Studies on synthesis, characterization, and functionalization of poly(3,4-dihydroxy-L-phenylalanine)

Article abstract of DOI:10.1246/cl.140032

In this paper, hyperbranched PDOPA polyester with precise structure was synthesized by thermal polycondensation. PDOPA showed good solubility, degradability, and biocompatibility. Functional polymers based on PDOPA were prepared by reactions of amine grou

Full text of DOI:10.1246/cl.140032

Received: January 18, 2014 | Accepted: February 4, 2014 | Web Released: February 8, 2014
CL-140032
Studies on Synthesis, Characterization, and Functionalization
of Poly(3,4-dihydroxy-L-phenylalanine)
Dongjian Shi,1,2 Rongjin Liu,1 Fengduo Ma,1 Daoyong Chen,2 Mingqing Chen,*1 and Mitsuru Akashi3
1The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Chemical and Material Engineering,
Jiangnan University, Wuxi 214122, P. R. China
2State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, P. R. China
3Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871
(E-mail: mqchen@jiangnan.edu.cn)
In this paper, hyperbranched PDOPA polyester with precise
(a)
O
C
C
O
O
O
C
N
O
C
O
structure was synthesized by thermal polycondensation. PDOPA
showed good solubility, degradability, and biocompatibility.
Functional polymers based on PDOPA were prepared by reac-
tions of amine groups in PDOPA with pyrenecarboxylic acid
and polylactide, respectively. Wide applications of the PDOPA
polyester are expected after modified with functional groups.
O
H3
C
C
O
Cl
N
O
O
HO
HO
HO
HO
H3
H3
C
C
O
O
N
N
NH2
COOH
(N-Phth)
Na2CO3
RT, 5 h
C5H5
RT, 24 h
COOH
C
O
COOH
DOPA
N-Phth-DOPA
ace-N-Phth-DOPA
O
O
H3
C
C
H3
C
C
O
O
O
O
O
O
N
(AcO)2O, AcONa
H2NNH2
NH2
OH
OH
n
C
200 °C, 5 h
n
C
O
H3
C
C
O
H3
C
C
O
O
P(N-Phth-DOPA)
PDOPA
f
f
g
c
d e
DMSO
C
C
Biobased polymers are a diverse class of materials that
have potential applications to adhesives, absorbents, lubricants,
cosmetics, drug delivery carriers, and food packaging products
in various fields.1 There are many biobased polymers such as
cellulose, poly(hydroxybutyrate) (PHB), poly(lactic acid) (PLA),
poly(amino acid)s, polysaccharides, and so on. One of the most
attractive biobased polymers is poly(amino acid).2 3,4-Dihy-
droxyphenylalanine (DOPA) is a specialized amino acid, which
is commonly found in many marine organisms and plant sources
and can be used for the treatment of neural disorders such as
Parkinson’s syndrome.3 DOPA has unique characteristics such
as easy crosslinking and strong wet adhesion properties with
metals, ceramics, organics, and polymers.4 In an effort to
duplicate these characteristics of DOPA in synthetic polymers,
DOPA was incorporated into synthetic polymers on side chains
or end groups to make them with good adhesive and mechanical
properties for application to imaging agents, surface treatments,
and coatings.5 Although the obtained polymers showed good
properties, some evidence suggest that catechols are oxidized to
o-quinones or semiquinone and undergo nucleophilic addition
reactions with primary amines such as lysine via Michael
addition.6 Thus, the exact structures of the resultant DOPA
derivatives or polymers are difficult to confirm and control.
Although Messersmith et al. synthesized polydopa with exact
structure by amidation reaction, the polymer existed with only
two or three repeat units, possibly due to the steric hindrance
of benzene groups.7 To widen applications of polydopa in
engineering and biomedicine, it is necessary to prepare func-
tional polydopa as a main chain with precise structure.
O
e O
HO
HO
g
a
N
c
b
(b)
(c)
h
b COOH
d
a
h
f
TFA-d
a
g
O
C
h
h
g
f
c d e
H3
C
e
O
O
O
O
b
N
c
OH
C
O
bC
n
d
a
c
H3
C
O
a
h
h
b
O
C
c d e
c d e
h
(d)
(e)
H3
C
e
O
NH2
OH
n
O
bC
a
H3
C
C
O
a
d
O
b
ppm
Figure 1. (a) Synthetic scheme for the PDOPA polymer and
1H NMR spectra of N-Phth-DOPA in DMSO-d6 (b), P(N-Phth-
DOPA) in TFA-d (c), PDOPA in acetone-d6 before degradation
(d), and PDOPA in acetone-d6 after degradation (e).
In order to obtain precise structure of PDOPA polyester, the
amine group in DOPA should be protected to avoid reacting with
carboxy groups during polymerization. Boc- or Fmoc-strategies
are most prominent today.9 However, the Boc- or Fmoc-
protected groups will be deprotected at temperatures above
180 °C, resulting in formation of a polymer mixture. Bergmann
and Zervas discovered an easily removable benzoxycarbonyl
protecting group,10 which is stable at high temperature. Thus,
N-(ethoxycarbonyl)phthalimide (N-Phth, 1.2 mol) was used to
react with DOPA (1 mol) in the presence of borax, as shown in
Figure 1a. With disappearance of absorbances of amine groups
In our previous reports, we have prepared a series of linear
and hyperbranched aromatic polyesters based on cinnamic acid
derivatives by thermal polycondensation, which showed high
performance, biocompatibility, and degradability.8 In this paper,
synthesis and characterization of hyperbranched poly(3,4-dihy-
droxy-L-phenylalanine) (PDOPA) polyester with precise struc-
ture is presented. In addition, pyrene and polylactide (PLA)
polymer chain as functional groups were introduced into
PDOPA side chains to prepare functional PDOPA, which might
widen PDOPA engineering and biomedical applications.
¹1
at 3100-3400 cm and appearance of ester absorbances at
1710 cm in FT-IR spectra (Figures S1a and S1b),11 amine
¹1
groups were confirmed to be protected by N-Phth. Peaks at 7.9
and 8.5 ppm in the H NMR spectrum (Figure 1b) assigned to
benzene groups in N-Phth, suggested the successful synthesis
of N-Phth-DOPA. Hydroxy groups in DOPA easily auto-oxidize
to form quinones, which causes loss of activity and turns the
product black. Thus, acetyl chloride was used to protect hydroxy
1
Chem. Lett. 2014, 43, 959–961 | doi:10.1246/cl.140032
© 2014 The Chemical Society of Japan | 959

(b)
(a)
p<0.05
groups before polymerization, and the structure of Ac-N-Phth-
DOPA was confirmed by the FT-IR spectrum (Figure S1c).
Then, Ac-N-Phth-DOPA (50 mmol) was heated at 200 °C using
acetic anhydride (10 mL) as a condensation reagent and sodium
acetate as a catalyst for transesterification. After purification in
methanol, a brown product P(N-Phth-DOPA) was obtained. The
1H NMR spectrum showed multiple peaks in the region of the
chemical shift of 6.9-7.2 ppm assigned to aromatic protons in
DOPA and a single peak at 2.5 ppm assigned to the acetylate end
group (Figure 1c). The N-Phth group can be cleaved in weak
alkaline solution, and thus deprotection of the amino groups in
P(N-Phth-DOPA) was performed in the present of hydrazine.
The FT-IR spectrum of PDOPA (Figure S1d) confirmed the
16
14
12
10
8
6
4
2
0
100
80
60
40
20
0
0
30 60 90 120 150 180
Time/min
Control
PDOPA
Figure 2. (a) Weight loss of PDOPA during hydrolytic
degradation. (b) Cell adhesion after 24 h on PDOPA film
(n = 6). Tissue culture polystyrene as a control sample.
Statistically significant differences using a two-sample t-test
for comparison.
¹1
presence of amide at 3050 and 3250 cm and disappearance of
Phth at 1710-1780 cm¹1. The 1H NMR spectrum (Figure 1d)
also confirmed the disappeared of Phth groups at 7.9-8.5 ppm to
obtain PDOPA polyester with active amine groups. The degree
of branching of PDOPA was 0.356 (Figure S2), which was
calculated from the 1H NMR spectrum (Figures 1c and 1d)
based on a reported method.12
O
O
(a)
(b)
O
O
1.0
0.8
0.6
0.4
0.2
0.0
m
NH2
HN
O
Pure Py
PDOPA
PDOPA-Py
O
C
n-m
800
600
400
200
0
O
The resultant PDOPA showed good solubility in acetone,
THF, DMF, DMSO, and other common organic solvents. This
result indicated that PDOPA could be easily processed and
further modified. Molecular weight (Mn) and distribution were
200
300
400
Wavelength/nm
500
600
360
400
440
480
520
560
600
Wavelength/nm
¹1
Figure 3. (a) UV-vis and (b) fluorescence spectra of PDOPA-
Py polyester (1 © 10¹5 mol L¹1).
determined by GPC in THF to be 11990 g mol and 1.89
(Figure S3), respectively. DSC showed Tg of PDOPA at about
106 °C and Tm higher than 200 °C (Figure S4), which were
higher than the values of some biobased polymers (such as
Tg PLA: 55 °C and Tm PLA: 160 °C).
reaction (Scheme S1a). 1H NMR spectrum (Figure S7) con-
firmed the structure of PDOPA-Py and the grafting degree of
Py was about 21.1%. The fluorescence of PDOPA-Py was
confirmed by UV-vis and fluorescence spectra, as shown in
Figure 3. Both UV-vis and fluorescence spectra showed the
special adsorptions of Py, suggesting Py was introduced into the
PDOPA polymer. Moreover, the fluorescence intensity of the
PDOPA-Py polymer was higher than pure Py and PDOPA
(Figure 3b). These results suggested the obtained PDOPA-Py
polymer was fluorescent.
Moreover, carboxy polylactide (PLA) was also a functional
group to react with PDOPA (Scheme S1b). The structure was
confirmed by 1H NMR spectra (Figure S8). The properties of
PDOPA-g-PLA are now being determined.
In conclusion, hyperbranched PDOPA polyester with
precise structure was prepared and confirmed to have good
solubility, biodegradability, and biocompatibility. PDOPA could
be modified by pyrene and PLA to obtain functional polymers
based on PDOPA. Since PDOPA has active amine groups,
PDOPA can be modified with many functional compounds or
polymers, such as cyclodextrins, chitosan, and so on. Therefore,
the PDOPA polyester can be a novel biopolymer to be used in
many fields.
Degradation of biobased polymers is a very important factor
for materials in biomedical applications. Therefore, the hydro-
lytic degradation of PDOPA was investigated in alkaline buffer
at pH 12 as an accelerated test. The weight of PDOPA decreased
significantly to approximately 28% after 30 min of hydrolysis
(Figure 2a), and to 83% after 150 min. These results suggested
that PDOPA has high degradability. The molecular weight of
remaining PDOPA after degradation was 11480 g mol¹1, which
is similar to that of PDOPA before degradation, as shown in
1
Figure S3. The H NMR spectrum (Figure 1e) of the remaining
PDOPA after degradation was the same as before degradation,
suggesting no structure change. To evaluate the degraded
structure, the product yielded by degradation in the supernatant
solution was analyzed by high-performance liquid chromatog-
raphy (HPLC). The results (Figure S5) showed monomer peak
together with other peaks presumably assigned to oligomers.
The mass of the degraded products in the supernant solution was
about 326.1 (Figure S6), which was approximately two DOPA
molecules. These results indicated that PDOPA degraded to
monomer and oligomers, and the oligomer might be due to
the melamine formation by DOPA auto-oxidation. We further
confirmed the cell compatibility of PDOPA through a cell
adhesion test using the MTT method. After L929 fibroblasts
(5 © 104 cells) were incubated on the PDOPA film for 24 h
at 37 °C in PBS, the cells adhered and grew to 14.2 © 104
cells (Figure 2b), and the cells on tissue culture polystyrene
(TCP, as a control sample) numbered 11.6 © 104 cells. There
was no significant difference in cell adhesion of PDOPA with
TCP.
This study was supported by the National Natural Science
Foundation of China (Nos. 21004029 and 51173072) and State
Key Laboratory of Molecular Engineering of Polymers (Fudan
University, K2011-08). The authors thank Dr. Koji Kadowaki
for cell adhesion experiments in Osaka University.
References and Notes
In order to prepare functional PDOPA, 1-pyrenebutyric acid
(Py) was employed to react with PDOPA through amidation
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Chem. Lett. 2014, 43, 959–961 | doi:10.1246/cl.140032
© 2014 The Chemical Society of Japan | 961