Synthesis, characterization and in vitro antimicrobial and biodegradability study of pseudo-poly(amino acid)s derived from N,N′-(pyromellitoyl)-bis- l-tyrosine dimethyl ester as a chiral bioactive diphenolic monomer

Article abstract of DOI:10.1007/s00726-010-0686-0

In this investigation N,N′-(pyromellitoyl)-bis-l-tyrosine dimethyl ester (7) as a chiral bioactive diphenolic monomer was prepared in three steps. The aim of this work was to obtain novel optically and biologically active pseudo-poly(amino acid)s (PAA)s that are more soluble in common organic solvents while maintaining their high thermal stability. Thus, several new, highly soluble, thermally stable, optically active and biodegradable PAAs containing different amino acid moieties in the main chain were prepared with moderate molecular weights via direct polycondensation using tosyl chloride, pyridine and N,N′-dimethylformamide as a condensing agent. The resulting novel polymers were characterized with FT-IR, ¹H-NMR, elemental and thermogravimetric analysis techniques. In addition, in vitro toxicity and biodegradability behavior of the diphenolic monomer 7, different synthetic diacids (3a-3e) and obtained PAAs, which were investigated in culture media, showed that the synthesized compounds and polymers derived from them are biologically active and biodegradable under a natural environment.

Full text of DOI:10.1007/s00726-010-0686-0

Amino Acids (2011) 40:611–621
DOI 10.1007/s00726-010-0686-0
ORIGINAL ARTICLE
Synthesis, characterization and in vitro antimicrobial
and biodegradability study of pseudo-poly(amino acid)s derived
from N,N0-(pyromellitoyl)-bis-L-tyrosine dimethyl ester as a chiral
bioactive diphenolic monomer
•
Shadpour Mallakpour Farhang Tirgir
•
Mohammad R. Sabzalian
Received: 26 February 2010 / Accepted: 30 June 2010 / Published online: 15 July 2010
Ó Springer-Verlag 2010
Abstract In this investigation N,N0-(pyromellitoyl)-bis-L-
tyrosine dimethyl ester (7) as a chiral bioactive diphenolic
monomer was prepared in three steps. The aim of this work
was to obtain novel optically and biologically active
pseudo-poly(amino acid)s (PAA)s that are more soluble in
common organic solvents while maintaining their high
thermal stability. Thus, several new, highly soluble, ther-
mally stable, optically active and biodegradable PAAs
containing different amino acid moieties in the main chain
were prepared with moderate molecular weights via direct
polycondensation using tosyl chloride, pyridine and N,N0-
dimethylformamide as a condensing agent. The resulting
Introduction
Bisphenols (BPs) are a group of chemical materials, which
consist of two phenolic rings joined together through a
bridging carbon or other chemical structures (Chen et al.
2002). BPs such as bisphenol-A (BPA, 2,2-bis(4-hydrox-
ydiphenyl)propane) are frequently used in manufacturing
high-performance materials, since their aromatic backbone
structures can significantly increase the stiffness and
mechanical strength of obtained polymers. It is used pri-
marily as a raw material for the production of polycar-
bonate plastics, epoxy resins and lacquer coatings (Sajiki
and Yonekubo 2004; Pressman et al. 2005). It is also
contained in thermographic and pressure-sensitive papers
and is used as the material for dental composites and
sealants (Joskow et al. 2006). The annual production of
BPA in the world exceeded 910 million pounds in the
beginning of the 1990s, and the amount has increased to
nearly 1 billion pounds in 2007 (Saal et al. 2007). How-
ever, due to its increased application, its demand has
greatly increased year by year. Therefore, the discharge of
a variety of BPs into the environment is estimated to
increase, and it seems that various BPs as well as BPA will
become widespread environmental pollutants in the near
future. As mentioned above, several researchers have
focused on the toxicity of BPA, especially its estrogenic
activity, and a number of data are available at present
(Vandenberg et al. 2007; Nakagawa and Tayama 2000;
Tsai 2006). Chen et al. (2002) paid much attention to the
toxicity of BPs. In their study, acute toxicity, mutagenicity
and estrogenicity of BPs were investigated. However, BPA
and other industrially used diphenols are cytotoxic and may
not be suitable candidate as monomers in medical implant
and biodegradable materials. So, there is a significant need
for a non-cytotoxic diphenolic monomer that could be used
1
novel polymers were characterized with FT-IR, H-NMR,
elemental and thermogravimetric analysis techniques. In
addition, in vitro toxicity and biodegradability behavior of
the diphenolic monomer 7, different synthetic diacids (3a–
3e) and obtained PAAs, which were investigated in culture
media, showed that the synthesized compounds and poly-
mers derived from them are biologically active and bio-
degradable under a natural environment.
Keywords Pseudo-poly(amino acid)s ꢀ Nontoxic
aromatic diol ꢀ L-tyrosine ꢀ Biodegradable
S. Mallakpour (&) ꢀ F. Tirgir
Department of Chemistry, Organic Polymer Chemistry Research
Laboratory, Isfahan University of Technology,
Isfahan 84156-83111, Islamic Republic of Iran
e-mail: mallak@cc.iut.ac.ir; mallak777@yahoo.com;
mallakpour84@alumni.ufl.edu
M. R. Sabzalian
Department of Agronomy and Plant Breeding,
College of Agriculture, Isfahan University of Technology,
Isfahan 84156-83111, Islamic Republic of Iran
123

612
S. Mallakpour et al.
as a building block in the design of biodegradable mate-
rials. This need was addressed by the development of some
tyrosine-based monomers (Pulapura and Kohan 1992;
Parth et al. 2009; Tangpasuthadol et al. 1997; Acunzo and
Kohn 2002; Aamer et al. 2009; Jack et al. 1996). L-Tyro-
sine is the only major natural nutrient amino acid con-
taining an aromatic hydroxyl group. L-Tyrosine is itself
used as a common dietary supplement, primarily because
of anecdotal reports of its ability to stimulate brain activity
for improved memory and mental alertness, to act as an
appetite suppressant, to control depression and anxiety and
to enhance physical performance. It has promise as a
valuable precursor compound for various industrial and
pharmaceutical applications (Eversloh et al. 2007; Shah
et al. 2009).
backbone linkages with nonamide bonds, such as imino-
carbonate, ester and carbonate bonds. This approach rep-
resents a logical extension of ‘‘pseudo’’-PAAs chemistry,
in which amino acids have been used as monomeric
building blocks in polymers that do not have the conven-
tional backbone structure found in peptides (Spatola 1983).
L-tyrosine-based ‘pseudo’-PAAs were introduced as a
novel class of polymeric biomaterials by Kohn and Langer
(1984, 1986). In view of the non-processibility of con-
ventional poly (L-tyrosine), which cannot be used as an
engineering plastic, recently, the synthesis of polymers was
reported using derivatives of tyrosine. These polymers are
degradable under physiological conditions and have sig-
nificantly improved engineering properties as compared to
most conventional PAAs (Tangpasuthadol et al. 2000).
Biodegradable ‘pseudo’-PAAs derived from tyrosine may
be used as an alternative to conventional non-degradable
polymers, such as polyethylene and polypropylene in the
fabrication of packaging films in the near future, and may
be a solution to the environmental problem (Hooper et al.
1998).
Poly(amino acid)s (PAA)s have been used as a simple
synthetic model for proteins in investigations relating to
antigenicity, enzyme specificity, DNA protein interactions,
protein folding and tertiary structure. In addition, PAAs
have been explored as high-performance textile fibers and
materials with nonlinear optical properties, as well as for
other specialty applications. During the 1980s, PAAs were
extensively explored as potential implant materials for
clinical applications including sutures and drug delivery
devices (Brocchini et al. 1997). When degradable polymers
are used as implant materials in patients, the potential
toxicity of the polymer degradation products and their
subsequent metabolites becomes a major anxiety. For this
reason, PAAs were particularly attractive candidates for
biomedical applications (Uhrich et al. 1999). Synthetic
PAAs that contain one or at most two different amino acid
residues were virtually non-immunogenic and were found
to degrade in vitro and in vivo to their respective amino
acid building blocks, which are nontoxic and natural
metabolites (Anderson et al. 1985). Furthermore, due to
their structural variability, a large number of PAAs were
available. Based on these considerations, it is not aston-
ishing that PAAs were intensively investigated as potential
implant material and biodegradable commercial polymers
(Anderson et al. 1974; Sidman et al. 1980). In spite of their
structural variability, however, PAAs tend to share a
number of common physicomechanical properties: most
PAAs are insoluble in common organic solvents and
degrade thermally in the molten state (Bamford et al.
1956). Due to the combination of these two properties,
most PAAs are unprocessable by conventional polymer
fabrication techniques, a disadvantage that has severely
limited their practical applicability. Since the insolubility
of PAAs and their high melting points are a reflection of
the strong interchain hydrogen bonds created by the
recurring amide backbone linkages, it is proposed that it
should be possible to improve the physicomechanical and
thermal properties of some PAAs by replacing the amide
Most general methods for preparation of ester bonds
comprise bulk polycondensation under elevated temperature
and solution step-growth polymerization in high-boiling
aprotic organic solvents (Fu and Liu 2008). Solution poly-
esterification using tosyl chloride (TsCl/dimethylformamide
(DMF)/pyridine (Py) as a condensing agent produces rather
high-molecular weight polymers from aromatic and ali-
phatic dicarboxylic acids and bisphenols (Higashi and Tobe
2001; Mallakpour and Kolahdoozan 2006).
In a previous work (Mallakpour et al. 2010), we reported
the synthesis of a derivative of tyrosine which can be
regarded as biological active diphenolic monomer and may
be employed as replacements for the industrially used
diphenols in the design of biodegradable and biological
materials. This study describes successful synthesis and
characterization of optically active PAAs via step-growth
polymerization reactions of different aliphatic diacids
derived from natural a-amino acids (3a–3e) with non-toxic
diphenolic monomer 7 under step-growth polymerization.
The use of dicarboxylic acids in direct polycondensation,
instead of their significantly more toxic though more
reactive derivatives, such as diacid chlorides, is one of the
main and important prerequisites for the study of such
polymer syntheses. To test this hypothesis, we synthesized
a series of pseudo-PAAs polymers in which natural
a-amino acids were linked together by nonamide bonds
such as ester linkage. In connection with our interest in
preparing optically active thermally modified polymers
(Mallakpour and Seyedjamali 2008; Mallakpour and
Zadehnazari 2010; Mallakpour and Rafiee 2008), herein
we also would like to describe the synthesis and charac-
terization of PAAs with main-chain chirality through the
123

PPAs from N,N0-(pyromellitoyl)-bis-L-tyrosine dimethyl ester as a diphenolic monomer
613
polycondensation reactions of N,N0-(pyromellitoyl)-bis-
dimethyl ester tyrosine (7) as a nontoxic aromatic diol
with N,N0-(pyromellitoyl)-bis-(L-phenylalanine) (3a), N,N0-
(pyromellitoyl)-bis-(L-leucine) (3b), N,N0-(pyromellitoyl)-
bis-(L-methionine) (3c), N,N0-(pyromellitoyl)-bis-(L-valine)
(3d) and N,N0-(pyromellitoyl)-bis-(L-alaine) (3e). In vitro
toxicity of BPA, synthetic aromatic diol 7 and different
aliphatic diacids (3a–3c) as well as obtained novel PAAs
were evaluated with Petri plate containing potato dextrose
agar (PDA).
atmosphere. Elemental analyses were performed by the
Iran Polymer and Petrochemical Research Institute, Teh-
ran, Iran. Melting points were taken with a Gallenham
melting point apparatus. Cultured samples in Petri plate
were imaged with a confocal digital camera (Canon
DS126181, Japan).
Synthesis of monomers
N,N0-(Pyromellitoyl)-bis-(L-a-amino acid)s (3a–3e) were
reported in our previous work (Faghihi et al. 2004a, b;
Mallakpour et al. 2002; Mallakpour and Habibi 2003;
Mallakpour and Shahmohammadi 2005).
Experimental
The brief procedure is as follows. Into a 25-mL, round-
bottomed flask, 4.58 9 10-2 mol of pyromellitic dianhydride
(1), 9.16 9 10-2 mol of L-a-amino acids (L-phenylalanine,
L-leucine, L-methionine, L-valine and L-alanin), 15 mL of
acetic acid and a stirring bar were placed. The mixture was
stirred at room temperature for 3 h and then refluxed for
10 h. The solvent was removed under reduced pressure and
then the mixture was poured into a mixture of 50 mL/5 mL
of cold distillated water/concentrated HCl and finally stirred
for 1 h. A white precipitate was formed, which was filtered
off, washed with water and dried under vacuum at 80°C to
give a compound (3a–3e). Recrystallization from methanol/
water gave white crystals.
Chemicals
All chemicals were purchased from Fluka Chemical Co.
(Buchs, Switzerland), Aldrich Chemical Co. (Milwaukee,
WI) and Riedel–deHaen AG (Seelze, Germany). Pyrom-
ellitic dianhydride (benzene-1,2,4,5-tetracarboxylic dian-
hydride) (1) (from Merck Chemical Co) was purified by
recrystallization from a mixture of acetic anhydride and
acetic acid (1:4). N,N0-dimethylformamide (DMF) was
dried over BaO and then was distilled under reduced
pressure.
Instruments
N,N0-(pyromellitoyl)-bis-L-tyrosine dimethyl ester (7)
was introduced for the first time for the application of a new
membrane-selective electrode based on the potentiometric
method for the determination of phenazopyridine as a drug
(Ensafi et al. 2010). The preparation and characterization of
this non-toxic and biologically active diol 7 have been
completely reported in a previous work (Mallakpour et al.
2010).
Nuclear magnetic resonance (1H-NMR, 500 MHz) spectra
and also carbon nuclear magnetic resonance (13C-NMR,
125 MHz) spectrum were recorded in DMSO-d6 solution
using a Bruker (Germany) Avance 500 instrument, at
Sharif University of Technology, Tehran, Iran. The FT-IR
adsorption spectra were recorded on a Nicole Impact 400D
IR spectrophotometer with KBr pallets. Vibration bands
were reported as wave number (cm-1). The band intensi-
ties were classified as weak (w), medium (m), strong (s) or
broad (br). Inherent viscosities of polymer solution (0.2%
w/v) in DMF were determined at 25°C by a standard
procedure using a Cannon–Fenske Routine Viscometer
(Cannon, Mainz, Germany). Specific rotations were mea-
sured by a Jasco polarimeter (Japan). Solubility of the
polymers was tested in various polar and non-polar sol-
vents. As much as 5 mg of the polymer was added to 1 mL
of different solvents. Thermogravimetric analysis (TGA)
data for the polymers were taken on Perkin-Elmer TGA
(Perkin-Elmer, Jugeshein, Germany) in nitrogen atmo-
sphere at a heating rate of 10°C min-1 at the Research
Institute of Petroleum Industry (Tehran, Islamic Republic
of Iran). Differential scanning calorimetry (DSC) data were
recorded on a DSC-PL-1200 instrument under nitrogen
atmosphere at a heating rate of 20°C min-1 under N2
Polymerization reactions
PAA8a–PAA8e were prepared by the following procedure:
For synthesis of PAA8a, a Py (0.20 mL, 2.4 9 10-3 mol)
solution of TsCl 0.185 g (9.75 9 10-4 mol), after 30 min
stirring at room temperature, was treated with DMF 0.07 g
(0.1 mL, 9.57 9 10-4 mol) for 30 min and the resulting
solution was added dropwise to a solution of diacid 3a,
0.10 g (1.95 9 10-4 mol) in Py (0.40 mL). The mixture
was maintained at room temperature for 30 min and then to
this mixture, 0.11 g (1.92 9 10-4 mol) diphenolic mono-
mer 7 was added as yellow powder and the whole solution
was stirred at room temperature for 30 min and then at
120°C for 4 h, respectively. As the reaction proceeded, the
solution became viscous. Then the viscous liquid was
precipitated in 15 mL of methanol to give 0.2 g (95%) of
PAA8a. PAA8b–PAA8e were prepared by a similar
123

614
S. Mallakpour et al.
procedure. The structures of some polymers were con-
firmed as PAAs with elemental analysis, FT-IR and
1H-NMR spectroscopy techniques. FT-IR spectra of all
polymers indicated the characteristic absorption peaks for
the imide ring at 1,775, 1,384 and 720 cm-1 because of the
symmetrical and asymmetrical carbonyl stretching.
study for the polycondensation reaction of diphenolic
monomer 7 and optically active diacids, Vilsmeier adduct
was used as a condensing agent for the polymerization
reaction of novel chiral diacids 3a–3e with aromatic
diphenolic monomer 7, of which a detailed mechanism is
illustrated in Scheme 3. Thus, sulfonium salt (I) was pre-
pared by dissolving TsCl in Py and stirring for 30 min
(aging time) followed by the addition of DMF and stirring
for 30 min until the Vilsmeier adduct (II) was formed, as
suggested previously (Higashi and Mitani 2000; Mallak-
pour and Kowsari 2006). The reaction mixture was added
to a solution of diacid in Py to produce activated diacid
(III). After a period of time, a powder of diphenolic
monomer 7 was added and the whole solution was main-
tained at elevated temperature for several hours. Polycon-
densation reaction was performed by varying the amount of
DMF, the molar ratio of TsCl/diacid, Py/diacid and the
time of heating. Under optimized conditions, polyesterifi-
cation of different chiral diacids with diphenolic monomer
7 was carried out as follow: TsCl was dissolved in Py at
room temperature and kept at this temperature for 30 min
according to previously reported procedures (Mallakpour
and Kowsari 2006; Mallakpour and Tirgir 2009). The yield
and viscosity of the resulting PAAs were affected by the
amount of DMF. From this data, it is clear that a ratio of
DMF/diacid should be about 7 (0.1 mL DMF) in order to
obtain polymers with high yield and moderate inherent
viscosity. Furthermore, addition of DMF did not improve
the molecular weights and the yields (Fig. 1). The effect of
ratio of TsCl/diacid on inherent viscosity and yield of the
resulting PAA8a is shown in Fig. 2. From this information,
an appropriate ratio of TsCl/diacid is 5 mol/mol. The effect
of reaction times on inherent viscosity and yield of the
obtained PAA8a is shown in Fig. 3. From this information,
a suitable reaction time of 4 h is required to get PAA8a
with high yield and inherent viscosity. The step-growth
polymerization reactions were also carried out at various
temperatures and a suitable temperature of 120°C was
obtained. The optimum conditions for the preparation of
PAA8a are summarized in Table 1. The synthesis and
some physical properties of these novel optically active
In vitro toxicity test
In vitro toxicity assessment has been widely used for recent
toxicity studies. Such assays provide rapid, cost-effective
and reliable results (Hayes et al. 2007; Bastioli 2005). In
vitro toxicity of BPA, optically active diphenolic monomer
7, different chiral aliphatic diacids (3a–3c) and PAAs were
evaluated based on the growth of different aliphatic diacids
(3a–3c), PAA8c and air-borne spores were co-cultivated
on the same Petri plates containing potato dextrose agar
(PDA) in three replications. Plates were incubated at
23–25°C for 4 weeks. Then colonial growth of fungal
saprophytes were noted and visually observed on each Petri
plate by digital camera (Canon DS126181).
Results and discussion
Monomer synthesis
Optically active diacids (3a–3e) were prepared according
to previous studies (Faghihi et al. 2004a, b; Mallakpour
et al. 2002; Mallakpour and Habibi 2003; Mallakpour and
Shahmohammadi 2005) and are shown in Scheme 1.
N,N0-(pyromellitoyl)-bis-L-tyrosine dimethyl ester (7)
was prepared according to a previous study (Mallakpour
et al. 2010) and is shown in Scheme 2.
Polymer synthesis
PAA8a–PAA8e were synthesized via direct polyconden-
sation reactions of an equimolar mixture of monomer 7
with several different optically active diacids, such as 3a,
3b, 3c, 3d and 3e in a system of TsCl/Py/DMF. In this
O
Scheme 1 Synthesis of
optically active diacids (3a–3e)
O
O
O
N
H
R
R
H
COOH
R
CH3COOH
Reflux
NH2
O
O
+
2
O
N
COOH
HOOC
H
O
2a-2e
O
O
1
3a-3e
CH2
CH2
CH3
CH2
CH2
CH
CH
R:
CH3
CH3
CH3
H3CS
CH3
a
b
c
d
e
123

PPAs from N,N0-(pyromellitoyl)-bis-L-tyrosine dimethyl ester as a diphenolic monomer
615
Scheme 2 Synthesis of N,N0-
(pyromellitoyl)-bis-L-tyrosine
dimethyl ester
O
C
O
HOOC
H2N
H3CO
H
H
SOCl2/MeOH
-15 oC
H3CO
ClH3N
C
H
Et3N/CH2Cl2
0 o
OH
OH
OH
H2N
C
4
6
5
L-Tyrosine methyl ester
L-Tyrosine
L-Tyrosine methyl ester hydrochloride
O
O
OCH3
H
O
O
H3CO
O
O
C
C
H
DMF
OH
HO
6
N
O
O
+
N
O
O
O
Reflux
O
1
7
Scheme 3 Mechanistic
representation of
CH3
CH3
..
N
O
H
C
polycondensation reaction of
diphenolic monomer 7 with
different chiral diacids using
TsCl/Py/DMF as a condensing
agent
:
N
N
S
Cl
O
Ar
S
Cl
O
-Py
O
Ar
(I)
TsCl
H3C
HOOC Ar'
-ArSO3H
COOH
Cl
N
CH
OSO2Ar
H3C
(II)
O
O
O
H2C
Cl
H3C
CH3
Cl
CH3
HO
Ar'' OH
N
C
O
Ar'
C
N
O
H
H
-DMF
-HCl
(III)
O
O
O
Ar'
Ar''
H2C
H3C
H2C
n
CH
CH3
PAA8a-PAA8e
2b
2a
Ar =
H3C
H2C
H3CS
2c
CH2
R:
O
R
H
O
N
H
R
N
Ar' =
CH
CH3
O
H3C
2e
O
3a-3e
H3C
2d
O
C
O
OCH3
H
O
H3CO
O
C
H
Ar'' =
N
N
O
O
7
123

616
S. Mallakpour et al.
Inherent
Table 1 The optimum conditions for the preparation of PAA8a
viscosity (dL/g)
Optimum condition
Yield (%)
0.5
0.45
0.4
100
90
80
70
60
50
40
30
20
10
0
TsCl/diacid (mol/mol)
Py/diacid (mol/mol)
DMF/diacid (mol/mol)
Aging time (min)
5
31
7
0.35
0.3
30
4
0.25
0.2
Reaction time (h)
0.15
0.1
Table 2 Synthesis and some physical properties of PAA8a–PAA8e
prepared using TsCl/DMF/Py
0.05
0
g (dL g-1 a
)
[a]2D5,a
Color
0.05
0.1
0.15
0.2
0.25
Diacid
Polymer
Yield (%)
DMF (ml)
3a
3b
3c
3d
3e
PAA8a
PAA8b
PAA8c
PAA8d
PAA8e
96
95
87
83
92
0.53
0.63
0.43
0.46
0.44
-11.36
-18.91
-24.33
-13.80
-25.41
W
Fig. 1 Effect of the amount of DMF added to TsCl on the inherent
viscosity and yield of PAA8a
W
OW
OW
W
Inherent viscosity
(dL/g)
Yiled (%)
W white, OW Off-white
a
0.45
0.4
100
95
90
85
80
75
70
Measured at a concentration of 0.5 dL g-1 in DMF at 25°C and
consisting of 2%W/V LiCl
0.35
0.3
0.25
0.2
The incorporation of chiral units into the polymer backbone
was confirmed by measuring the specific rotations of poly-
mers with sodium source lamps (Table 2). The specific rota-
tion of polymers based on different diacids showed random
changes. As shown in Table 2, all of the polymers show
optical rotations and are therefore optically active.
0.15
0.1
0.05
0
1
2
3
4
5
6
TsCl/diacid (mol/mol)
Polymer characterization
Fig. 2 Effect of the amount of TsCl added to diacid on the inherent
viscosity and yield of PAA8a
The obtained polymerswere characterized by FT-IR, 1H-NMR
spectroscopy techniques and elemental analyses. The results
are shown in Tables 3 and 4. All of these PAAs exhibited
absorption at 1,380 and 728 cm-1 showing the presence of the
imide heterocycle in these polymers (Table 3). The 1H-NMR
spectrum (500 MHz) of PAA8c is shown in Fig. 4. In the
1H-NMR spectrum ofthis polymer, appearance of the methoxy
protons (OCH3) at 3.66 ppm as a single peak indicates the
presence of ester groups in the polymer side chain. The protons
of the two chiral centers appeared as two peaks in the range of
5.25–5.26 and 5.37–5.38 ppm, respectively. The resonance of
aromatic protons appeared in the range of 6.80–7.24 ppm.
Elemental analysis data of the resulting polymers are also in
good agreement with calculated values of carbon, hydrogen
and nitrogen in the polymers (Table 4).
Inherent
viscosity (dL/g)
Yield (%)
0.54
0.52
0.5
98
96
94
92
90
88
86
84
82
0.48
0.46
0.44
0.42
2
3
4
5
Time of reaction
Fig. 3 Effect of reaction time on the inherent viscosity and yield of
PAA8a
Thermal properties
PAA8b–PAA8e are listed in Table 2. The inherent vis-
cosities of the polymers under optimized condition were in
the range of 0.43–0.63 dL g-1 and the yields were 83–96%.
The thermal properties of some PAAs were evaluated by
means of thermal gravimetric analysis (TGA). Thermal
stability of the polymers was studied based on 5 and 10%
123

PPAs from N,N0-(pyromellitoyl)-bis-L-tyrosine dimethyl ester as a diphenolic monomer
617
Table 3 1H-NMR and FT-IR characterization of PAAs
Polymer Spectra data
PAA8a FT-IR peaks (cm-1): 3,031 (w), 1,776 (m), 1,725 (s), 1,605 (w), 1H-NMR peaks (500 MHz, DMSO-d6, TMS) (ppm): d 3.20–3.23
1,508 (m, sh), 1,455 (m), 1,383 (s), 1,363 (s), 1,195 (m, br),
1,113 (m), 917 (w), 728 (m), 701 (m)
(m, 8H), 3.68 (s, 6H), 5.28–5.29 (dd, 2H, J1 = 10.73 Hz,
J2 = 3.77 Hz), 5.54–5.58 (dd, 2H, J1 = 10.73 Hz,
J2 = 4.80 Hz), 6.80–6.82 (m, 2H, Ar–H), 6.90 (s, 4H, Ar–H),
7.14–7.24 (m, 12H, Ar–H), 8.19 (s, 4H, Ar–H)
PAA8b FT-IR Peaks (cm-1): 2,959 (m), 1,777 (m), 1,725 (s), 1,508 (m), 1H-NMR Peaks (500 MHz, DMSO-d6, TMS) (ppm): d 0.85–0.90
1,456 (w,br), 1,382 (s), 1,362 (m), 1,198 (m, br), 1,114 (w),
1,018 (m), 917 (w), 728 (m)
(s, 6H), 0.95–0.97 (s, 6H), 1.51 (s, 2H), 1.93 (s, 2H), 2.11 (s,
2H), 3.31 (s, 4H), 3.66 (s, 6H), 5.25 (s, 4H), 6.86 (s, 4H, Ar–H),
7.17 (s, 4H, Ar–H), 8.15 (s, 2H, Ar–H), 8.30 (s, 2H, Ar–H)
PAA8c FT-IR Peaks (cm-1): 2,917 (m), 1,776 (m), 1,725 (s), 1,507 (m), 1H-NMR Peaks (ppm): (500 MHz, DMSO-d6, TMS): d 1.99–2.04
1,437 (w, br), 1,382 (s), 1,362 (m), 1,196 (m, br), 1,169 (w, br), (m, 10H), 2.37–2.58 (m, 4H), 3.31 (s, 4H), 3.66 (s, 6H), 5.25 (m,
1,112 (m), 1,018 (w), 728 (m)
2H), 5.37–5.38 (m, 2H), 6.84 (s, 4H, Ar–H), 7.18 (s, 4H, Ar–H),
8.16 (s, 2H, Ar–H), 8.33 (s, 2H, Ar–H)
PAA8d FT-IR Peaks (cm-1): 2,917 (m), 1,776 (m), 1,725 (s), 1,508 (m), 1,438 (w, br), 1,383 (s), 1,363 (m), 1,196 (m, br), 1,112 (w), 1,018
(w), 917 (w), 728 (m)
PAA8e FT-IR Peaks (cm-1): 2,924 (w), 2,853 (w), 1,776 (w), 1,725 (s), 1,594 (w), 1,508 (m), 1,436 (m, br), 1,384 (m), 1,235 (s), 1,116 (m),
1,017 (m), 917 (w), 834 (m), 728 (m), 701 (m)
Table 4 Elemental analysis of PAA8a and PAA8b
Polymer Formula Elemental analysis (%)
Table 5 Thermal behavior of PAA8a and PAA8b
Polymer Decomposition temperature (°C) Char yieldc (%) Tgd(°C)
C
H
N
T5a
Tb10
PA8Aa
PAA8b
(C58H40N4O16
)
Calculated 66.41
3.83
3.77
4.52
4.44
5.34
5.47
5.71
5.66
PAA8a 363
PAA8b 328
a
390
341
30
15
116
80
MW(1,048.96) Found
65.34
Calculated 63.67
Found 62.81
(C52H44N4O16
)
Temperature at which there was 5% weight loss
MW(980.92)
b
Temperature at which there was 10% weight loss recorded by TGA
at a heating rate of 10°C min-1 in a nitrogen atmosphere
c
Percentage weight of material left undecomposed after TGA anal-
ysis at a maximum temperature of 800°C in a nitrogen atmosphere
d
Glass transition temperature was recorded at a heating rate of
20°C min-1 in a nitrogen atmosphere
the range of 80–116°C, respectively (Table 5). The pure
poly(L-tyrosine) showed a slight slope change around
185°C (Gupta and Lopina 2004), which implied that it
hardly underwent glass transition. For PAA8a and PAA8b,
thermal degradation temperatures were observed above 390
and 341°C, respectively. Compared to pure poly(L-tyrosine)
(200°C), these obtained polymers showed potential for
avoiding the risk of thermal degradation. The lower Tg
values and high thermal degradation temperature values
suggested a broad thermal processing temperature range.
Hence, the L-tyrosine-based PAAs could be considered as
biomaterial with significant engineering advantages.
Fig. 4 1H-NMR (500 MHz) spectrum of PAA8c in DMSO-d6 at RT
weight loss (T5, T10) of the polymers. The thermoanalysis
data of these polymers are summarized in Table 5.
According to Table 5, it can be concluded that the resulting
polymers are thermally stable. The differential scanning
calorimetry (DSC) technique was used to determine the Tg
of the polymers. The heating rate was kept at 20°C min-1
under a flow of nitrogen. Figure 5 shows the DSC curves for
PAA8a and PAA8b. These polymers showed Tg values in
Solubility of PAAs
The solubility of PAA8a–PAA8e was tested quantitatively
in various solvents. All of the PAAs are soluble in organic
polar solvents such as DMF, N,N0-dimethyl acetamide
123

618
S. Mallakpour et al.
Table 6 Solubility properties of PAAs
Solvent
PAA8a PAA8b PAA8c PAA8d PAA8e
DMF
?
??
???
??
???
-
??
???
???
???
-
??
???
???
???
-
??
???
??
???
-
2%LiCl-DMF ???
NMP
??
??
-
DMAC
CH2Cl2
CHCl3
H2O
-
-
-
-
-
-
-
-
-
-
DMSO
CH3CN
HOAC
EtOAC
MeOH
EtOH
???
-
???
-
???
-
???
-
???
-
-
-
-
?
-
-
-
-
-
-
-
-
-
-
-
Acetone
THF
-
-
-
-
-
-
-
-
-
-
H2SO4
??
??
???
???
???
Solubility: measured at a polymer concentration of 5 mg mL-1
?, Soluble at boiling temperature of the solvent; ??, soluble at
boiling temperature of the water bath; ???, soluble at RT; , par-
tially soluble at RT; -, insoluble
a period of 2, 3 and 4 weeks, respectively. Figure 7g–i on
culture media PDA shows that synthetic diphenolic
monomer 7 was almost completely covered and colonized
by saprophytic fungi and they formed densely populated
areas after 2, 3 and 4 weeks, respectively. As shown in
Fig. 7j, k, PAA8c was wholly colonized by saprophytic
fungi similar to diphenolic monomer 7 after 2 and 4 weeks,
respectively. Figure 7l shows very clear images of fungal
colonial growth in the presence of PAA8c. In contrast,
BPA on the media was not invaded after 2, 3 and 4 weeks
(Fig. 7d, h and f, respectively). It seems that BPA has toxic
properties for the mentioned fungal growth than synthetic
diphenolic monomer 7 at the period of experimental time,
and it may have lower degradation rate under the soil burial
conditions (Mallakpour et al. 2010).
Fig. 5 DSC thermograms of PAA8a and PAA8b under N2
atmosphere and a heating rate of 20°C min-1
(DMAc), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrol-
idone (NMP) and H2SO4 at room temperature and are
insoluble in solvents such as chloroform, methylene chlo-
ride, methanol, ethanol and water (Table 6).
Fungal biodegradation
Figure 6a–c shows an overview of diacids 3a, 3b and 3c
cultured on Petri plates and colonized by saprophytic fungi.
Figures 6d, f, 7g–i, show colonial growth of fungi on the
diacid derived from L-phenylalanine (3a) and L-methionine
(3b), respectively. This may indicate biodegradability of
these diacids in the presence of fungal saprophytes. On the
contrary, the diacid derived from L-leucine was not invaded
by the fungi under the same condition (Fig. 6j–l). Inter-
estingly, the L-leucine-derived diacid (3b) is intensively
hydrophobic (Fig. 6k) possibly due to alkyl groups (iso-
propyl groups) preventing enzymatic activity and growth
of microorganisms.
Conclusions
The challenge of this research is the use of aromatic
hydroxyl group of L-tyrosine amino acid as a new chiral
nontoxic diphenolic monomer 7 candidate for the prep-
aration of optically active PAAs. In designing of this
monomer, some notification is considered. The protecting
groups used to block the NH2 and COOH of tyrosine
have a significant impact on the properties of the
obtained diphenolic monomer 7 and then on the resulting
Fungal growth profiles of BPA as a blank (Mallakpour
et al. 2010), diphenolic monomer 7 and PAA8c in Petri
plate containing culture media are shown in Fig. 7a–c after
123

PPAs from N,N0-(pyromellitoyl)-bis-L-tyrosine dimethyl ester as a diphenolic monomer
619
Fig. 6 Whole Petri plates
containing diacids 3a, 3b and 3c
cultivated and incubated with
saprophytic fungi at 23–25°C
after 1 (a), 2 (b) and 4 weeks
(c), respectively. Colonial
growth around synthetic diacid
3a (d, e and f) after 2, 3 and
4 weeks in Petri plates,
respectively, and colonial
growth around 3c (g, h and i) at
23–25°C, after 2, 3 and weeks,
respectively. Also inhibited
growth of saprophytic fungi
incubated with 3b (j, k and l)
after 2, 3 and 4 weeks in Petri
plates, respectively
polymers, such as thermal stability due to the presence of
imide linkages, solubility because of the existing pendant
and polar methyl ester group in the side chain, and
biodegradability and biological activity due to the pres-
ence of L-tyrosine amino acids in the main chain of the
synthetic materials. Therefore, the combination of these
different properties within one single design in the aro-
matic diol backbone was used to synthesis N,N0-(pyro-
mellitoyl)-bis-L-tyrosine dimethyl ester, which may be
considered as a nontoxic diphenolic monomer derived
from L-tyrosine in three steps. This monomer is more
thermally stable than BPA because its melting point (mp)
is 168°C, while the MP of BPA is 158°C. This monomer
is soluble in most organic solvents, while BPA has lower
solubility; diphenolic monomer 7 is biologically active,
while BPA has toxic properties and is not biodegradable
under microbial effect. This type of biologically active
monomer has a valuable potential for development of
new bioactive polymers with biodegradable properties.
Here also, a series of new optically active PAAs having
N,N0-(pyromellitoyl)-bis-L-tyrosine dimethyl ester and
N,N0-(pyromelitoyl)-bis-L-a-amino acids moieties were
synthesized by the direct polycondensation method.
Polymerization was performed by the reaction of chiral
diacids containing some a-amino acids in the main chain
with diphenolic monomer 7 using TsCl/DMF/pyridine as
condensing agent. The resulting PAAs are thermally
stable and are readily soluble in common organic sol-
vents. In vitro toxicity studies also suggest that polymeric
products have no inhibition effect against microbial
growth at lower and high concentrations. Hence, these
multiblock copolymers with good cytocompatibility are
123

620
S. Mallakpour et al.
Fig. 7 Whole Petri plates
containing diphenolic monomer
7, PAA8c and BPA cultivated
and incubated with saprophytic
fungi at 23–25°C after 1 (a), 2
(b) and 4 weeks (c),
respectively. Also inhibited
growth of saprophytic fungi
incubated with BPA (d–
f) versus their colonial growth
around synthetic diphenolic
monomer 7 (g, h and i) after 2, 3
and 4 weeks in Petri plates,
respectively, and colonial
growth around PAAc (j and k)
at 23–25°C, after 2 and
4 weeks, respectively. High
resolution photographs of
colonial growth of fungal on
PAA8c (l)
Anderson JM, Spilizewski KL, Hiltner A (1985) In: Williams DF (ed)
Biocompatibility of tissue analogs. CRC Press, Boca Raton,
pp 67–88
favorable candidates as biodegradable and biologically
active polymers.
Bamford CH, Elliot A, Hanby WE (1956) Synthetic polypeptides.
Academic Press, New York
Bastioli C (2005) Handbook of biodegradable polymers. Smithers
Rapra Technology, Shropshire
Brocchini S, Schachter DM, Kohn J (1997) Amino acid derived
polymers for use in controlled delivery systems of peptides.
Therapeutic protein and peptide formulation and delivery. ACS
Symp Ser 675(9):154–167
Acknowledgments We wish to express our gratitude to the
Research Affairs Division, Isfahan University of Technology (IUT),
for partial financial support. Further financial support from National
Elite Foundation (NEF) and Center of Excellency in Sensors and
Green Chemistry (IUT) is gratefully acknowledged. We also extend
our thanks to Mr. M. Hatami for the helpful discussions.
Chen MY, Ike M, Fujita M (2002) Acute toxicity, mutagenicity, and
estrogenicity of bisphenol-A and other bisphenols. Environ
Toxicol 17:80–86
References
Ensafi AA, Mallakpour S, Doozandeh F, Allafchian AR, Tirgir F
(2010) Highly selective potentiometric sensor for determining
phenazopyridine hydrochloride in biological fluids using N,N0-
(pyromellitoyl)-bis-L-tyrosine dimethyl ester. Anal Lett
(accepted)
Aamer KA, Genson KL, Kohn J, Becker ML (2009) Impact of
polymer-bound iodine on fibronectin adsorption and osteoblast
cell morphology in radiopaque medical polymers: tyrosine-
derived polycarbonate blends as a model system. Biomacromol-
ecules 10:2418–2426
Eversloh TL, Nicole C, Santos S, Stephanopoulos G (2007) Perspec-
tives of biotechnological production of L-tyrosine and its
applications. Appl Microbiol Biotechnol 77:751–762
Faghihi K, Foroughifar N, Mallakpour S (2004a) Facile synthesis ofnovel
optically active poly(amide-imide)s derived from N,N0-(pyromelli-
toyl)-bis-L-alanine diacid chloride, tetrahydropyrimidinone and
Acunzo F, Kohn J (2002) Alternating multiblock amphiphilic
copolymers of PEG and tyrosine-derived diphenols. 1. Synthesis
and characterization. Macromolecules 35:9360–9365
Anderson JM, Gibbons DF, Martin RL, Hiltner A, Woods R (1974)
In: Hulbert SF, Levine SN, Moyle DD (eds) Prostheses and
tissue: the interface problem. Wiley, New York, pp 197–207
123

PPAs from N,N0-(pyromellitoyl)-bis-L-tyrosine dimethyl ester as a diphenolic monomer
621
tetrahydro-2-thioxopyrimidine by microwave-assisted polyconden-
sation. Iranian Polym J 13:93–99
bis-S-valine diacid chloride and aromatic diamines under micro-
wave irradiation and classical heating. Iranian Polym
14:473–483
J
Faghihi K, Zamani K, Mirsamie A, Mallakpour S (2004b) Facile
synthesis of novel optically active poly(amide-imide)s contain-
ing N,N0-(Pyromellitoyl)-bis-L-phenylalanine diacid chloride and
5,5-disubstituted hydantoin derivatives under microwave Irradi-
ation. J Appl Polym Sci 91:516–524
Mallakpour S, Tirgir F (2009) Preparation and characterization of
new thermally stable and optically active polyesters by direct
polycondensation reaction promoted by Vilsmeier adduct.
e-Polymers, no. 108
Fu C, Liu Z (2008) Syntheses of high molecular weight aliphatic
polyesters in 1-alkyl-3-methylimidazolium ionic liquids. Poly-
mer 49:461–466
Gupta AS, Lopina ST (2004) Synthesis and characterization of
L-tyrosine based novel polyphosphates for potential biomaterial
applications. Polymer 45:4653–4662
Hayes A, Bakand S, Winder C (2007) Novel In vitro exposure
techniques for toxicity testing and biomonitoring of airborne
contaminants. In: Marx U, Sandig V (eds) Drug testing in vitro:
breakthroughs and trends in cell culture technology. Wiley,
Berlin, pp 103–124
Higashi F, Mitani K (2000) Preparation of copolyesters from diols
and bisphenols by the solution polycondensation with TsCl/
DMF/Py as a condensing agent. J Polym Sci A Polym Chem
38:1270–1276
Mallakpour S, Zadehnazari A (2010) Microwave irradiation as a
versatile tool for increasing reaction rates and yields in synthesis
of optically active polyamides containing flexible L-leucine.
Amino Acids 38:1369–1376
Mallakpour S, Hajipour AR, Habibi S (2002) Microwave-assisted
synthesis of new optically active poly(ester-imide)s contain-
ing N,N0-(pyromellitoyl)-bis-L-phenylalanine moieties. J Appl
Polym Sci 86:2211–2216
Mallakpour S, Tirgir F, Sabzalian MR (2010) Synthesis and structural
characterization of novel biologically active and thermally stable
poly(ester-imide)s containing different natural amino acids
linkages. J Polym Res. doi:10.1007/s10965-010-9427-z
Nakagawa Y, Tayama S (2000) Metabolism and cytotoxicity of
bisphenol A and other bisphenols in isolated rat hepatocytes.
Arch Toxicol 74:99–105
Higashi F, Tobe AA (2001) A new polycondensation involving
dicarboxylic acids with differently activated carboxyl groups by
TsCl/DMF/Py. Macromol Chem Phys 202:745–749
Hooper KA, Macon ND, Kohn J (1998) Comparative histological
evaluation of new tyrosine-derived polymers and poly (L-lactic
acid) as a function of polymer degradation. J Biomed Mater Res
41:443–454
Jack Ch, Jose LC, Kenneth JK, Harold A, Kenneth SJ (1996) Canine
bone response to tyrosine-derived polycarbonates and poly(L-
lactic acid). J Biomed Mater Res 31:35–41
Joskow R, Barr DB, Barr JR, Calafat AM, Needham LL, Carol R
(2006) Exposure to bisphenol A from bis-glycidyl dimethacry-
late-based dental sealants. Am Dent Assoc 137:353–362
Kohn J, Langer R (1984) New approach to the development of
bioerodible polymers for controlled release applications employ-
ing naturally occurring amino acids. Polym Mater Sci Eng
51:119–121
Kohn J, Langer R (1986) Poly(iminocarbonates) as potential bioma-
terials. Biomaterials 7:176–182
Mallakpour S, Habibi S (2003) Microwave-promoted synthesis of
new optically active poly(ester-imide)s derived from N,N0-
(pyromellitoyl)-bis- L-leucine diacid chloride and aromatic diols.
Eur Polym J 39:1823–1829
Mallakpour S, Kolahdoozan M (2006) Preparation and characteriza-
tion of novel optically active poly(amide-ester-imide)s based on
bis(p-aminobezoic acid)-N-trimellitylimido-S-valine via direct
polyesterification. Iran Polym J 15:307–315
Mallakpour S, Kowsari E (2006) Synthesis of novel optically active
poly(ester imide)s by direct polycondensation reaction promoted
by tosyl chloride in pyridine in the presence of N,N-dimethy-
formamide. J Appl Polym Sci 101:455–460
Parth NS, Rachel LM, Stephanie TL, Yang HY (2009) Electrospin-
ning of L-tyrosine polyurethanes for potential biomedical
applications. Polymer 50:2281–2289
Pressman EJ, Johnson BF, Shafer SJ (2005) Advances in polycar-
bonates. ACS Symp Ser 898(3):22–38
Pulapura S, Kohan J (1992) Tyrosine-derived polycarbonates: back-
bone-modified pseudo-poly (amino acids) designed for biomed-
ical applications. Biopolymers 32:411–417
Saal FSV, Akingbemi BT, Belcher SM et al (2007) Chapel hill
bisphenol A expert panel consensus statement: integration of
mechanisms, effects in animals and potential to impact human
health at current levels of exposure. Reprod Toxicol 24:1–26
Sajiki J, Yonekubo J (2004) Leaching of bisphenol A (BPA) from
polycarbonate plastic to water containing amino acids and
its degradation by radical oxygen species. Chemosphere
55:861–867
Shah PN, Puntel AA, Lopina ST, Yun YH (2009) Development and in
vitro cytotoxicity of microparticle drug delivery system for
proteins using L-tyrosine polyphosphate. Colloid Polym Sci
287:1195–1205
Sidman KR, Schwope AD, Steber WD, Rudolph SE, Poulin SB
(1980) Biodegradable, implantable sustained release systems
based on glutamic acid copolymers. J Membrane Sci 7:277–291
Spatola AF (1983) In: Weinstein B (ed) Chemistry and biochemistry
of amino acids, peptides, and proteins. Marcel Dekker, New
York, pp 267–357
Tangpasuthadol V, Shefer A, Yu CH, Zhou J, Kohan J (1997)
Thermal properties and enthalpy relaxation of tyrosine-derived
polyarylates. J Appl Polym Sci 63:1441–1448
Tangpasuthadol V, Pendharkar S, Peterson M, Kohn J (2000)
Hydrolytic degradation of tyrosine-derived polycarbonates, a
class of new biomaterials. Part II: 3-yr study of polymeric
devices. Biomaterials 21:2379–2387
Tsai WT (2006) Human health risk on environmental exposure to
bisphenol-A: a review. J Environ Sci Health Part A 24:225–255
Uhrich KE, Cannizzaro SM, Langer RS, Shakesheff KM (1999)
Polymeric systems for controlled drug release. Chem Rev
99:3181–3198
Mallakpour S, Rafiee Z (2008) Microwave-induced synthesis of new
optically active and soluble polyamides containing pendent 4-(2-
phthalimidiylpropanoyl amino)-benzoylamino-groups. Amino
Acids 37:665–672
Mallakpour S, Seyedjamali H (2008) Synthesis and characterization
of novel organosoluble and optically active aromatic polyesters
containing L-methionine and phthalimide pendent groups. Amino
Acids 34:531–538
Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV (2007)
Human exposure to bisphenol
24:139–177
Mallakpour S, Shahmohammadi MH (2005) Synthesis of new optically
active poly(amide-imide)s derived from N,N0-(pyromellitoyl)-
A
(BPA). Reprod Toxicol
123