Zn(OAc)2-catalyzing ring-opening polymerization of N-carboxyanhydrides for the synthesis of well-defined polypeptides

Article abstract of DOI:10.3390/molecules23040760

Despite notable progress, the fabrication of well-defined polypeptides via controlled ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCAs) using convenient catalysts under mild conditions in a relatively short polymerization time i

Full text of DOI:10.3390/molecules23040760

molecules
Article
Zn(OAc)2-Catalyzing Ring-Opening Polymerization
of N-Carboxyanhydrides for the Synthesis of
Well-Defined Polypeptides
Yanzhao Nie 1, Xinmei Zhi 1, Haifeng Du 2,* and Jing Yang 1,
*
ID
1
State Key Laboratory of Chemical Resource Engineering, Beijing Key Laboratory of Bioprocess, College of
Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China;
nieyanzhao126@126.com (Y.N.); zxmlucky903@163.com (X.Z.)
Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Molecular Recognition and
Function, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
Correspondence: haifengdu@iccas.ac.cn (H.D.); yangj@mail.buct.edu.cn (J.Y.); Tel.: +86-010-62652117 (H.D.);
+86-010-64427578 (J.Y.)
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 13 March 2018; Accepted: 23 March 2018; Published: 26 March 2018
Abstract: Despite notable progress, the fabrication of well-defined polypeptides via controlled
ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCAs) using convenient
catalysts under mild conditions in a relatively short polymerization time is still challenging. Herein,
an easily obtained catalyst system composed of zinc acetate and aniline was explored to mediate
the fast ROP of
γ-benzyl-L-glutamate-N-carboxyanhydride (BLG-NCA) monomer, to produce
poly( -benzyl-L-glutamates) (PBLGs) with controllable molecular weights and narrow dispersity.
γ
Considering the excellent cooperative action of zinc acetate and a broad scope of aniline derivatives
with different functional groups to control ROP of BLG-NCA, this method may offer a useful platform
enabling the rapid generation of end-functionalized PBLG and block copolymers for numerous
biomedical applications.
Keywords: Lewis pair polymerization; polymerization catalysis; polymer synthesis; polypeptides;
ring-opening polymerization
1. Introduction
Polypeptides, as an important class of biopolymers, have found extensive applications in diverse
biomedical areas, such as serving as scaffolds for tissue engineering, matrices for drug delivery,
and responsive materials for biosensors [
structures has thus attracted the intense attention of chemists. The ring-opening polymerization
(ROP) of -amino acid N-carboxyanhydrides (NCAs) has witnessed a rapid growth and has become
an important method for polypeptide synthesis since Deming and co-workers made a breakthrough by
using transition metal complexes as active species [ ]. Some organometallic (such as rare earth metal
catalysts [ ]) and metal-free initiators (e.g., hexamethyldisilazanes [ 13], tetrafluoroborate [14,15]
and secondary amine-assisted primary amines [16 19]) or polymerization systems [20 24] were
1–5]. The synthesis of polypeptides with well-defined
α
6,7
8
9–
–
–
successively explored in the past decades. Despite these advances, some problems still await solution,
such as the difficult access to initiators or catalysts, sensitivity of the catalysts to moisture and air,
and long polymerization times. Therefore, it is highly desirable to develop simple and readily accessible
catalysts to realize the controlled synthesis of polypeptides under mild conditions.
The combination of Lewis acids (LAs) and bases (LBs) for cooperative activation of small
molecules has attracted huge interest since the seminal work of Stephan [25–32]. In the past decade,
this concept has shown remarkable versatility, and a wide range of Lewis pairs have been developed
Molecules 2018, 23, 760; doi:10.3390/molecules23040760
www.mdpi.com/journal/molecules

Molecules 2018, 23, 760
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for the activation of small molecules, catalytic hydrogenation or hydrosilylation, and new reactivity
development [33 40]. Recently, very promising results have also been reported in polymerization
catalysis [41,42]. Indeed, Lewis pairs of aluminum-based Lewis acids and N-heterocyclic carbenes or
phosphines have been explored in the polymerization of acrylic monomers [43 49]. Lactones have been
precisely polymerized by highly interacting Lewis pairs for the synthesis of aliphatic polyesters [50 54].
–
–
–
As a continuation of our ongoing interest in Lewis pairs, we have recently reported a controlled ROP
of NCAs using a frustrated Lewis pair of bulky bis-(2,4,6-tris-trifluoromethylphenyl)-fluoroborane
(Fmes2BF) and aniline (Scheme 1) [55]. During the polymerization, the bulky Fmes2BF can capture the
ω
-amine end of a propagating chain to form a new Lewis pair intermediate, efficiently suppressing
side reactions. Nevertheless, Fmes2BF lacks the ability to activate monomers, thus resulting in long
polymerization times. Moreover, the complex preparation of boron derivatives for the synthesis of
well-defined polypeptides might be also challenging.
Scheme 1. The reported boron-based strategy and the new approach proposed in this study. ROP-
ring-opening polymerization.
Zinc alkoxides are known to efficiently propagate the ROP of cyclic esters and lactones [56–58].
Among a multitude of zinc complexes, zinc acetate (Zn(OAc)2) is readily available and stable under
ambient conditions in the presence of moisture and air. However, to the best of our knowledge,
the low-cost zinc acetate has seldom been reported to catalyze the polymerization of monomers.
Aiming to develop simple and efficient catalytic polymerization systems, herein, the combination of
zinc acetate and aniline/its analogues is explored for the controlled ROP of γ-benzyl-L-glutamate
N-carboxyanhydrides (BLG-NCAs) (Scheme 1). Such a combination of zinc acetate and aniline
exhibits exciting catalytic polymerization efficiency for BLG-NCA and produces well-defined
poly(γ-benzyl-L-glutamates) (PBLGs) with controlled molecular weights and low dispersity in
relatively short polymerization time. Moreover, zinc acetate tolerates well a broad spectrum of
aniline analogues to promote the controlled ROP of BLG-NCA. In particular, combinations of zinc
acetate and functionalized anilines derivatives offer an attractive approach for easily preparing PBLGs
with special groups at the amino end of the polymer backbone to further satisfy the needs of numerous
biological applications. For example, in the presence of zinc acetate, amantadine, an anti-influenza
virus drug, can initiate BLG-NCA to afford well-defined drug-conjugated polypeptides.

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2. Results and Discussion
2.1. Controlled ROP of BLG-NCAMediated by Zn(OAc)2 and Aniline
Herein, aniline was selected as an example of such an initiator that contains a primary
amine but has low nucleophilicity in comparison with the reported benzylamine or n-hexylamine.
The polymerization of BLG-NCA initiated by aniline was firstly investigated in dichloromethane
(DMC), N,N-dimethylformamide (DMF) and tetrahydrofuran (THF), respectively. Nearly 100%
monomer conversion was achieved in 5.0 h in THF or DMF, and the obtained molecular weight
(Mn) was much higher than the calculated ones (runs 5 and 8 in Table 1). When the solvent was
changed to DCM with a polar, non-coordinating solvent, the polymerization was rapidly completed
in 1.0 h (run 2) with measured Mn (1.35
×
104), but with a molecular weight distribution (Ð) of
1.36. When Zn(OAc)2 and aniline cooperatively initiated BLG-NCA to polymerize in THF or DMF,
the polymerization time was extended to 24 h (run 5 vs. run 6 in Table 1) and abroader Ð was obtained
(run 8 vs. run 9). To our satisfaction, when the cooperative catalysis using Zn(OAc)2 and aniline in
a molar ratio of 1 to 1 was performed in DCM, the polymerization proceeded completely in 3.0 h to
afford PBLG with a dramatic decrease in Ð to 1.16 (Table 1, run 3).
Table 1. Polymerization of BLG-NCA in different solvents a.
Time c
(h)
Conv.
Mn.cal × 10
−4 e
−4 f
Mn,mea × 10
Zn(OAc)2:Aniline:BLG-NCA b
Ð f
Run
Solvent
d
(g/mol)
(g/mol)
1
2
3
4
5
6
7
8
9
DCM
DCM
DCM
THF
THF
THF
DMF
DMF
DMF
0:0:50
0:1:50
1:1:50
0:0:50
0:1:50
1:1:50
0:0:50
0:1:50
1:1:50
24.0
1.0
3.0
24.0
5.0
24.0
24.0
5.0
0
-
-
-
100%
100%
0
100%
86%
0
1.10
1.10
-
1.10
0.95
-
1.35
0.90
-
3.50
1.64
-
1.36
1.16
-
1.35
1.31
-
100%
100%
1.10
1.10
4.18
1.57
1.35
1.70
8.0
a
Carried out at 25 ◦C. b Indicating that the feeding molar ratio of Zn(OAc)2, aniline and
γ
-benzyl-L-glutamate-
N-carboxyanhydride (BLG-NCA). c Indicate polymerization time. Determined by monitoring the change of NCA
d
anhydride absorption peak at 1785 cm-1 in FT-IR spectroscopy, and polymerization time was contained in the
e
f
bracket. Calculated by [BLG-NCA]/[aniline]
represents molecular weight distribution.
×
(MNCA-44)
×
monomer conversion. Determined by GPC, Ð
Subsequently, several commercial available zinc salts were tested in this polymerization of
BLG-NCA in DCM. Zinc chloride (ZnCl2) was found to inhibit the polymerization (Table 2, runs 1),
and zinc trifluoromethane sulfonate (Zn(OTf)2) gave only 58% conversion of BLG-NCA in 20 h
(Table 2, run 2). Interestingly, Zn(OAc)2
a similar result, but in a relatively shorter reaction time (Table 2, run 3). Under the same conditions,
no polymerization occurred at all when Zn(OAc)2 2H2O was used alone. Therefore, the combination
of Zn(OAc)2 2H2O and aniline was thus crucial to observe efficient and controlled polymerization
of BLG-NCA. Due to the sparing solubility of Zn(OAc)2 2H2O in DCM, some suspended material
was observed during the polymerization. The loading amount of Zn(OAc)2 2H2O was therefore
investigated. As shown in Table 2, a decrease in the molar ratio of Zn(OAc)2 2H2O and aniline from
·
2H2O in place of Zn(OAc)2combined with aniline exhibited
·
·
·
·
·
2:1 to 0.25:1 still gave good control over the polymerization (runs 5–7), and the polymerization time of
BLG-NCA was only slightly shortened from 2.5 h to 2.0 h with 99% monomer conversion. When the
ratio of Zn(OAc)2
·
2H2O and aniline was set to 0.1:1, at which some insoluble Zn(OAc)2 2H2O could
·
be still observed, the polymerization went rapidly, but resulted in a relatively broad molecular weight
distribution (run 4 in Table 2 and Figure 1A).
Kinetic studies were conducted for the polymerization of BLG-NCA with varying ratios of
Zn(OAc)2
·
2H2O and aniline, to investigate the effects of the amount of Zn(OAc)2
·
2H2O on chain initiation
and propagation. The progress of the polymerization was monitored in situ by IR at fixed time intervals

Molecules 2018, 23, 760
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(0.5 min), and a plot of ln([M]0/[M]t) versus time with gradually increasing slopes was observed (Figure 1B).
Two stages were noted in the polymerization process: the early stage of the reaction with low initiation
rate constant (ki) reflected from the slope and the propagation stage toward the end of the reaction
with high propagation rate constant (kp). This was possibly associated with the lower nucleophilicity
of aniline as initiator in comparison with the propagating species in the presence of Zn(OAc)2
With an increase in the molar amount of Zn(OAc)2 2H2O from 0.1 to 0.5 relative to aniline, the initiation
rate and the propagation rate were correspondingly decreased, whereas, upon further enhancing the
amount of Zn(OAc)2 2H2O in the range between 0.5 and 2.0, similar polymerization kinetics were detected,
which was attributed to the poor solubility of Zn(OAc)2·2H2O in DCM.
·2H2O.
·
·
Table 2. Polymerization of
γ-benzyl-L-glutamate-N-carboxyanhydride (BLG-NCA) catalyzed by aniline
and various zinc salts in DCM a.
−4 e
−4 d
Mn,mea × 10
Mn.cal × 10
Zinc Salts:Aniline:BLG-NCA b
Run
Zinc Salts
Time (h)
Conv.c
Ð e
(g/mol)
(g/mol)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
ZnCl2
Zn(OTf)2
1:1:50
1:1:50
1:1:50
0.1:1.50
0.25:1:50
0.5:1:50
2:1:50
1:1:25
1:1:75
1:1:100
1:1:150
20
20
2.5
1.0
2.0
2.5
2.5
2.5
3.0
0
58
0
—
—
0.64
1.10
1.10
1.10
1.10
1.10
0.56
1.65
2.20
3.30
4.40
1.10
2.20
0.23
0.90
0.95
0.95
0.95
0.95
0.45
1.76
2.59
2.76
4.24
0.98
2.48
1.27
1.16
1.36
1.17
1.15
1.16
1.16
1.16
1.13
1.14
1.12
1.18
1.18
Zn(OAc)2
Zn(OAc)2
Zn(OAc)2
Zn(OAc)2
Zn(OAc)2
Zn(OAc)2
Zn(OAc)2
Zn(OAc)2
Zn(OAc)2
Zn(OAc)2
Zn(OAc)2
Zn(OAc)2
·
·
·
·
·
·
·
·
·
·
·
·
2H2O
2H2O
2H2O
2H2O
2H2O
2H2O
2H2O
2H2O
2H2O
2H2O
2H2O
2H2O
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
4.5
5.0
8.0
1:1:200
2.0 + 2.0 g
2.0 + 3.0 g
1:1:(25 + 25)f
1:1:(50 + 50)f
Carried out at 25 ◦C.
Indicating that the feeding molar ratio of Zn(OAc)2 2H2O, aniline and
·
a
b
γ
-benzyl-L-glutamate-N-carboxyanhydride (BLG-NCA). c Determined by monitoring the change of NCA anhydride
absorption peak at 1785 cm−1 in FT-IR spectroscopy. d Calculated by [BLG-NCA]/[aniline]
(MNCA-44) monomer
×
×
e
f
conversion. Determined by GPC, Ð represents molecular weight distribution. BLG-NCA monomer was added in
g
the two sequences. Polymerization time for complete conversion of monomer in each sequence.
The feeding ratios of BLG-NCA and the combination of Zn(OAc)2
under the optimal condition (25 C, 1.0 mL DCM, [BLG-NCA] = 0.75 M and [Zn(OAc)2
·
2H2O and aniline were studied
2H2O]/[aniline]
◦
·
= 1/1), as well. As the amount of BLG increased from 25 to 200, the polymerization could proceed
completely to furnish the corresponding PBLG with the enhanced molecular weight and narrow
distributions (Table 2, runs 8–12). Indeed, the polymerization could be carried out with two sequential
additions of BLG-NCA (25/25 and 50/50) without any chain breaking reactions (runs 13 and 14 in
Table 2 and Figure S2), and the obtained molar mass of the resultant PBLG were identical to those
from the same amount of BLG in one batch addition. When BLG was polymerized in the presence of
Zn(OAc)2 2H2O and aniline with a ratio of 1:1:50, the molar mass of PBLG versus time was further
·
monitored by GPC measurements. As a result, it was found that Mn of the obtained PBLG increased
linearly with the reaction time (Figure 1C,D), and the low Ð was maintained in the range of 1.16–1.18.
This indicated that the a combination of Zn(OAc)2
over BLG-NCA polymerization under mild conditions in relatively short polymerization time.
·
2H2O and aniline exhibited an excellent control

Molecules 2018, 23, 760
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Figure 1.
molar ratio of Zn(OAc)2
of -benzyl-L-glutamate-N-carboxyanhydride (BLG-NCA) initiated by Zn(OAc)2
system with varying molar ratio of Zn(OAc)2 2H2O equal. to aniline; ( ) GPC profiles of PBLG
depending on the different monomer conversion.
(
A
) GPC curves of poly(
γ
-benzyl-L-glutamates) (PBLG) synthesized with varying the
) Kinetics of the ring-opening polymerization (ROP)
2H2O and aniline
·
2H2O and aniline. (
B
γ
·
·
C
(D
) Mn and Ð vs. BLG-NCA conversion.
◦
[BLG-NCA]/[Zn(OAc)2·2H2O]/[aniline] = 50/1/1, [BLG-NCA] = 0.75 M, at 25 C in DCM.
The structure of the obtained PBLG was carefully analyzed by 1H-NMR (Figure S3). Because
benzyl and amide signals from the repeating units of PBLG appeared in the range of 7.20 and 8.50 ppm,
it was difficult to explicitly discriminate the presence of aniline at the chain end of the resultant PBLG.
Thus, the polymer afforded at the feeding ratio of Zn(OAc)2 2H2O, aniline and BLG-NCA as 1:1:25 was
·
further analyzed by MALDI-TOF mass spectrometry. As shown in Figure 2A, a series of peaks at 219n
+ 92 + 1 + 23 (n denotes the number of the repeating units) were assigned to be n(BLG-NCA–CO2) +
C6H5NH + H + Na+ (Figure 2A). Thus the major product of BLG-NCA polymerization was the desired
polypeptide bearing aniline via the amide bond to one chain end and new generated amine group
on the other chain end. Meanwhile, additional distributions of peaks of very low intensities, such as
6584.2 Da in Figure 2B, was determined to be PBLG terminated with a pyroglutamate unit, which was
resulted from intramolecular cyclization via attack of the amine end-group on the benzyl ester group
as reported [59].
We attempted to monitor the structural change of aniline and the polymerization process of
BLG-NCA in the presence of Zn(OAc)2 by NMR, but failed, probably due to the poor solubility of
Zn(OAc)2
the results suggest that Zn(OAc)2
of BLG-NCA substantially which indicated the presence of an interaction between Zn(OAc)2
and aniline at the initiation stage. Moreover, combined with the relatively short polymerization time,
it can be speculated that Zn(OAc)2 2H2O also plays a vital role to activate the BLG-NCA monomer,
promoting the overall polymerization rate. These two words were separated with space.
·
2H2O in DCM. Although the exact polymerization mechanism remains unclear at present,
2H2O and aniline acted cooperatively to better control the ROP
2H2O
·
·
·

Molecules 2018, 23, 760
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Figure 2. MALDI-TOF mass spectrum (A) and its enlarged region (B) of the poly(γ-benzyl-L-
glutamates) (PBLG) accompanied by the proposed PBLG structures and their measured masses ionized
with sodium or potassium ion.
2.2. Available Scope of Aniline Analogues
Aiming at extending the scope of such ROP dual catalysis, our efforts were subsequently devoted
to studying the tolerance of Zn(OAc)2
or withdrawing substitutes (Scheme 2). All these polymerizations proceeded smoothly using
combinations of Zn(OAc)2 2H2O and the corresponding amines, and exhibited good control over
the polymerization of BLG-NCA (Table 3). Remarkably, Zn(OAc)2 2H2O and sterically congested
·
2H2O with aniline derivatives bearing either electron-donating
·
·
anilines, such as Ana-1, were tolerated well in the polymerization, resulting in a narrow dispersity
of 1.13 (run 1in Table 3). When Ana-2 and Ana-3 bearing electron-donating substituents were used
in the presence of Zn(OAc)2
comparison with the use of only the corresponding aniline derivatives. The aniline analogues 4~6
with electron-withdrawing groups were also suitable to combine with Zn(OAc)2 2H2O to efficiently
·
2H2O, PBLG with lower Ð was afforded (run 2 and 3 in Table 3) in
·
control the ROP of BLG-NCA with a dramatic decrease in Ð from 1.63 to 1.29 (run 6 in Table S2 vs.
run 6 in Table 3). In addition, the fluorescent 1-aminopyrene (Ana-7) was selected to cooperate with
Zn(OAc)2
(in the absence of Zn(OAc)2
fluorescence peaks of the resultant PBLG were clearly detected at 385 nm and 405 nm and attributed to
the characteristic signal of pyrene ring (Figure S13), indicating the aminopyrene group on the PBLG
·
2H2O for BLG-NCA polymerization, and the obtained Ð was clearly decreased from 1.38
2H2O, run 7 in Table S1 and Figure S10) to 1.18 (Table 3, run 7). Moreover,
·
structure. Also, Zn(OAc)2 2H2O showed good tolerance for alkynyl-functionalized anilines (Ana-8) to
·
promote the ROP of BLG-NCA with narrow dispersity (Ð = 1.26) (Table 3, run 8). Such versatile carbon
terminus of the resulting PBLG provided a great opportunity to link with other functional molecules
or block polymers through alkyne-thiol reaction or alkyne-azide click-chemistry.
Scheme 2. Chemical structures of aniline analogues investigated in this study.

Molecules 2018, 23, 760
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Table 3. Polymerization results of γ-benzyl-L-glutamate-N-carboxyanhydride (BLG-NCA) catalyzed
by a combination of Zn(OAc)2·2H2O and various aniline analogues in DCM a.
−4 c
−4 d
Mn,exp × 10
Mn,mea × 10
Time (h) b
Ð d
Run
Analogues
(g/mol)
(g/mol)
1
2
3
4
5
Ana-1
Ana-2
Ana-3
Ana-4
Ana-5
Ana-6
Ana-7
Ana-8
Ana-9
Ana-10
2.5
2.5
2.5
2.5
2.0
6.5
3.5
2.5
2.5
1.0
1.10
1.10
1.10
1.11
1.10
1.11
0.57
1.10
0.57
1.11
1.01
0.87
0.95
0.97
1.18
1.62
1.14
0.97
0.56
2.80
1.13
1.10
1.11
1.31
1.21
1.29
1.18
1.26
1.23
1.13
6
7 e
8
9 e
10
a
Performed by molar ratio of Zn(OAc)2 2H2O, aniline analogues and BLG-NCA with 1:1:50 except run 7 and
·
run 9 at 25 ◦C. b The polymerization time for 99%monomer conversion. Calculated by ([aniline analogues]-1)
+ [BLG-NCA]/[aniline analogues]
molecular weight distribution. [Zn(OAc)2·2H2O]/[aniline analogues]/[M] = 1/1/25.
c
d
×
(MNCA-44)
×
monomer conversion. Determined byGPC, Ð represents
e
2.3. Synthesis of Functional PBLG with Well-Defined Structure
Based on the preparation of aniline derivative Ana-9 containing 2-bromoisobutyryl group (Figure S14),
the combination of Zn(OAc)2 2H2O and Ana-9 initiated the polymerization of BLG-NCA and exhibited
·
satisfactory control over this polymerization, with a Mn of 5600 and low dispersity (Ð = 1.23) (run 9 in
Table 3). Subsequently, such 2-bromoisobutyryl-capped PBLG as macroinitiator further initiated MEA
monomer to polymerize via atom transfer radical polymerization (ATRP, Figure 3A). The strong proton
signals at 3.34 and 3.57 ppm (denoted as 18 and 19 in Figure 3B) associated with methyl and methylene
around the ether group of the side chain indicated the presence of PMEA blocks in the final polymer. GPC
measurements exhibited Mn of 13,500 with unimodal and relatively narrow distribution (Ð = 1.30) without
macroinitiator (Figure 3C), confirming the successful preparation of the block copolymer.
Figure 3.
PBLG-b-PMEA; (C) GPC curves of PBLG-b-PMEA.
(A) Synthesis route of block polymer PBLG-b-PMEA; (B)
1H-NMR spectrum of

Molecules 2018, 23, 760
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In fact, some drug molecules bearing amine groups could efficiently control the polymerization of
BLG-NCA in the presence of Zn(OAc)2 2H2O, as well. For example, amantadine (Aman, Ana-10 in
·
Scheme 2), an anti-influenza virus drug, was itself capable of initiating the polymerization of BLG-NCA,
but an ill-defined polymer was obtained with broad dispersity (run 10 in Table S1 and Figure S15).
Whereas, the polymerization was well controlled in the presence of Aman and Zn(OAc)2 2H2O, and the
·
obtained Ð dramatically dropped from 1.48 to 1.13 (Table 3, run 10). The Aman-capped PBLG dissolved
in TFA was further reacted with HBr (33 wt % in acetic acid) for 30 min to remove the benzyl protection
groups (Figure 4A).The chemical structure of the resultant copolymer was confirmed by 1H-NMR
measurements. As shown in Figure 4B, comparing the integral between the benzyl protons (denoted
as 6,
δ δ = 3.98 ppm),
= 5.06 ppm) and the methine proton of the backbone (denoted as 2 and 20,
the afforded amphiphilic polymer Aman-capped PBLG-co-PLG had 50% remaining benzyl groups.
In an aqueous environment, such a copolymer could self-assemble via hydrophobic forces to form
Aman-containing nanocarriers (Figure 4C). The apparent average size was about 110 nm measured
by DLS (inset in Figure 4C). Besides, when Aman-capped PBLG was incubated in the mixture of
HBr and TFA for longer times, water-soluble Aman-conjugated PLG was successfully fabricated via
the complete removal of the benzyl groups from the PBLG structure (Figure S16). This provided
an efficient way to prepare water-soluble drug-conjugates with well-defined structure and enhance the
solubility of a hydrophobic drug in a physiological environment.
Figure 4.
spectrum of Aman-capped PBLG-co-PLG; (
Aman-capped PBLG-co-PLG with 50% benzyl groups content. Its DLS diagram is in the inset.
(A
) Synthesis of Aman-conjugated poly(
γ-benzyl-L-glutamates) (PBLG); (B)
1H-NMR
C
) TEM image of the self-assembled morphology from

Molecules 2018, 23, 760
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3. Materials and Methods
3.1. Materials
Anhydrous DMC, DMF and THF purchased from J&K Scientific Ltd. (Beijing, China) were directly
used without treatment. All aromatic amines except aniline were also purchased from J&K. Aniline
was purchased from Sinopharm Chemical (Beijing, China). All liquid amines were dried with KOH
for overnight, followedby distillation in vacuum at least three times. p-Anisidine and p-toluidine
were recrystallized at least three times. H-Glu(OBn)-OH was purchased from GL Biochem Ltd.
(Shanghai, China) and used as received. Amantadine (Aman) ordered from Alfa Aesar (Beijing, China)
was used without further treatment. 4-Alkyl aniline purchased from J&K was recrystallized twice
from dichloromethane. BLG-NCA was synthesized according to the reported procedure [60], followed
by recrystallization four times prior to use. Zinc chloride (ZnCl2), zinc trifluoromethanesulfonate
(Zn(OTf)2), zinc acetate and zinc acetate dihydrate (Zn(OAc)2 2H2O) purchased from Alfa Aesar were
·
used without treatment. Trifluoroacetic acid (TFA) and HBr (33wt% in acetic acid) were ordered from
J&K.2-Bromo-2-methylpropionic acid 4-ester aniline was prepared as report [61]. 2-Methoxyethyl
acrylate (MEA) from Aldrich (Beijing, China) was distilled under vacuum and kept below 5 ◦C prior
to use.
3.2. Characterization
The chemical structures of the monomer were characterized by 1H-NMR carried out on a 400 MHz
NMR instrument (Bruker Corporation, Rheinstetten, Germany) at room temperature using CDCl3
as solvent. The chemical shifts were measured against the solvent signal of CDCl3 (δ = 7.26 ppm)
as internal standard, respectively. GPC measurements of the synthetic polypeptides were carried
out on an Agilent LC 1260 instrument (Agilent Technologies Inc., Calfornia, USA) equipped with
a differential refractive-index detector. Aguard column and two 7.5
columnswere used. The measurements were performed using THF (containing 0.02 M LiBr) as
eluent (flow rate of 1.0 mL/min at 35 ◦C), and polystyrene standards were employed for calibration.
×
300 mm PLgel MIXED-C
Polymer solutions at the concentration of 4.0 mg/mL were injected at an injection volume of 80 µL.
In situ IR study of NCA polymerization was carried out by using a ReactIRTM 15 equipped with a
MCT Detector from Mettler Toledo AutoChem (Zurich, Swiss). The DiComp (diamond) probe was
connected via AgX 6 mm
×
2 m fiber (silver halide). Spectra were taken from 2000 to 650 cm−1 at
4 wavenumber resolution and the automatic sampling interval was 0.5 min. The calibrated curves
for calculating the conversion of BLG-NCA during polymerization were plotted by the intensity
ratio of the peaks at 1785 cm−1 (anhydride of monomer) to that at 1731 cm−1 (benzyl carbonyl
group), based on preparing the mixture solution of monomer BLG-NCA and polymer PBLG with the
different molar ratio. In the process of polymerization, the polymerization solution was taken out
from the system, and spotted on the KBr plate for scanning by FT-IR (Nicolet Inc., Madisen, WI, USA).
The accumulation rate was 16 times with 4 wavenumber resolution). The obtained intensity ratio at
1785 and 1731 cm−1 was compared with the calibrated curves (Figure S1 in Supplementary Materials)
to afford the monomer conversion. Transmission electron microscopy (TEM) was performed on a
HT7700 instrument (HitachiHigh-Technologies Corporation, Tokyo Japan) with 100 KV. Dynamic laser
scattering (DLS) were measured at 660 nm by using a laser light scattering spectrometer (ZEN3600,
Malvern Inc., Malvern, Britain) equipped with Zetasizer software. Desorption/ionization-time of flight
mass spectrometer (MALDI-TOF MS) experiments were carried out on a Bruker Autoflex III (laser
frequency 100 Hz, 355 nm and detector voltage of 1689 V). A Hitachi F-4600 Fluorescence instrument
was used to measure the steady-state fluorescence emission of pyrene-capped PBLG.
3.3. Typical Polymerization Procedure
All polymerizations were carried out in a Vigor glovebox with a nitrogen atmosphere. The given
amount of Lewis acid and aniline was stirred in 1.0 mL anhydrous CH2Cl2 for 10 min, followed by

Molecules 2018, 23, 760
10 of 13
adding 197.4 mg BLG-NCA (0.75 mmol). The mixture was kept stirring at 25 ◦C, and a trace amount
of the reaction mixture was taken out from the system at a predetermined time for monitoring the
monomer conversion by IR spectroscopy. The final reaction solution was precipitated in anhydrous
methanol, and washed with anhydrous methanol twice. The obtained polymers were dried in vacuum.
3.4. Synthesis of Diblock Polymer PBLG-Block-PMEA
A Schlenk flask equipped with a magnetic stir bar was charged with MEA monomer (195.2 mg,
1.5 mmol), PBLG macroinitiator (109 mg, 0.02 mmol), PMDETA (4.2 mg, 0.024 mmol), CuBr (2.9 mg,
0.02 mmol) and 1.0 mL dry and degassed dioxane. The flask was further degassed through three
freeze-pump-thaw cycles. After the reaction system was immersed in an oil bath at 80 ◦C for 24 h,
the polymerization solution was diluted with CHCl3, then purified through a neutral alumina column
to remove copper. The concentrated reaction solution was dropwise added into cold diethyl ether,
and the obtained polymer was dried in vacuum for 12 h.
3.5. Preparation of Aman-Conjugated Poly(
PBLG-co-PLG)
γ-benzyl-L-glutamate)-co-poly(L-glutamic acid) (Aman-Conjugated
In a round bottom flask with a glass stopper and a stirrer bar, Aman-conjugated PBLG (200 mg)
dissolved in 1.0 mL TFA was mixed with 2 equiv. of HBr (33 wt % in acetic acid) relative to per carbonyl
group for 30 min. Then, the solution was poured into a large excess of cold diethyl ether, and a white
1
precipitate was afforded in a yield of75%. H-NMR (DMSO-d6,
δ
ppm): 7.61–8.80 (NHCO), 7.01–7.42
(Ph-H), 4.80–5.20 (Ph-CH2), 3.60–4.42 (OCCHNH), 1.80–2.90 (Aman and CH2). Aman-conjugated
poly(L-glutamic acid) (Aman-conjugated PLG) was obtained through a similar procedure except that
1
the reaction time was extended to 6 h. H-NMR (DMSO-d6,
(OCCHNH), 1.80–2.90 (Aman and CH2).
δ ppm): 7.61–8.80 (NHCO), 3.60–4.42
3.6. Preparation of Nanoparticles from Amphiphilic Copolymer Aman-Conjugated PBLG-co-PLG
The preparation of nanoparticles was carried out by slowly adding 0.5 mL DMF containing 5.0 mg
Aman-conjugated PBLG-co-PLG into 10.0 mL ultrapure water. The nanoparticles solution was dialyzed
against ultrapure water in a dialysis bag with a molecular weight cutoff of 3500 for 48 h, during which
dialysis water was refreshed every 6 h. The concentration of the resulting nanoparticles was fixed at
0.5 mg/mL.
4. Conclusions
In summary, a simple operation and efficient approach for the preparation of well-defined
synthetic polypeptides was developed using a combination of Zn(OAc)2
·
2H2O and anilines.
Significantly, the tolerance of Zn(OAc)2 2H2O to a wide range of amine initiators paved the way
·
for the convenient synthesis of highly desirable polypeptides with important functional groups,
which would meet the requirements of synthetic polypeptides as biomaterials. Future work in our
laboratory will seek to study the polymerization mechanism. The properties of the ensuing copolymers
and drug-conjugated polymers will also be investigated to gain better knowledge on the influence of
their functionality.
Supplementary Materials: The following are available online, Figure S1: Calibrated curve of BLG-NCA
−1
conversion vs the peak intensity ratio at 1785 cm and 1731 cm−1; Figure S2. GPC curves of PBLG prepared in
1
the sequential addition. (A) 25/25; (B) 50/50.; Figure S3. H-NMR spectrum of PBLG catalyzed by Lewis pair
of Zn(OAc)2 2H2O and aniline; GPC profiles of PBLG initiate by Ana-1 (Figure S4), Ana-2 (Figure S5), Ana-3
·
(Figure S6), Ana-4 (Figure S7), Ana-5 (Figure S8), Ana-6 (Figure S9), Ana-7 (Figure S10), Ana-8 (Figure S11) and
Ana-9 (Figure S12) with or without Zn(OAc)2 2H2O; Figure S13. Fluorescent spectrum of PBLG initiated by a
combination of Zn(OAc)2
2H2O with 1-aminopyrene; Figure S14. 1H-NMR (A) and 13C NMR (B) spectra of
2-bromo-2-methylpropionic acid 4-nitrophenyl ester; Figure S15. GPC profiles of PBLG initiate by Ana-10 with or
without Zn(OAc)2 2H2O; Figure S16. 1H-NMR spectrum of Aman-capped PLG; Table S1. Polymerization results
·
·
·

Molecules 2018, 23, 760
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of BLG-NCA catalyzed by various aniline analogues without Zn(OAc)2
route of Ana-9.
·
2H2O in CH2Cl2; Scheme S1. Synthesis
Acknowledgments: This work was supported by National Natural Science Foundation of China (NSFC, Grant
No. 21374005, U1663227), Beijing National Laboratory for Molecular Sciences (BNLMS20150127), the Fundamental
Research Funds for the Central Universities (PYBZ1702).
Author Contributions: Haifeng Du and Jing Yang conceived and designed the experiments and wrote
the paper; Yanzhao Nie performed the experiments and analyzed the data; Xinmei Zhi contributed
reagents/materials/analysis tools
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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