Full text of DOI:10.1002/1616-5195(20010801)1:6<266::AID-MABI266>3.0.CO;2-K

266
Macromol. Biosci. 2001, 1, 266–269
Communication: A new class of biodegradable polyam-
pholytes, poly[(aspartic acid)-co-lysine], were synthesized
by thermal polycondensation of aspartic acid and lysine
under reduced pressure and subsequent hydrolysis. Poly-
merization conditions were optimized to yield maximal
water-soluble poly(succinimide-co-lysine) with high
molecular weight (1608C/3.5 h). The succinimide/lysine
ratio in the polyampholytes could be adjusted by their
feed ratio. Characterization of the poly(succinimide-co-
lysine) by 1H NMR revealed that x-amine and carboxylic
groups in lysine participated in the polymerization, leav-
ing a-amino groups as pendant cationic moieties.
Synthetic route for the production of amphoteric poly[(aspar-
tic acid)-co-lysine].
Synthesis of Biodegradable Amphoteric Poly[(aspartic
acid)-co-lysine] by Thermal Polycondensation
H. L. Jiang,* G. P. Tang, K. J. Zhu
Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
The State Key Laboratory of Functional Polymer Materials for Absorption and Separation, Nankai University,
Tianjin 300071, China
E-mail: opl-jhl@emb.zju.edu.cn
are few reports on the co-polycondensation of aspartic
acid and lysine to give biodegradable amphoteric poly-
[(aspartic acid)-co-lysine] [P(ASP-co-LYS)] (Scheme 1).
Such biodegradable polyampholytes are promising for
the preparation of absorbable polyelectrolyte complexes
with high pH-sensitivity for controlled protein release by
complexation with polyanions.[9–10] In this work, the
synthesis and characterization of [P(ASP-co-LYS)] are
reported.
Introduction
Poly(amino acid)s, such as poly(aspartic acid), poly(glu-
tamic acid) and polylysine, have always been synthesized
by polymerization of N-carboxyanhydrides of a-amino
acids, i.e., the NCA method. However, the NCA method
has a cost disadvantage and a production problem
because the pendant reactive groups carried by the amino
acids should be protected prior to polymerization, then
deprotected under harsh conditions to give poly(amino
acids). In addition, phosgene, diphosgene or triphosgene
is needed for the synthesis of N-carboxyanhydrides.[1]
There is considerable research into the production of
poly(succinimide) by the thermal polycondensation of
aspartic acid.[2–4] Compared with the NCA method, ther-
mal polycondensation has the advantages of simple
operation and low cost. In addition, other amino acids,
such as glysine, ornithine and x-amino acid can also be
incorporated to the polymer main chain by simple co-
polycondensation.[5–8] However, to our knowledge, there
Experimental Part
Materials and Measurements
d,l-Aspartic acid (ASP) and l-lysine (LYS) were purchased
from Weikang Amino Acid Corp (Shanghai). 1H NMR spec-
tra were obtained with a Bruker DMX500 NMR spectro-
meter operating at 500 MHz using D2O as a solvent and tet-
ramethylsilane (TMS) as the internal standard. Infrared (IR)
spectra were recorded on a Bruker Vector 22 Spectrometer.
Macromol. Biosci. 2001, 1, No. 6
i WILEY-VCH Verlag GmbH, D-69451 Weinheim 2001
1616-5187/2001/0608–0266$17.50+.50/0

Synthesis of Biodegradable Amphoteric Poly[(aspartic acid)-co-lysine] by Thermal ...
267
Results and Discussion
Synthesis and Characterization of P(SI-co-LYS)
It was observed that significant water-insoluble products
were generated after the thermal co-polycondensation of
aspartic acid and lysine at 1808C for 3.5 h, which could
not dissolve in common organic solvents, such as, N,N-
dimethylformamide, dimethyl sulfoxide and trifluroacetic
acid. From their insoluble characteristic, it may be con-
cluded that the water-insoluble species are the crosslink-
ing products which were generated by the nucleophilic
reaction of the pendant amino groups presented in lysine
units with succinimides in the polymer main chain during
polymerization.[8] The water-soluble parts were precipi-
tated into methanol to give a yellowish viscous product.
It should be noted that the two monomers could dissolve
in methanol/water mixed solution (90:10, v/v), ensuring
the purity of the precipitated products. After drying under
Scheme 1. Synthetic route for the production of amphoteric
poly[(aspartic acid)-co-lysine].
1
vacuum, a hard lump was obtained. H NMR characteri-
Samples were either film cast in chloroform onto NaCl plates
or pressed into KBr pellets. Molecular weights of the poly-
mers were estimated using gel permeation chromatography
(GPC, Waters GPC Model 208) on a bank of Waters col-
umns (Ultrahydrogel 2000, 500 and 120). The eluant was
water containing 0.1 m sodium nitrate and 5 mmol sodium
azide; the flow rate was 0.8 ml N min–1 and the temperature
was 458C.
zation of the polymers confirms their chemical structures
as P(SI-co-LYS) (Scheme 1), which is described in the
following section.
In order to obtain maximal water-soluble polymers
with relatively high molecular weight, the polymerization
conditions were optimized. The effects of temperature on
copolymerization of ASP and LYS (20/80 feed ratio in
weight) are shown in Table 1. It can be seen that raising
polymerization temperature results in an increase in the
molecular weight (MW) of the water-soluble copolymers
at the expense of their yields. Due to both the low fraction
of water-insoluble species and the moderate MW of
water-soluble copolymers at 1608C, the following poly-
merization was fixed under such conditions. Table 2
shows the effect of ASP/LYS feed ratio on the polymer
yield, MW and SI/LYS ratio in the—copolymers at 1608C.
It can be seen that the copolymer Mw varies from 15000
to 46000. In addition, the SI/LYS ratio is always lower
Polycondensation
A typical procedure for the co-polycondensation of ASP
with LYS is as follows: ASP (0.25 g, 1.88 mmol), LYS
(0.75 g, 5.14 mmol) and 85% o-phosphoric acid (0.4 g,
3.51 mmol) were mixed for 15 min at room temperature
manually. The mixture was heated at 1608C for 3.5 h under
reduced pressure (ca. 5 mmHg) using a rotary evaporator.
The reaction system changed from syrupy heterogeneous
liquid to a glassy solid as the reaction proceeded. After com-
pletion of the reaction, the pale brown glassy product was than the ASP/LYS feed ratio indicating that LYS can be
dissolved at room temperature by adding 5 ml of distilled
water. The solution was filtered and adjusted to pH10.0 with
diluted NaOH, then poured dropwise into methanol (50 ml)
to yield a viscous precipitate. The purifying step described
above was repeated twice and the precipitate was dried at
room temperature under reduced pressure to give a yellowish
lump of poly(succinimide-co-lysine) (P(SI-co-LYS)).
easily incorporated into the copolymers. In addition, the
GPC traces show a single peak, indicating the absence of
homopolymers of ASP and LYS (results not shown).
Table 1. The effect of polymerization temperature on percent
of water-insoluble products, yield and MW of water-soluble
poly(SI-co-LYS). ASP/LYS feed ratio (in weight) was fixed at
20/80, polymerization time was 3.5 h.
Alkali Hydrolysis
Temperature
Water-insoluble Water-soluble copolymer
copolymer percent
8C
The hydrolysis of P(SI-co-LYS) was carried out as follows:
the pH of the aqueous polymer solution was adjusted to 10.8
with 0.1 n NaOH solution. The solution was stirred for 1 h,
then acidified to ca. pH8.0 by adding 35% aqueous HCl and
finally poured into methanol. The precipitate was filtered
and dried at room temperature under reduced pressure to
yield a yellowish powder of P(ASP-co-LYS).
—
—
Yield
%
–––––––––––––––
%
Mw
Mn
140
160
180
200
0.6
2.2
38.1
78.2
32
79
58
16
13000 5600
34000 12000
38000 14000
44000 19000

268
H. L. Jiang, G. P. Tang, K. J. Zhu
Table 2. The effect of ASP/LYS feed ratio on yield, MW of
water-soluble poly(SI-co-LYS) and SI/LYS ratio in the copo-
lymers. Polymerization was conducted at 1608C for 3.5 h.
—
—
b)
Yielda)
%
Polymer
Mw
Mn SI/LYS
ratioc)
P(SI-co-LYS) 81:19d)
P(SI-co-LYS) 62:38
P(SI-co-LYS) 42:58
P(SI-co-LYS) 32:68
P(SI-co-LYS) 22:78
52
59
71
78
79
15000 4800 72:28
17000 5400 58:42
39000 16000 36:64
46000 21000 25:75
34000 12000 11:89
Scheme 2. Chemical structure of poly(succinimide-co-lysine)
with a-amino groups in lysine participating in the copolymeriza-
tion.
a)
b)
Calculated by weight of water-soluble copolymer/weight of
theoretic copolymer.
Determined by GPC, distilled water as solvent and dextran as
nation of peaks obatined from the literature and the above
results, is shown in Figure 1.[6, 7] At ca. 2.25 ppm in all the
NMR spectra, no peaks of methylene protons presented
as * in Scheme 2 are observable, a result which can be
produced only when the a-amino groups in LYS partici-
pate in the copolymerization. However, the occurrence of
peaks at b that can be designated as the methine protons
reveals that it is the x-amino groups in LYS taking part
in the co-polycondensation.
standard.
c)
d)
In mole, calculated from 1H NMR spectra.
Feed ratio in mole corresponding to that in weight of 80:20,
60:40, 40:60, 30:70 and 20:80, respectively.
Alkali Hydrolysis
P(SI-co-LYS) was further hydrolyzed in NaOH solution
to give amphoteric poly[(aspartic acid)-co-lysine]. Fig-
ure 2 shows 1H NMR spectrum of poly(ASP-co-LYS)
20:80. In comparison with the NMR spectrum of P(SI-
co-LYS) 20:80, new peaks can be observed at 4.40, 4.57
and 2.46–2.72 ppm. The two former can be designated as
methine protons in ASP units and the latter as methylene
protons in ASP units. In addition, the peak at 3.02–
3.20 ppm resulting from methylene protons in SI units
disappears, indicating the complete hydrolyzation of SI
units. It was observed that precipitates were generated
upon mixing poly(aspartic acid) (PASP) and P(SI-co-
LYS) 40:60 within the pH range from 2.5–9.0, suggest-
ing the formation of PASP/P(SI-co-LYS) complexes. In
contrast, a mixture of PASP and LYS was homogeneous
within the pH range 1–14, which further confirms the
incorporation of cationic groups into the polymers. How-
Figure 1. 1H NMR spectra of poly(succinimide-co-lysine) with
ASP/LYS feed ratio (wt.-%): (1) 20/80, (2) 40/60, (3) 60/40, (4)
80/20.
1
Figure 1 shows the H NMR spectra of water-soluble
copolymers obtained with different ASP/LYS feed ratios.
It can be seen that with the increase in the ASP/LYS feed
ratio the peak intensities at a and c increase, while the
peaks at b, e, f and g gradually diminish, which indicates
that the former two result from protons in SI units and the
four latter can be attributed to those in LYS units. The
peak at d shows no apparent variation in intensity with
changes in the ASP/LYS feed ratio, suggesting it origi-
nated from protons in both SI and LYS units. The desig-
Figure 2. 1H NMR spectrum of poly[(aspartic acid)-co-lysine]
20:80. D2O was the solvent and tetramethylsilane the internal
standard.

Synthesis of Biodegradable Amphoteric Poly[(aspartic acid)-co-lysine] by Thermal ...
269
[1] H. R. Kricheldorf, “a-Aminoacid-N-carboxy-anhydride
ever, precipitates can only be produced within pH range
2.5–4.8 for the mixtures of PASP and P(ASP-co-LYS)
40:60, indicating the amphoteric nature of P(ASP-co-
LYS). The entrapment of proteins into and protein release
behavior from polyanion/P(ASP-co-LYS) complexes are
under investigation.
and Related Heterocycles”, Springer-Verlag, Berlin 1987,
pp. 3–58.
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Acknowledgement: Funding of this research by the Zhejiang
Provincial Natural Science Foundation of China is gratefully
acknowledged.
[6] T. Kakuchi, M. Shibata, S. Matsunami, T. Nakato, M.
Tomida, J. Polym. Sci., Part A 1997, 35, 285.
[7] M. Tomida, T. Nakato, M. Kuramochi, M. Shibata, S. Mat-
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Received: January 21, 2001
Revised: May 25, 2001
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[10] H. L. Jiang, K. J. Zhu, J. Appl. Polym. Sci. 2001, 80, 1416.