.5), whereas for
other metal cations, important from the environmental pollu-
tion point of view (Pb2þ, Cd2þ, Hg2þ, Cu2þ, Fe3þ, and Mn2þ),
log b are usually higher than 10. More detailed analysis of
the values of the overall stability constants showed that
selectivity of investigated gels decreases in the order: Hg2þ
The mechanism of release was also analyzed in terms of the
Higuchi model55 extended by Peppas and Ritger:56
Mt
¼ ktn;
(2)
M
1
where Mt and M are the masses of drug released at time t
1
and 1, respectively, k is a constant incorporating character-
istics of the macromolecular network system and drug, and
n is the diffusional exponent. Information about the release
mechanism can be gained by fitting the release data (for the
first 60% of fractional release) and comparing the values of
n to the semiempirical values for various geometries.56 For a
cylindrical geometry, values of n up to 0.45 correspond to a
purely Fickian diffusion mechanism. Values of n greater than
0.89 indicate a relaxation controlled-release mechanism, and
n values between 0.45 and 0.89 indicate an anomalous
ꢃ Cu2þ ꢃ Cd2þ ꢄ Pb2þ ꢄ Fe3þ ꢃ Mn2þ ꢃ Mg2þ ꢄ Ca2þ
.
Efficiency of Release of Amino Acids
The presence of free a-amino acid groups attached to the
polymer network also opens up the possibility of further
modification of the gel by introducing compounds that con-
tain an amino acid groups and can cocomplex metal ions
with the polymer network. The compounds used to modify
546
JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY 2012, 50, 542–550

JOURNAL OF
POLYMER SCIENCE
ARTICLE
WWW.POLYMERCHEMISTRY.ORG
Synthesis of Copper Complexes of Acryloyl and
Methacryloyl Derivatives of Ornithine and Lysine
To a solution of NaOH (2.35 g, 58.7 mmol, in 55 mL of
water), amino acid monohydrochloride (29.6 mmol) was
added. Next, a solution of CuSO4ꢇ5H2O (3.75 g, 15 mmol, in
55 mL of water) was added and the resulting deep blue so-
ꢅ
lution was cooled to 10 C. Acryloyl or methacryloyl chloride
(37.5 mmol) and 2 M NaOH (15 mL) were simultaneously
added dropwise, maintaining the pH at 9–10. After the addi-
tion was complete (ca. 30 min), the reaction mixture was
allowed to reach room temperature and was stirred over-
night. The blue precipitate was filtered and washed succes-
sively with water (50 mL ꢆ 2), ethanol (50 mL ꢆ 2), and
diethyl ether (50 mL). The precipitate was air dried for 24 h.
N-d-Acryloyl Ornithine Copper Complex (3)
Yield 62% (blue solid), IR (KBr): 3278, 3077, 2934, 2878,
1655, 1615, 1552, 1482, 1455, 1398, 1338, 1313, 1279,
1252, 1237, 1141, 1092, 993, 961, 808, 784, 675, 582, 430.
FIGURE 5 The relationship ln(Mt/M
) versus ln(t) for 20%
1
NIPA-AcLys hydrogel.
release mechanism. The relationships between ln(Mt/M )
N-d-Methacryloyl Ornithine Copper Complex (4)
1
and ln(t) for two values of pH are shown in Figure 5. The
values for diffusional exponent n value for pH 2.1 and 10.4
are 0.68 and 0.61, respectively. The obtained values of n
indicate an anomalous release mechanism that is in line with
the proposed above release mechanisms.
Yield 76% (blue solid), IR (KBr): 3291, 3257, 3059, 2930,
2878, 1652 1615, 1537, 1483, 1453, 1395, 1325, 1276,
1213, 1141, 1088, 925, 806, 792, 754, 707, 651, 579, 431.
N-e-Acryloyl Lysine Copper Complex (5)
Yield 84% (blue solid), IR (KBr): 3283, 3152, 3065, 296,
2868, 1655, 1619, 1541, 1474, 1462, 1402, 1383, 1319,
1308, 1259, 1250, 1237, 1147, 1099, 985, 954, 921, 806,
779, 729, 667, 589, 538, 440.
EXPERIMENTAL
Instrumentation
NMR spectra were recorded with a Varian Unity Plus 200
MHz spectrometer for D2O solutions using residual H2O as
the internal reference. Mass spectra were recorded with an
ESI/MS Mariner (PerSeptive Biosystem) mass spectrometer.
Reactions were monitored by TLC (silica gel 60 F254, 0.2
mm, Merck), and the visualization was effected with UV. Col-
umn chromatography was performed on silica gel (Merck,
230–400 mesh). HPLC was performed using a LaChrom
Merck instrument equipped with a Metachem (Varian) Pola-
ris C-18A column (5 lm, 250 mm ꢆ 4.6 mm) and C-18 Phe-
nomenex precolumn; Diode Array Detector: (190–400 nm),
integration of the signal at 215 nm; isocratic elution, 1.0
mL/min of water: acetonitrile (90:10). Infrared spectra were
obtained with a Perkin-Elmer 2000 FTIR spectrometer.
N-e-Methacryloyl Lysine Copper Complex (6)
Yield 76% (blue solid), IR (KBr): 3274, 3153, 2945, 2935,
2868, 1652, 1618, 1529, 1473, 1461, 1401, 1379, 1312,
1280, 1259, 1215, 1146, 1096, 1010, 917, 871, 80, 779, 729,
666, 587, 491, 447, 424.
Synthesis of Acryloyl and Methacryloyl Derivatives of
Ornithine and Lysine
To a stirred suspension of well-powdered amino acid–copper
complex (2.3 mmol) in water (25 mL), thioacetamide (2.5
mmol) was added. After stirring for 20 min, the mixture was
adjusted to pH 9 and the copper sulfide precipitate formed
was filtered through a celite pad giving colorless filtrate. The
water was evaporated, and the residue was dissolved in
MeOH/CF3COOH (2% CF3COOH in MeOH) and precipitated
with Et2O. The crude product was recrystallized from
MeOH/Et2O.
Materials
NIPA (97%), BIS (99%), ammonium persulfate (APS, 99.99%),
N,N,N0,N0-tetramethylethylenediamine (TEMED, 99.5%), L-orni-
thine monohydrochloride salt (99%), L-lysine monohydro-
chloride salt (98%), acryloyl chloride (96%), methacryloyl
chloride (96%), and sodium borohydride (98%) were pur-
chased from Aldrich and Fluka. Ethylenediaminetetraacetic
acid disodium salt dihydrate (EDTA, 99%), sodium hydroxide
(NaOH, 99%), copper (II) sulfate pentahydrate (CuSO4.5H2O,
98%), and thioacetamide (98%) were purchased from POCh
(Poland). All chemicals were used as received except for NIPA,
which was recrystallized twice from the benzene/hexane mix-
ture (90:10 v/v). All solutions were prepared using high-pu-
rity water obtained from a Milli-Q Plus/Millipore purification
system (water conductivity: 0.056 lS/cm).
N-d-Acryloyl Ornithine (7)
Yield 82% (white solid), 1H NMR (500 MHz, D2O): d 6.19–
6.33 (m; 2H), 5.79 (dd; J ¼ 10 Hz, J ¼ 1.5 Hz; 1H), 3.79 (t; J
¼ 6.25 Hz; 1H), 3.30–3.40 (m; 2H), 1.86–1.98 (m; 2H), 1.59–
1.75 (m, 2H). 13C NMR (50 MHz, D2O): d177.20, 171.25,
132.60, 129.86, 57.11, 41.40, 30.53, 26.91. IR (KBr): 3302,
3083, 2945, 2872, 2629, 2123, 1656, 1626, 1608, 1583,
1556, 1516, 1461, 1448, 1408, 1382, 1358, 1328, 1243,
1204, 1135, 987, 954, 837, 803, 750, 723, 706, 671, 575,
547, 438, 412. High-resolution mass calcd for C8H14N2O3Na
[M þ Na]þ: 209.0902. Found: 209.0894.
WWW.MATERIALSVIEWS.COM
JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY 2012, 50, 542–550
547

JOURNAL OF
POLYMER SCIENCE
ARTICLE
WWW.POLYMERCHEMISTRY.ORG
N-d-Methacryloyl Ornithine (8)
Yield 83% (white solid), H NMR (200 MHz, D2O): d 5.66 (s;
NIPA-MAcOrn
1
Yield 77% (white solid), IR (KBr): 3439, 3074, 2974 2937,
2877, 2119, 1658, 1554, 1460, 1388, 1367, 1277, 1236,
1172, 1131, 974, 898, 883, 838,646, 537.
1H), 5.42 (s, 1H), 3.74 (t; J ¼ 5.9 Hz; 1H), 3.28 (t; J ¼ 6.7
Hz, 2H), 1.90 (s; 3H), 1.75–1.95 (m; 2H), 1.55–1.70 (m; 2H).
13C NMR (50 MHz, D2O): d174.52, 172.08, 139.20, 121.05,
54.50, 38.95, 27.91, 24.39, 17.76. IR (KBr): 3299, 2947,
2662, 2107, 1656, 1618, 1582, 1537, 1465, 1449, 1416,
1352, 1328, 1280, 1222, 1173, 1153, 920, 825, 743, 721,
700, 663, 562, 535, 466, 431. High-resolution mass calcd for
C9H16N2O3Na [M þ Na]þ: 223.1059. Found: 223.1064.
NIPA-AcLys
Yield 76% (white solid), IR (KBr): 3440, 3074, 2974, 2936,
2877, 2125, 1657, 1548, 1460, 1388, 1368, 1276, 1173,
1131, 976, 927, 883, 838, 654, 532.
NIPA-MAcLys
Yield 72% (white solid), IR (KBr): 3440, 3074, 2974, 2936,
2876, 2111, 1651, 1548, 1460, 1388, 1367, 1276, 1232,
1172, 1131, 971, 927, 883, 839, 655, 537.
N-e-Acryloyl Lysine (9)
Yield 65% (white solid), H NMR (200 MHz, D2O): d 6.10–6.35
1
(m; 2H), 5.72 (dd; J ¼ 9.2 Hz, J ¼ 2.8 Hz, 1H), 3.73 (t; J ¼ 6.1
Hz, 1H), 3.26 (t; J ¼ 6.8 Hz, 2H), 1.75–2.00 (m; 2H), 1.50–1.70
(m; 2H), 1.25–1.50 (m; 2H). 13C NMR (50 MHz, D2O): d174.67,
168.58, 130.11, 127.16, 54.61, 39.04, 30.10, 28.04, 21.86. IR
(KBr): 3302, 3073, 2944, 2867, 1656, 1627, 1605, 1582, 1521,
1443, 1410, 1360, 1325, 1247, 1202, 1134, 982, 836, 800,
722, 670, 536, 444, 419. High-resolution mass calcd for
C9H16N2O3Na [M þ Na]þ: 223.1059. Found: 223.1031.
Determination of Amount of Amino Acid Incorporated
into Polymeric Network
The amount of amino acid incorporated into the polymeric
network of the gels was estimated from the NMR spectra.
The gel samples for the NMR experiments were prepared by
swelling the pieces of dry polymeric network in D2O in NMR
tubes. The 1H NMR spectra were recorded after 1 day. In
1
each case of H NMR spectra of polymeric networks contain-
N-e-Methacryloyl Lysine (10)
ing lysine or ornithine units, the region between 2.5 and 4.5
ppm was used for the calculations. The distinguishable signal
at 3.2 ppm (representing the d-protons or e-protons of orni-
thine and lysine, respectively) and overlapped signals at
3.75–4.1 ppm (representing a-protons from amino acid, pro-
tons from the isopropyl group, or methylene protons from
the linker, respectively) were used for estimating the amino
acid loading content into the network.
1
Yield 62% (white solid), H NMR (200 MHz, D2O): d 5.65 (s;
1H), 5.41 (s; 1H), 3.70 (t; J ¼ 6.0 Hz, 1H), 3.24 (t; J ¼ 6.3
Hz, 2H), 1.89 (s; 3H), 1.75–2.00 (m; 2H), 1.50–1.70 (m; 2H),
1.30–1.50 (m; 2H). 13C NMR (50 MHz, D2O): d 174.82,
171.97, 139.27, 120.90, 54.74, 39.18, 30.19, 28.16, 21.89,
17.78. IR (KBr): 3322, 2947, 2926, 2864, 2606, 2146, 1654,
1619, 1581, 1519, 1446, 1415, 1359, 1351, 1325, 1217,
1204, 1162, 1135, 980, 917, 863, 812, 799, 736, 721, 666,
532, 442, 416. High-resolution mass calcd for C10H18N2O3Na
[M þ Na]þ: 237.1215. Found: 237.1198.
12 < b >
Ygel
¼
100%;
(4)
< a; c; d >
Synthesis of Gels Containing Lysine and Ornithine Units
The amino acid groups, which can interact with heavy met-
als, were introduced into poly(N-isopropylacrylamide) net-
work by simple free-radical solution copolymerization. The
total concentration of NIPA, derivatives of amino acid, and
BIS was kept constant at 700 mM. The pregel solutions
were degassed, and the polymerization was initiated and
accelerated by APS (1.88 mM) and TEMED (32 mM) and
where <b> denotes the area of the signal at 3.2 ppm and
<a, c, d> is the area between 3.75 and 4.10 ppm, respectively.
To make the equation more precise, the area of the methylene
protons from the linker should be divided by 2 to reflect the
presence of two protons in this residue; however, the corre-
sponding proton signals are not sufficiently well separated.
Fortunately, the error is very small, as the mole fraction of the
linker was kept at a low level. The estimated percentages of
incorporated amino acids are in good accordance with the
molar fraction of monomers used in the polymerization pro-
cess. Data for NIPA-MAcOrn gels are shown in Table 1.
ꢅ
carried out at 5 C for 20 h. The gels were synthesized with
the mole fraction of the amino acid derivative (Ac/MAcLys/
Orn) equal to Ysolution in the pregel solution. Ysolution is
defined as:
nAc=MAcLys=Orn
Typical spectra of polymer networks based on methacryloyl
L-ornithine with differing amounts of incorporated ornithine
moieties are shown in Figure 6. With an increase in orni-
thine loading, both the signal at 3.2 ppm and the overlapped
signals at 3.75 ꢈ 4.10 ppm increase.
Ysolution
¼
100%
(3)
nNIPA þ nAc=MAcLys=Orn þ nBIS
In this article, Ysolution ¼ 2, 5, 10, 20, and 0%. The mole frac-
tion of BIS was kept constant at 1%. The gels were synthe-
sized in a glass tube of inner diameter, do, 7.7 mm. After syn-
thesis, the gel samples were immersed into pure water to
remove excess reagents and finally dried.
Examination of Amino Acid Release
The drug release properties were evaluated using the 20%
NIPA-AcLys gel and phenylalanine. The gels synthesized in a
7.7-mm-diameter glass pipe were cut to obtain uniform pieces
ꢀ 4 mm in diameter (mass of the dry polymer was ꢀ 2 mg).
The gel samples were immersed in a 10-mL solution of phe-
nylalanine and copper–ion complexes of stoichiometry 1:1.
NIPA-AcOrn
Yield 91% (white solid), IR (KBr): 3439, 3075, 2974, 2936,
2877, 2115, 1659, 1550, 1460, 1388, 1368, 1278, 1172,
1131, 977, 928, 883, 839, 647, 534.
548
JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY 2012, 50, 542–550

JOURNAL OF
POLYMER SCIENCE
ARTICLE
WWW.POLYMERCHEMISTRY.ORG
TABLE 1 Content of Amino Acid in the Pregel Solutions and of Amino Acid Incorporated into
Polymeric Network of the Gels (Estimated from 1H NMR Spectra)
2% NIPA-MAcOrn
5% NIPA-MAcOrn
10% NIPA-MacOrn
20% NIPA-MAcOrn
Ysolution
Ygel
2.0%
2.6%
5.0%
6.4%
10.0%
11.1%
20.0%
21.0%
The solution of copper complexes with phenylalanine was
prepared by mixing 2 mM solution of copper with 2 mM solu-
tion of phenylalanine. Both solutions were prepared in acetate
buffer of pH 5.0. To decrease the amount of free copper ions
(aqua complexes) in the final solution, a 10% excess of phe-
nylalanine was used in the mixing process. Then, the solution
was separated from the precipitated blue crystals (1:2 com-
plexes) and used to introduce the amino acid to the gel. The
loading process was considered to be completed after 2 h;
then, the entire gel samples turned a uniform dark blue color.
Next, the gel samples were immersed three times into 10 mL
of pure water to remove unbound species and placed in a 3-
mL solution of various pH (from 2.1 to 10.4) where the
release process was investigated.
Examination of Copper Sorption
For the examination of sorption properties, the gels with 0, 5,
10, and 20% content of AcOrn were selected. The synthesized
ꢅ
gels were dried gently, at ꢀ 60 C, to constant mass and then
ground. To estimate the sorption capacity, 50 mg of dry poly-
mer was mixed with 10 mL of metal ion solution (pH 5.0,
concentration range 5–140 mg/L). The mixtures were shaken
for 2 h; then, the metal concentration in the aqueous solutions
was determined by atomic absorption spectrophotometry. The
sorption capacity, qe (mg/g), was estimated as follows: qe ¼
[(Co ꢁ Ce)V]/m, where Co and Ce are the initial and equilib-
rium concentrations (mg/L) of metal ion in the aqueous solu-
tion, respectively, V is the volume of the metal ion solution,
and m is the weight of the sorbent.
The amount of released phenylalanine was determined by
chromatography. The working calibration plot used in the
determination of phenylalanine was as follows: y ¼ 4.05 ꢆ
108c ꢁ 567, R ¼ 0.9999.
The regression equation of copper solution working curve
was y ¼ 0.0224x þ 0.345, R ¼ 0.9982.
CONCLUSIONS
We have shown that hydrogels containing free a-amino acid
groups can be easily prepared by free-radical polymerization
of acryloyl and methacryloyl derivative of ornithine and ly-
sine. Copper-complex formation followed by thioacetamide
reaction were found to give a convenient and scalable proce-
dure for preparation of amino acid derivatives (acryloyl and
methacryloyl amino acids). The volume of the gel depends
on the amount of amino acid moieties incorporated into the
polymer network and on the presence of metal ions. Metal
ions that have the ability to create complexes with a-amino
acid groups lead to the shrinking of the gels. Investigation of
the sorption of Cu2þ by the a-amino acid groups revealed
that the adsorption process proceeds in accordance with the
Langmuir isotherm. The shrinking of the polymeric network
due to sorption of some metal ions leads to better mechani-
cal properties of the gel, so it could be useful in the removal
of the gel from the solution, for instance, for further regener-
ation. Nevertheless, the obtained results show that the equi-
librium adsorption capacities are relatively high. Additionally,
the presence of free amino acid groups attached to the poly-
mer network offers a possibility of loading the gel with com-
pounds which can cocomplex the metal ions. These com-
pounds could be released from the gel by appropriate
alteration of the environmental pH. For phenylalanine, we
have shown that the mechanism and the amount of released
amino acid strongly depend on pH. The amount of released
amino acid at medium pH ¼ 2.1 is approximately double
that for pH in the range of 5.5–10.4. Additionally, at pH 2.1,
the gel samples lose their dark blue color and become more
swelled, whereas in a more basic medium the samples
shrink and the blue color becomes darker. Because some
FIGURE 6 1H NMR spectra of methacryloyl L-ornithine-based
hydrogels.
WWW.MATERIALSVIEWS.COM
JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY 2012, 50, 542–550
549

JOURNAL OF
POLYMER SCIENCE
ARTICLE
WWW.POLYMERCHEMISTRY.ORG
26 Hiratani, H.; Alvarez-Lorenzo, C.; Chuang, J.; Guney, O.;
Grosberg, A. Y.; Tanaka, T. Langmuir 2001, 17, 4431–4436.
molecules important from the medical, pharmaceutical, or
therapeutic points of view have free amino acid groups (e.g.,
glutathione), such gels with free amino acid groups are also
interesting from the standpoint of drug delivery systems.
27 Yu, Y. Q.; Tian, H. J.; Chang, X. Z. Asian J. Chem. 2008, 20,
4803–4812.
28 Lokitz, B. S.; Convertine, A. J.; Ezell, R. G.; Heidenreich, A.;
Li, Y.; McCormick, C. L. Macromolecules 2006, 39, 8594–8602.
This work was supported by Grant IP2010016870 from the
Ministry of Science and Higher Education and by Warsaw
University Grant BW-191208.
29 He, C.; Zhao, C.; Guo, X.; Guo, Z.; Chen, X.; Zhuang, X.; Liu,
S.; Jing, X. J. Polym. Sci. Part A: Polym. Chem. 2008, 46,
4140–4150.
30 Patra, T.; Pal, A.; Dey, J. Langmuir 2010, 26, 7761–7767.
REFERENCES AND NOTES
31 Sailaja, G.; Nowshuddin, S.; Rao, M. N. A. Tetrahedron Lett.
2004, 45, 9297–9298.
1 Zhang, J. T.; Huang, S. W.; Liu, J.; Zhuo, R. X. Macromol.
Biosci. 2005, 5, 192–196.
32 Roeske, R.; Stewart, F. H. C.; Stedman, R. J.; duVigneaud, V.
J. Am. Chem. Soc. 1956, 78, 5883–5887.
2 Mahkam, M. J. Biomed. Mater. Res. B 2005, 75, 108–112.
33 Taylor, U. F.; Dyckes, D. F.; Cox, J. R. Int. J. Pept. Protein
Res. 1982, 19, 158–161.
3 Hyk, W.; Karbarz, M.; Stojek, Z.; Ciszkowska, M. J. Phys.
Chem. B 2004, 108, 864–868.
34 Scott, J. W.; Parker, D.; Parrish, D. R. Synth. Commun. 1981,
11, 303–314.
4 Wang, L.; Liu, M.; Gao, C.; Ma, L.; Cui, D. React. Funct.
Polym. 2010, 70, 159–167.
35 Wiejak, S.; Masiukiewicz, E.; Rzeszotarska, B. Chem. Pharm.
Bull. 2001, 49, 1189–1191.
5 Hussain, T.; Ranjha, N. M.; Shahzad, Y. Des. Monomers
Polym. 2011, 14, 233–249.
36 Wunsch, E.; Fries, G.; Zwick, A. Chem. Ber. 1958, 91,
542–547.
6 Yeghiazarian, L.; Arora, H.; Nistor, V.; Montemagno, C.; Wies-
ner, U. Soft Matter 2007, 3, 939–944.
37 Zahn, H.; Zuber, H.; Ditscher, W.; Wegerle, D.; Meienhofer,
J. Chem. Ber. 1956, 89, 407–414.
7 Konishi, Y.; Asai, S.; Midoh, Y.; Oku, M. Sep. Sci. Technol.
1993, 28, 1691–1702.
38 Bunegar, M. J.; Dyer, U. C.; Green, A. P.; Gott, G. G.; Jaggs,
C. M.; Lock, C. J.; Mead, B. J. V.; Spearing, W. R.; Tiffin, P. D.;
Tremayne, N.; Woods, M. Org. Process Res. Dev. 1998, 2,
334–336.
8 Ogawa, K.; Kokufuta, E. Langmuir 2002, 18, 5661–5667.
9 Tumturk, H.; Karaca, N.; Demirel, G.; Sahin, F. Int. J. Biol.
Macromol. 2007, 40, 281–285.
10 Karbarz, M.; Gniadek, M.; Stojek, Z. Electroanalysis 2009, 21,
1363–1368.
39 Petit, L. D. Pure Appl. Chem. 1984, 56, 247–292.
40 Kiss, T.; Sovago, I.; Gergely, A. Pure Appl. Chem. 1991, 63,
597–638.
11 Klis, M.; Karbarz, M.; Stojek, Z.; Rogalski, J.; Bilewicz, R.
J. Phys. Chem. 2009, 113, 6062–6067.
41 Berthon, G. Pure Appl. Chem. 1995, 67, 1117–1240.
42 Langmuir, I. J. Am. Chem. Soc. 1916, 38, 2221–2295.
43 Freundlich, H. M. F. J. Phys. Chem. 1906, 57, 385–471.
12 Liang, Y.; Song, S.; Yao, H.; Hu, N. Electrochim. Acta 2011,
56, 5166–5173.
13 Zhang, Y.; Ji, H. F.; Brown, G. M.; Thundat, T. Anal. Chem.
2003, 75, 4773–4777.
44 Tempkin, M. I.; Pyzhev, V. Acta Phys. Chem. USSR 1940, 12,
327–356.
14 Bashir, R.; Hilt, J. Z.; Elibol, O.; Gupta, A.; Peppas, N. A.
Appl. Phys. Lett. 2002, 81, 3091–3093.
45 Redlich, O.; Peterson, D. L. J. Phys. Chem. 1959, 63,
1024–1026.
15 Tokuyama, H.; Yanagawa, K.; Sakohara, S. Sep. Purif. Tech-
nol. 2006, 50, 8–14.
46 Sips, R. J. Chem. Phys. 1948, 16, 490–495.
16 Tokuyama, H.; Iwama, T. Langmuir 2007, 23, 13104–13108.
47 Stafiej, A.; Pyrzynska, K. Sep. Purif. Technol. 2007, 58,
49–52.
17 Yamashita, K.; Nishimura, T.; Ohashi, K.; Ohkouchi, H.;
Nango, M. Polym. J. 2003, 35, 545–550.
48 Lee, P. L.; Sun, Y. C.; Ling, Y. C. J. Anal. At. Spectrom. 2009,
24, 320–327.
18 Ju, X. J.; Chu, L. Y.; Liu, L.; Mi, P.; Lee, Y. M. J. Phys.
Chem. B 2008, 112, 1112–1118.
49 Dakova, I.; Karadjova, I.; Ivanov, L.; Georgeva, V.; Evtimova,
B.; Georgev, G. Anal. Chim. Acta 2007, 584, 196–203.
19 Kristesen, T. E.; Hansen, T. Eur. J. Org. Chem. 2010, 17,
3179–3204.
50 Caykara, T.; Inam, R. J. Appl. Polym. Sci. 2003, 89,
2013–2018.
20 Karbarz, M.; Pyrzynska, K.; Romanski, J.; Jurczak, J.; Stojek,
Z. Polymer 2010, 51, 2959–2964.
51 Cavus, S.; Gurdag, G. Ind. Eng. Chem. Res. 2009, 48,
2652–2658.
21 Bignotti, F.; Penco, M.; Sartore, L.; Peroni, I.; Mendichi, R.;
Casolaro, M.; D’Amore, A. Polymer 2000, 41, 8247–8256.
52 Chen, C. Y.; Chiang, C. L.; Chen, C. R. Sep. Purif. Technol.
2007, 54, 396–403.
22 Casolaro, M.; Paccagnini, E.; Mendichi, R.; Ito, Y. Macromo-
lecules 2005, 38, 2460–2468.
53 El-Hamshary, H.; El-Garawany, M.; Assubaie, F. N.; Al-Eed,
M. J. Appl. Polym. Sci. 2003, 89, 2522–2526.
23 Casolaro, M.; Bottari, S.; Ito, Y. Biomacromolecules 2006, 7,
1439–1448.
54 Sovago, I.; Kiss, T.; Gergely A. Pure Appl. Chem. 1993, 65,
1029–1080.
24 Karbarz, M.; Pulka, K.; Misicka, A.; Stojek, Z. Langmuir 2006,
22, 7843–7847.
55 Higuchi, T. J. Pharm. Sci. 1963, 52, 1145–1149.
25 Karbarz, M.; Romanski, J.; Michniewicz, K.; Jurczak, J.; Sto-
jek, Z. Soft Matter 2010, 6, 1336–1342.
56 Peppas, N.; Ritger, P. L. J. Controlled Release 1987, 5,
37–42.
550
JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY 2012, 50, 542–550