Chemosynthesis of poly(ε-lysine)-Analogous polymers by microwave-assisted click polymerization

Article abstract of DOI:10.1021/bm1013662

Poly(ε-lysine) (ε-PL)-analogous click polypeptides with not only similar R-amino side groups but also similar main chain to ε-PL were chemically synthesized for the first time through click polymerization from aspartic (or glutamic)-acidbased dialkyne and

Full text of DOI:10.1021/bm1013662

ARTICLE
pubs.acs.org/Biomac
Chemosynthesis of Poly(ε-lysine)-Analogous Polymers
by Microwave-Assisted Click Polymerization
Jinshan Guo,† Ying Wei,† Dongfang Zhou,† Pingqiang Cai,‡ Xiabin Jing,† Xue-Si Chen,† and Yubin Huang*,†
†State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,
Graduate School of the Chinese Academy of Sciences, Renmin Str. 5625, Changchun 130022, P. R. China
‡Department of Materials Science and Engineering, Jilin University, Changchun 130022, P. R. China
S Supporting Information
b
ABSTRACT: Poly(ε-lysine) (ε-PL)-analogous click polypepti-
des with not only similar R-amino side groups but also similar
main chain to ε-PL were chemically synthesized for the first time
through click polymerization from aspartic (or glutamic)-acid-
based dialkyne and diazide monomers. With microwave-assist-
ing, the reaction time of click polymerization was compressed
into 30 min. The polymers were fully characterized by NMR,
ATR-FTIR, and SEC-MALLS analysis. The deprotected click
polypeptides had similar pKa value (7.5) and relatively low
cytotoxicity as ε-PL and could be used as substitutes of ε-PL in
biomedical applications, especially in endotoxin selective re-
moval. Poly(ethylene glycol) (PEG)-containing alternating co-
polymers with R-amino groups were also synthesized and characterized. After deprotection, the polymers could be used as
functional gene vector with PEG shadowing system and NCA initiator to get amphiphilic graft polymers.
’ INTRODUCTION
media. Moreover, the triazole rings resulted by CuAAC could
perfectly imitate the functions of amide bonds even including
spatial structures for the similar atomic spacing and dipole
between the two.23,24 This interesting discovery makes it possible
for chemical synthesis of R-amino side groups containing poly-
mer to imitate ε-PL more similarly through click chemistry.
Until now, there were many attainments on click polymerization
to get linear copolymers with triazole units in the main chain,25
regardless of A-B strategies26-32 or A-A and B-B strategies.15-17
Also, there were some reports concerning click chemistry-synthe-
sized polypseudopeptides with amino side groups. However, the
amino side groups were mostly ε-amino groups;29,32 although the
starting monomers were based on natural amino acid, they needed
to be functionalized by unnatural amino acids, such as N3-Phe27,28
and azido-(L)-Lys (Boc),31,32 which were difficult to synthesize, or
the main chains contained ester bonds.36 Moreover, conventional
click polymerizations were time-consuming.26-30,33-39 Recently,
microwave-assisted click chemistry emerged as the time-saving
requirement, which could greatly shorten the reaction time of click
polymerization.31,32
As a kind of unusual cationic, naturally occurring homopoly-
(amino acid), biosynthesized poly(ε-lysine) (ε-PL) that bears
R-amino groups on the side chains is composed of a linkage
between the ε-amino groups and carboxyl groups of L-lysine.1
Compared with chemically synthesized poly(L-lysine) (PLL,
composed of linkage between the R-amino groups and carboxyl
groups of L-lysine) (pKa = 9 to 10), ε-PL is edible and nontoxic
with lower pKa (7.6).1 The existence of R-amino groups in ε-PL
seems play the important role of sterilization, antisepsis, gene
transfection, and endotoxin removal applications. However, for
the difficulties of getting large cyclic monomers (nine-member
N-carboxyl-ε-amino acid anhydride (ε-NCA) or oenantho-
lactam) of L-lysine as well as the poor solubility of R-amino
protecting L-lysine in organic solvents, the chemosynthesis of
ε-PL has not been reported. Although polymers baring R-amino
side groups were already synthesized from aspartic (or glutamic)
acid anhydride and diol through the prepolymerization and
subsequent polycondensation,2-4 the synthesis of anhydride
and polycondensation were both water-removal procedures that
are difficult to control. The so-formed ester bonds were greatly
different from the amide bonds in ε-PL and liable to degrade.
Recently, click chemistry,5 especially copper(I)-catalyzed 1,3-
dipolar azide-alkyne cycloaddition (CuAAC)6-22 attracted our
attention. This kind of reaction is tolerable to water, oxygen, and
a wide range of functionalities and proceeded with almost
quantitative yields under mild conditions in different reaction
In this work, we first report the design and synthesis of a series
of ε-PL-analogous polypeptides and PEG-containing alternating
copolymers with R-amino groups on the side chains by the
Received: November 16, 2010
Revised:
January 14, 2011
Published: February 08, 2011
r
2011 American Chemical Society
737
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|

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Scheme 1. Click Polymerization of Diazide and Dialkyne Monomers 2-5, 6a, and 6b
combination of the A-A and B-B strategy click polymerization
Technology. Hemoglobin (bovine erythrocytes, BR) was imported and
packaged by Shanghai Kayon Biological Technology. Propargylamine
and propargyl bromide were from Tianzunzezhong chemical limited
corporation (Nanjing, China). BOP reagent and trifluoroacetic acid
(CF3COOH) were from GL Biochem (Shanghai, China). Hydrobromic
acid in glacial acetic acid (HBr/AcOH, 33%, w/v) was purchased from
Alfa Aesar and used without further purification. Copper(I) bromide was
washed with glacial acetic acid, followed by washing with methanol and
diethyl ether. After drying under vacuum, it was kept under nitrogen
atmosphere before use. Tetrahydrofuran (THF) was dried by refluxing
with sodium and then distilled after being immersed with CaH2 for more
than 2 weeks. Triethylamine (TEA) was dried by refluxing with CaH2
and then distilled. All other reagents were commercially available and
used without further purification.
with microwave-assisting technique (Scheme 1).
’ EXPERIMENTAL SECTION
General. N-Benzyloxycarbonylaspartic acid (Z-Asp) and N-benzy-
loxycarbonylglutamic acid (Z-Glu) were synthesized according to
traditional peptide synthesis. 3-Azidopropanamine was synthesized by
adapting synthetic procedures described previously.11,40 Poly(ethylene
glycol) (PEG) with an average Mn of 950-1050 (1a) and 1900-2200
(1b), respectively, was purchased from Sigma-Aldrich. ε-Polylysine (ε-
PL) was provided by Zhejiang Silver-Elephant bioengineering. Standard
endotoxins (ETs) were purchased from National Institute for the
Control of Pharmaceutical and Biological Products (Beijing, China).
Tachypleus (or Limulus) amebocyte lysate (TAL or LAL) (with a
detection limit of 0.03 EU/mL) and TAL (LAL) reagent water
(endotoxin concentration <0.005 EU/mL) were from Zhanjiang Bo-
kang Ocean Creature Company. Bovine serum albumin fraction V (BSA,
99%) was from Sigma-Aldrich. γ-Globulins (from bovine blood, 99%)
and cytochrome c (from bovine heart, mol wt: 12 327 Da, 95%) were
imported from Sigma and packaged by Beijing Solarbio Science &
The microwave reactions were carried out in an MCR-3 microwave
reactor (Yuhua Instruments, Zhengzhou, China). The pocket-sized pH
meter used in pKa determination was from Hanna instruments company
1
(Italy). H NMR and 13C NMR spectra were recorded on a Bruker
AVANCE DRX 300 or 400 spectrometer in CDCl3, D2O, or DMSO-d6.
The MALDI-TOF/TOF mass spectra were recorded on an autoflexIII
smart beam mass-spectrometer of Bruker Daltonics. Differential scanning
738
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calorimetry (DSC) and thermal gravimetric analysis (TGA) were
taken by Perkin-Elmer at a heating rate of 20 °C/min under a nitrogen
atmosphere. We performed size exclusion chromatography-multiangle
laser light scattering (SEC-MALLS) analysis by combining a Water-515
GPC equipped with Waters Styragel HMW6E column (eluent: 10 mM
LiBr/DMF, flow rate: 1.0 mL/min, 40 °C) and a DAWN EOS MALS
detector (Wyatt Technology, laser wavelength: 690.0 nm). Attenuated
total reflection Fourier transform infrared (ATR-FTIR) spectra were
measured with a Bruker Vertex 70 spectrometer. Concentration of
endotoxin was assayed by a Limulus test involving turbidimetric time
assay at 450 nm with Toxinometer BET-16 (Tianda Tianfa, Tianjin,
China) at 37 °C. Protein concentration was determined by absorbencies
at 280 nm (proteins other than cytochrome c) or 410 (cytochrome c) or
406 nm (hemoglobin) using a UV-2450 spectrometer (Shimadzu,
Japan) with minimum wavelength resolution of 0.2 nm.
Synthesis of Dialkyne and Diazide Monomers. Synthesis of
N-Benzyloxycarbonylaspartic Dipropargyldiamide (Dipropargyl Z-Asp)
(2). Z-Asp (1.33 g, 5 mmol) and dried triethylamine (TEA) (4 mL, 28
mmol) were dissolved in 30 mL of dried THF under a nitrogen atmosphere
with stirring. After the addition of propargylamine (1.4 mL, 20 mmol) and
BOP reagent (5.30 g, 12 mmol), the reaction mixture was kept stirring
under nitrogen atmosphere for 24 h. The solvent was removed under
reduced pressure, and the residual was dissolved in DMF. The DMF
solution was poured in 1 M KHSO4 aqueous solution. The crude product
was filtered and washed completely with water, 5% NaHCO3 aqueous
solution, and water and finally dried under vacuum at 40 °Cfor24htogivea
white solid (1.55 g, yield 91%). 1H NMR (Figure S1 in Supporting
Information) (300 MHz; DMSO-d6; δ): 2.4 (2H, m, CHCH2CO), 3.08
(2H, t, CtCH), 3.83 (4H, br, CH2CtCH), 4.37 (1H, t, CHCO), 5.0(2H,
s, CH2Ph), 7.35 (5H, br, Ph), 7.42, 8.28, 8.39 (3H, m, 3 CONH). 13C
NMR (Figure S2 in the Supporting Information) (101 MHz; DMSO-d6;
δ): 28.0 (CH2CtCH), 37.2 (CHCH2CO), 51.4 (CHCO), 65.5
(CH2Ph), 73.0 (CtCH), 80.7 (CtCH), 127.7-128.3 and 136.9 (Ph),
155.7 (NHCOO), 168.9 (NHCOCH), 170.9 (NHCOCH2). MALDI-
TOF/TOF, C18H19N3O4 [MþNa]þ calcd: 364.1, observed: 364.1;
[MþK]þ calcd: 380.2, observed: 380.1.
TOF, C18H25N9O4 [MþNa]þ calcd: 454.2, observed: 154.2; [MþK]þ
calcd: 470.3, observed: 470.1.
Synthesis of N-Benzyloxycarbonylglutamic Di(3-azidopropyl)diamide
(Diazido Z-Glu) (5). Monomer 5 was obtained using Z-Glu and 3-azido-
propanamine according to the same procedure for the synthesis of
compound 2 with a yield of 91%; here 3-azidopropanamine was also
1
three-folds excess. H NMR (Figure S1 in the Supporting Information)
(300 MHz; DMSO-d6; δ): 1.61-1.86 (4Hþ2H, m, CH2CH2N3 and
CHCH2CH2CO), 2.09 (2H, t, CHCH2CH2CO), 3.09 (4H, m, CH2N3),
3.32 (4H, t, CH2CH2CH2N3), 3.90 (1H, t, CHCO), 5.01 (2H, s, CH2Ph),
7.35 (5H, br, Ph), 7.40, 7.83-7.86, 7.93-7.96 (3H, m, 3 CONH). 13C
NMR (Figure S2 in the Supporting Information) (101 MHz; DMSO-d6;
δ): 27.9 (CH2CH2CO), 28.4 (CH2CH2N3), 31.8 (CH2CO), 35.8
(CH2NH), 48.4 (CH2N3), 54.6 (CHCO), 65.4 (CH2Ph), 127.7-128.3,
137.0 (Ph), 155.9 (NHCOO), 171.5 (NHCOCH and NHCOCH2).
MALDI-TOF/TOF, C19H27N9O4 [MþNa]þ calcd: 468.2, observed:
468.2; [MþK]þ calcd: 484.3, observed: 484.2.
Synthesis of Dialkyne-Terminated PEG (6a, 6b). Dialkyne-termi-
nated PEGs (6a, 6b) were synthesized by adapting the synthetic
procedure previously described.41 In general, 1 equiv of PEG (1a, 1b)
was reacted with the excess amount of propargyl bromide (40 equiv) and
NaOH powder (40 equiv) in toluene for 15 h at 50 °C. The solvent was
removed under vacuum, and the residual was dissolved in water. The
solution was extracted with dichloromethane twice. After being dried
with MgSO4, the final product was obtained by precipitation in diethyl
ether with a yield of 84%. NMR date for 6b (Figure S3 in the Supporting
Information): 1H NMR (400 MHz; CDCl3; δ): 2.46 (t, CtCH), 3.65-
3.72 (br, CH2CH2O), 4.20 (d, CH2CtCH). 13C NMR (101 MHz;
CDCl3; δ): 58.0 (CH2CtCH), 70.2 (CH2CH2O), 74.4 (CtCH), 77.9
(CtCH). The average molecular weights of 6a and 6b were calculated
from 1H NMR as 1025 and 2152 g/mol, respectively.
Microwave-Assisted Click Polymerizations. Synthesis of
Click Polypeptide 7. The synthesis of click polypeptide was carried
out in a microwave reactor using CuBr as catalyst according to
literature.30-32,38,39 In general, a solution of dipropargyl Z-Asp-
(compound 2, 170.5 mg, 0.5 mmol), diazido Z-Asp(compound 4,
215.5 mg, 0.5 mmol), and CuBr (7 mg, 0.05 equiv) in N2-purged
DMF (1 mL) was placed in the microwave reactor and was irradiated at
100 °C for 30 min under a nitrogen atmosphere. The clear solution was
turned into a turbid gel. The gel was dissolved in 1 mL of DMF, and the
solution was poured in 0.1 M HCl (50 mL). The precipitant was
centrifuged, and washed with 0.1 M HCl and water. After lyophilization,
the membrane-like white product 7 was obtained with a yield of 90%
(347 mg). 1H NMR (Figure 1) (300 MHz; DMSO-d6; δ): 1.87-1.89
(4H, br, 2 CH2CH2NH), 2.45 (4H, m, CHCH2CO), 3.03 (4H, br,
triazole-CH2CH2CH2NH), 3.95 (2H, br, CHCO), 4.30 (8H, br,
triazole-CH2 (CH2)2 NH and triazole-CH2NH), 5.00 (4H, s, CH2Ph),
7.31-7.33 (10H, br, Ph), 7.45-7.47, 7.92, 8.06 (6H, m, CONH), 8.34,
8.43 (2H, d, the H in the two triazole). 13C NMR (101 MHz; DMSO-d6;
δ): 29.8 (triazole-CH2CH2CH2NH), 35.8 (triazole-(CH2)2CH2NH),
37.5, 37.8 (CHCH2CO), 47.0 (triazole-CH2NH and triazole-CH2
(CH2)2 NH), 51.8, 52.0 (CHCO), 65.5 (CH2Ph), 122.9 (CdCH on
triazole), 127.7, 127.8, 128.3, and 136.9 (Ph), 144.7 (CdCH on
triazole), 155.7 (NHCOO), 169.2, 169.4, 171.1, and 171.2 (CONH
on the main chain). ATR-FTIR (cm-1): 3288 (CONH), 3064
(triazole), 1711 (OCONH of Z groups), 1657 (CONH in the main
chain), 740, 698 (Ph).
Synthesis of N-Benzyloxycarbonylglutamic Dipropargyldiamide
(Dipropargyl Z-Glu) (3). Monomer 3 was obtained using Z-Glu and
propargylamine according to the same procedure as that for the
synthesis of compound 2 with a yield of 90%. 1H NMR (Figure S1 in
the Supporting Information) (300 MHz; DMSO-d6; δ): 1.68-1.84
(2H, d, CHCH2CH2CO), 2.09 (2H, t, CHCH2CH2CO), 3.00-3.07
(2H, m, CtCH), 3.80 (4H, m, CH2CtCH), 3.93 (1H, t, CHCO), 4.97
(2H, s, CH2Ph), 7.32 (5H, br, Ph), 7.38-7.41, 8.22, 8.33 (3H, m, 3
CONH). 13C NMR (Figure S2 in the Supporting Information) (101
MHz; DMSO-d6; δ): 27.7 (CH2CH2CO), 28.0 (CH2CtCH), 31.6
(CH2CO), 54.3 (CUHCO), 65.5 (CH2Ph), 72.9 (CtCH), 81.1
(CtCH), 127.7-128.4, 137.0 (Ph), 156.0 (NHCOO), 171.3, 171.4
(NHCOCH and NHCOCH2). MALDI-TOF/TOF, C19H21N3O4
[MþNa]þ calcd: 378.2, observed: 378.1; [MþK]þ calcd: 394.2,
observed: 394.1.
Synthesis of N-Benzyloxycarbonylaspartic Di(3-azidoproyl)diamide
(Diazido Z-Asp) (4). Monomer 4 was obtained using Z-Asp and
3-azidopropanamine according to the same procedure as that for the
synthesis of compound 2 with a yield of 89%; here 3-azidopropanamine
was three-fold excess. 1H NMR (Figure S1 in the Supporting In-
formation) (300 MHz; DMSO-d6; δ): 1.60 (4H, m, CH2CH2N3),
2.33-2.44 (2H, m, CHCH2CO), 3.09 (4H, t, CH2N3), 3.32 (4H, t,
CH2CH2CH2N3), 4.30 (1H, t, CHCO), 5.01 (2H, s, CH2Ph), 7.33 (5H,
br, Ph), 7.40, 7.84-7.87, 7.92-7.96 (3H, m, 3 CONH). 13C NMR
(Figure S2 in the Supporting Information) (101 MHz; DMSO-d6; δ):
28.4 (CH2CH2N3), 36.0 (CH2NH), 37.9 (CH2CO), 48.3 (CH2N3),
52.0 (CHCO), 65.5 (CH2Ph), 127.1-128.3, 137.0 (Ph), 155.7
(NHCOO), 169.3 (NHCOCH), 171.1 (NHCOCH2). MALDI-TOF/
Synthesis of Click Polypeptide 8. Click polypeptide 8 was obtained
using dipropargyl Z-Asp (compound 2, 170.5 mg, 0.5 mmol) and
diazido Z-Glu (compound 5, 222.5 mg, 0.5 mmol) according to the
same procedure for the synthesis of polypeptide 7 with a yield of 89%
1
(350 mg). H NMR (300 MHz; DMSO-d6; δ): 1.70-1.90 (6H, br,
CH2CH2CH2NH and CH2CH2CO), 2.14 (2H, br, CH2CH2CO), 2.45
(2H, m, CHCH2CO), 3.04 (4H, br, triazole-(CH2)2 CH2NH), 3.93,
739
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127.7, 128.3, and 137.0 (Ph), 144.7 (CdCH on triazole), 155.9
(NHCOO), 17.4, 171.6 (CONH on the main chain).
Synthesis of PEG-Containing Alternating Copolymers. Copolymers
11a, 11b, 12a, and 12b were synthesized using the same procedure. Here
the synthesis of 11a was shown below as an example. Dialkyne-
terminated PEG 1000 (6a) (216 mg, more than 0.21 mmol), compound
4 (86.2 mg, 0.2 mmol), triethylamine (28 μL, 0.5 equiv), and CuBr
(3 mg, 0.05 equiv) were mixed in N2-purged DMF (2 mL) and settled in
the microwave reactor. The reaction mixture was irradiated at 100 °C for
30 min under a nitrogen atmosphere. The solution was poured in diethyl
ether (containing a little amount of triethylamine to remove copper
ions). The precipitant was centrifuged and washed with diethyl ether.
Copolymer 11a was finally obtained after being dried under vacuum.
Because the structures of 11b and 12b were similar to that of 11a and
12a, respectively, only the NMR data and spectra of 11a and 12a are
given below:
1
NMR date of 11a (Figure S4 in the Supporting Information): H
NMR (300 MHz; DMSO-d6; δ): 1.87-1.89 (4H, br,
2
CH2CH2CH2NH), 2.45 (2H, m, CHCH2CO), 300-3.02 (4H, br, 2
triazole-(CH2)2 CH2NH), 3.28-3.47 (85.0 H, br, CH2CH2O of PEG),
4.10 (0.69H, s, CH2 that attach to the alkyne terminal of PEG), 4.28
(5H, br, triazole-CH2 (CH2)2 NH and CHCO), 4.48 (4H, s, triazole-
CH2O), 4.98 (2H, s, CH2Ph), 7.28 (5H, br, Ph), 7.40, 7.90 (3H, m,
CONH), 8.02 (2H, br, the H in the two triazole). 13C NMR (101 MHz;
DMSO-d6; δ): 29.8 (triazole-CH2CH2CH2NH), 35.6, 35.8 (triazole-
(CH2)2 CH2NH), 37.7 (CHCH2CO), 46.9 (triazole-CH2 (CH2)2
NH), 51.9 (CHCO), 57.4 (CtCH of the alkyne terminal), 63.5
(triazole-CH2O), 65.5 (CH2Ph), 68.5, 68.9, 69.7 (CH2CH2O of PEG),
77.0 (CtCH of the alkyne terminal), 123.9 (CdCH on triazole), 127.6,
128.2 136.8 (Ph), 143.8 (CdCH on triazole), 155.6 (NHCOO), 1693,
171.1 (CONH on the main chain).
Figure 1. 1H NMR spectra of click polypeptide 7 (A) before and (B)
after deprotection.
4.28-4.40 (10H, d, triazole-CH2NH, COCHCH2CO, COCH (CH2)2
CO and triazole-CH2 (CH2)2NH), 5.01 (4H, s, CH2Ph), 7.34 (10H, br,
Ph), 7.43-7.45, 7.96, 8.06 (6H, m, CONH), 8.35, 8.44 (2H, d, the H in the
two triazole). 13C NMR (101 MHz; DMSO-d6; δ): 27.8 (CH2CH2CO),
29.8 (triazole-CH2CH2CH2NH), 31.7 (CH2CH2CO), 35.7 (triazole-
(CH2)2CH2NH), 37.7 (CHCH2CO), 47.0 (triazole-CH2NH and tria-
zole-CH2 (CH2)2 NH), 51.9 (CHCH2CO), 54.4 (CH (CH2)2CO), 65.5
(CH2Ph), 123.2 (CdCH on triazole), 127.0, 127.6, 128.2, and 136.9 (Ph),
144.9 (CdCH on triazole), 155.7, 155.9 (NHCOO), 169.4, 171.1, and
171.4 (CONH on the main chain).
1
NMR date of 12a (Figure S5 in the Supporting Information): H
NMR (300 MHz; DMSO-d6; δ): 1.69-1.90 (6H, br, CH2CH2CH2NH
and CH2CH2CO), 2.10 (2H, br, CH2CH2CO), 3.01 (4H, br,
triazole-(CH2)2 CH2NH), 3.29-3.50 (85.0 H, br, CH2CH2O of PEG),
3.90 (1H, br, CHCO), 4.10 (0.46H, d, CH2 that attach to the alkyne
terminal of PEG), 4.30 (4H, t, triazole-CH2 (CH2)2 NH), 4.47 (4H, s,
triazole-CH2O), 4.98 (2H, s, CH2Ph), 7.25-7.30 (5H, br, Ph), 7.39,
7.41, 7.88 (3H, m, CONH), 8.04 (2H, br, the H in the two triazole). 13C
NMR (101 MHz; DMSO-d6; δ): 27.7 (CH2CH2CO), 29.8 (triazole-
CH2CH2CH2NH), 31.7 (CH2CH2CO), 35.7 (triazole-(CH2)2 CH2-
NH), 46.9, 47.1 (triazole-CH2 (CH2)2 NH), 54.5 (CHCO), 63.5
(triazole-CH2O), 65.4 (CH2Ph), 68.5, 68.9, 69.7 (CH2CH2O of PEG),
123.9 (CdCH on triazole), 127.6, 128.3, 136.9 (Ph), 143.9 (CdCH on
triazole), 155.9 (NHCOO), 171.5 (CONH on the main chain).
Deprotection of Click Polypeptide. As an example, the click
polypeptide 7 (400 mg, containing 1 mmol Z groups) was dissolved in
CF3COOH (6 mL) with the addition of 33% HBr/AcOH (4 mL, about
10 equiv to Z groups). The mixture was stirred at 0 °C under a nitrogen
atmospheres for 2 h. After that, the solution was concentrated and
poured in diethyl ether. The precipitant was dissolved in water and
dialyzed against water using a dialysis tube with MW cutoff of 3500 Da
for 3 days and then freeze-dried. Deprotected click polypeptide 7
Synthesis of Click Polypeptide 9. Click polypeptide 9 was obtained
using dipropargyl Z-Glu (compound 3, 177.5 mg, 0.5 mmol) and diazido
Z-Asp (compound 4, 215.5 mg, 0.5 mmol), according to the same
procedure for the synthesis of polypeptide 7 with a yield of 90% (354
mg). 1H NMR (300 MHz; DMSO-d6; δ): 1.70-1.90 (6H, br,
CH2CH2CH2NH and CH2CH2CO), 2.16 (2H, br, CH2CH2CO),
2.40 (2H, m, CHCH2CO), 3.04 (4H, br, triazole-(CH2)2 CH2NH),
4.00, 4.30 (10H, d, triazole-CH2NH, COCH (CH2)2 CO, triazole-CH2
(CH2)2 NH and COCHCH2CO), 5.01 (4H, s, CH2Ph), 7.32-7.34
(10H, br, Ph), 7.45-7.47, 7.96, 8.07 (6H, m, CONH), 8.32, 8.43 (2H, d,
the H in the two triazole). 13C NMR (101 MHz; DMSO-d6; δ): 27.9
(CH2CH2CO), 29.9 (triazole-CH2CH2CH2NH), 31.9 (CH2CH2CO),
35.9 (triazole-(CH2)2CH2NH), 37.7 (CHCH2CO), 47.3 (triazole-
CH2NH and triazole-CH2 (CH2)2 NH), 51.9 (CHCH2CO), 54.7
(CH (CH2)2CO), 65.6 (CH2Ph), 123.2 (CdCH on triazole), 127.7,
128.4, and 137.0 (Ph), 145.2 (CdCH on triazole), 155.9, 156.0
(NHCOO), 169.4, 171.2, and 171.8 (CONH on the main chain).
Synthesis of Click Polypeptide 10. Click polypeptide 10 was ob-
tained using dipropargyl Z-Glu (compound 3, 177.5 mg, 0.5 mmol) and
diazido Z-Glu (compound 5, 222.5 mg, 0.5 mmol), according to the
same procedure for the synthesis of polypeptide 7 with a yield of 90%
1
(DCP7) was obtained with a yield of 85% (227 mg). H NMR (300
1
MHz; D2O; δ): 1.91-1.97 (4H, m, CH2CH2CH2NH), 2.70, 2.76 (4H,
d, CHCH2CO), 3.01-3.04 (4H, br, triazole-(CH2)2 CH2NH), 4.05,
4.13 (2H, d, COCH), 4.24-4.33 (8H, br, triazole-CH2NH and triazole-
CH2 (CH2)2 NH), 7.74 (2H, s, triazole). 13C NMR (Figure S6 in the
Supporting Information) (101 MHz; D2O; δ): 28.3 (triazole-
CH2CH2CH2NH), 34.0, 34.2 (CHCH2CO), 35.8, 36.0 (triazole-
(CH2)2CH2NH), 47.3, 47.4 (triazole-CH2NH and triazole-CH2
(CH2)2 NH), 49.9 (COCH), 123.4 (CdCH on triazole), 143.8
(CdCH on triazole), 170.0 (CONH on the main chain). ATR-FTIR
(cm-1): 3244 (NH2), 3071 (triazole), 1657 (CONH in the main chain).
(360 mg). H NMR (300 MHz; DMSO-d6; δ): 1.75-1.92 (8H, br,
CH2CH2CH2NH and CH2CH2CO), 2.14 (4H, br, CH2CH2CO), 3.03
(4H, br, triazole-(CH2)2 CH2NH), 3.91-4.00 (2H, m, COCH (CH2)2
CO), 4.30 (8H, br, triazole-CH2NH and triazole-CH2 (CH2)2 NH),
5.01 (4H, s, CH2Ph), 7.34 (10H, br, Ph), 7.44-7.46, 7.90-7.92, 8.05
(6H, m, CONH), 8.32, 8.43 (2H, d, the H in the two triazole). 13C NMR
(101 MHz; DMSO-d6; δ): 27.9 (CH2CH2CO), 29.9 (triazole-
CH2CH2CH2NH), 31.7 (CH2CH2CO), 35.8 (triazole-(CH2)2-
CH2NH), 47.0, 47.2 (triazole-CH2NH and triazole-CH2 (CH2)2
NH), 54.4, 54.6 (COCH), 65.5 (CH2Ph), 122.9 (CdCH on triazole),
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Synthesis of Thermal DCP7. To investigate the effect of Cu ions
residual, thermal DCP7 was synthesized through thermal click poly-
merization, followed by deprotection procedure. The synthesis of thermal
click polypeptide 7 was carried out in a microwave reactor but without
CuBr catalyst. In general, a solution of dipropargyl Z-Asp(compound 2,
170.5 mg, 0.5 mmol) and diazido Z-Asp(compound 4, 215.5 mg, 0.5
mmol) in N2-purged DMF (1 mL) was placed in the microwave reactor
and was irradiated at 120 °C for 75 min under a nitrogen atmosphere.
The postprocessing and following deprotection procedure were the
same as click polypeptide 7. 1H NMR (300 MHz; D2O; δ): 1.58-1.65
and 1.88-1.97 (4H, d, CH2CH2CH2NH), 2.70, 2.80 (4H, br,
CHCH2CO), 3.04-3.4 (4H, br, triazole-(CH2)2 CH2NH), 3.76, 3.83
(br, 2xH, 1, 4-triazole-CH2NH), 4.05, 4.13 (2H, d, COCH), 4.24-4.33
([8-2x]H, br, 1, 5-triazole-CH2NH and triazole-CH2 (CH2)2 NH),
7.43 (2xH, s, 1,4-triazole), 7.77 ([2-2x]H, s, triazole).
pKa Determination of Deprotected Click Polypeptide
7. The pKa of deprotected click polypeptide 7 (DCP7) was determined
by potentiometric titration. In general, DCP7 (13.4 mg, with 50 mmol
amino groups) was dissolved in distilled water (10 mL) using a beaker
equipped with a pH meter. Then, NaOH solution (2.5 mM) was
dropwisely added to the solution, and the pH values of the solution
accompanied by the addition volume of NaOH solution were recorded
and analyzed according to the method below (eq 1).
Figure 2. TGA curves of polymers 7-10, 11a, 11b, 12a, and 12b in N2.
½-NH3þꢀ a ½Hþꢀ þ ½-NH2ꢀ
½Hþꢀ½-NH2ꢀ
Ka ¼
½-NH3þꢀ
When
1
V ¼ Va, ½-NH2ꢀ ¼ ½-NH3þꢀ
2
So
Ka ¼ ð½HþꢀÞ
, pKa ¼ ðpHÞ
ð1Þ
1 = 2Va
1 = 2Va
Here [-NH3þ] and [-NH2] stand for concentration of the amino
groups in ionic state and molecular state.
Va stands for the volume of the added NaOH solution at the end point
of titration, which was determined by the pH-V curve as well as the
d(pH)/dV-V curve (the maximum in the d(pH) /dV-V curve)
described in Figure S6 of the Supporting Information.
Figure 3. Cytotoxicity study of (A) ε-PL, (B) DCP7, (C) thermal
DCP7, and (D) PLL at different polymer concentrations against L929
fibroblasts evaluated by MTT assay after 48 h.
measured on a microplate reader. Each sample was measured at least six
times, and the average results are shown in Figures 3 and 4.
Cytotoxicity of Deprotected Click Polypeptide 7. The
relative cytotoxicity of DCP7 was assessed with a methyl tetrazolium
(MTT) assay against L929 mouse fibroblast. Biosynthesized ε-PL and
chemically synthesized PLL were used as the positive and negative
controls, respectively. Because DCP7 could have the possibility of
containing a trace amount of the copper ion residual, which would
poison cells, microwave-assisted thermal click polymerizations (120 °C,
75 min) without Cu (I) catalyst were also taken to obtain the
deprotected click polypeptide 7 without Cu ion (thermal DCP7) for
the cytotoxicity assessment. The weighed dry samples (ε-PL, DCP7,
thermal DCP7, and PLL) were sterilized by ultraviolet (UV) and then
dissolved in RPMI 1640 culture medium. Subsequently, 100 μL of L929
cells in RPMI 1640 culture medium at a density of 5000 cells per well was
added to each well in a 96-well plate. Cells were incubated for 48 h in an
incubator (37 °C, 5% CO2), followed by the addition of the sample-
containing culture medium to make the final sample concentrations of 1,
0.5, and 0.25 mg mL-1, respectively. After another 24 and 48 h of
incubation, the cells were observed using an inverted microscope
(Nikon-2000U). We added 20 μL of MTT stock solution (5 mg mL-1
in PBS) to each well of the plate for an additional 4 h of incubation. The
purple formazan produced by active mitochondria was solubilized using
200 μL of DMSO. The optical density (OD) at 492 nm in each well was
Endotoxin Selective Removal Properties of DCP7. To
assess the endotoxin-selective removal properties of DCP7, epoxy-
containing cross-linked polystyrene (PS-EO) microspheres was pre-
pared by dispersion polymerization of epoxypropyl methacrylate (EM),
styrene (S), and divinyl benzene (DVB). (See the Supporting Informa-
tion and Scheme S1.) DCP7, ε-PL (as positive control), and PLL (as
negative control) were then immobilized to PS-EO microspheres
through the reaction between amino groups and epoxy groups. (See
the Supporting Information and Scheme S2.) The amino-group con-
tents of DCP7-, ε-PL-, or PLL-immobilized PS-EO microspheres were
determined by ninhydrin technique according to literature42 to be 17.5,
13.0, and 14.5 μmol/g, respectively. The endotoxin adsorption and
protein recovery properties of the DCP7-, ε-PL-, or PLL immobilized
microspheres with about equal total amino content (DCP7-immobilized
PS-EO microspheres: 17.5 μmol/g ꢁ 10 mg; ε-PL-immobilized PS-EO
microspheres: 13.0 μmol/g ꢁ 11 mg; PLL-immobilized PS-EO micro-
spheres: 14.5 μmol/g ꢁ 11 mg) were investigated independently. The
endotoxin adsorption process was described below: typically, the
adsorbent was first treated with 0.2 M NaOH solution, normal saline,
and ethanol to remove the original pyrogen and other impurities. The
adsorbent was then dispersed by ultrasonic in 1 mL of endotoxin
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Figure 4. Optical images of the incubated L929 fibroblast cells. Blank1, Blank2: without polymer (blank); A1, A2: in 0.5 mg mL-1 of ε-PL; B1, B2: in
0.5 mg mL-1 of DCP7; C1, C2: in 0.5 mg mL-1 of thermal DCP7; and D1, D2: in 0.25 mg mL-1 of PLL for 24 h (Blank1, A1, B1, C1, and D1) and 48 h
(Blank2, A2, B2, C2, and D2).
solution in TAL (LAL) reagent water with a concentration of 10 EU/
mL. The mixture was oscillated at 25 °C for 15 min. The adsorbent was
centrifuged, and the endotoxin concentration of the supernatant was
measured by a Limulus test. The protein recoveries of different polyca-
tion (ε-PL, DCP7, or PLL) immobilized PS-EO microspheres were
determined as below: after pretreatment, as described in the endotoxin
adsorption process, the adsorbent was mixed with 1 mL of protein
solution in distilled water. After oscillating at 25 °C for 15 min, the
adsorbent was centrifuged, and the protein concentration of the super-
natant was determined by UV measurement. As an example, the
endotoxin selective removal properties from hemoglobin solution of
different polycation (ε-PL, DCP7, or PLL)-immobilized PS-EO micro-
spheres were determined as below: hemoglobin solution that contained
∼4500 EU/mL endotoxin was prepared; then, the pretreated adsorbent
was mixed with 1 mL of the above-mentioned solution. After oscillating
at 25 °C for 15 min, the adsorbent was centrifuged. The endotoxin and
hemoglobin concentration of the supernatant were measured. All
endotoxin adsorption and protein recovery experiments were repeated
three times, and the results were averaged.
many difficulties. To mimic the structure of ε-PL, we must obtain
a kind of polymers with not only R-amino side groups but also
similar main chain structure to ε-PL. Because of the interesting
feature of the triazole groups that it can imitate the character of
amide bonds in some cases, click chemistry was chosen as a useful
tool to construct the ε-PL-analogous polymers.
Monomer Synthesis. To apply the click polymerization
strategy, we should obtain that kind of monomers with diazide
or dialkyne groups on both ends of the molecular structures.
R-Amino side group is another necessary structural factor. For
these reasons, aspartic acid and glutamic acid, which have one
R-amino group and two carboxyl groups, were selected as starting
reactants, and the amino groups of them were protected by
benzyloxycarbonyl groups to give N-benzyloxycarbonylaspartic
acid (Z-Asp) and N-benzyloxycarbonylglutamic acid (Z-Glu).
The diazide and dialkyne monomers 2-5 were synthesized by
amide condensation of Z-Asp and Z-Glu with 3-azidopropana-
mine or propargylamine in anhydrous THF in the presence of
BOP reagent and triethylamine with high yields (∼90%). Highly
purified monomers could be obtained conveniently because the
main byproduct of this reaction was a kind of salt, which could be
conveniently washed away with water. Dialkyne-terminated PEG
6a, 6b with different molecular weights was also prepared by the
reaction of PEG (molecular weight of 1a and 1b were 1000 and
2000 Da, respectively) and large excess of propargyl bromide (40
fold) using NaOH as catalyst. All diazide and dialkyne mono-
mers, including 2-5 and 6a, 6b, were characterized by 1H and
13C NMR (Figures S1-S3 in Supporting Information), indicat-
ing high purity. Monomers 2-5 were also analyzed by mass
spectroscopy.
’ RESULTS AND DISCUSSION
Biosynthesized ε-PL has a lot of advantages over chemically
synthesized PLL, especially in the application of selective re-
moval of endotoxin from protein solution. Endotoxin
(lipopolysaccharide (LPS)), is the constituent of cell walls of
Gram-negative bacteria, which largely exists in bioproducts, such
as peptides and proteins. The removal of endotoxin even in
nanogram quantities from drugs and fluids before injection is
critical because of its potent biological activities causing pyro-
geneic and shock reactions in mammals. Endotoxin is an amphi-
pathic molecule that has both an anionic region and a
hydrophobic region. The removal of endotoxin mainly takes
use of its amphipathic properties. In the case of removing
endotoxin from protein solution, the adsorption of protein is
commonly unavoidable. It seems that the low pKa (7.6) of ε-PL
makes it possible to decrease the interaction with protein,
especially the acidic protein like BSA.43-45 However, the large
scale production of ε-PL using biosynthesis technique is incon-
venient. The purification procedures are complicated, and the
molecular weight of ε-PL is uneasy to control. As chemists, we
have been seeking a route to synthesize chemically ε-PL or its
substitutes for many years. To synthesize ε-PL straightly from R-
amino protected L-lysine or its large cyclic monomers would have
Click Polypeptides. The click polypeptides were synthesized
through click polymerizations of dialkyne 2 or 3 and diazide 4 or
5 monomers. The equal equivalence of purified dialkyne 2 or 3
and diazide 4 or 5 monomers was mixed together in DMF under
a nitrogen atmosphere with CuBr as catalyst (0.05 equiv accord-
ing to alkyne or azide groups). Polymerizations could be
successfully processed and finished within 30 min in a microwave
reactor. The obtained click polypeptides 7-10 were character-
ized by 1H and 13C NMR as well as SEC-MALLS analyses.
1
As an example, H NMR spectra of click polypeptide 7 is
presented in Figure 1. The polyaddition reactions between
dialkyne and diazide monomers were confirmed by the appear-
ance of the characteristic signal of triazole rings at 8.34 and 8.43
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Table 1. Characteristics of Click Peptide 7-10 and PEG-
Containing Multi-Block Copolymers11a,11b and 12a,12b
semicrystalline polymers and had similar thermal decomposi-
tion behavior as that of the click polypeptides 7-10 (Figure 2
and Table 1). Replacement of the starting monomers from
diazido Z-Asp to diazido Z-Glu showed little effect on the Tm
values of the copolymers. However, the Tm values of the
copolymers were much lower than that of the normal PEG
polymer with similar molecular weight (Tm of PEG 20000 is
61 °C). We deduced that the existence of the triazole groups
and the huge protecting groups hindered the ordered arrange-
ment of the small PEG chains, resulting in the decrease in the
crystallinity. It was interesting that when PEG 2000 was used
instead of PEG 1000, the copolymers with lower molecular
weight would be obtained (11b and 12b). Extended reaction
time to 75 min would have little effect on the molecular
weight. We supposed that the longer chains of PEG 2000
would provide the more serious shielding circumstance, mak-
ing it more difficult for the alkyne groups on PEG to encounter
with the diazido monomers during the same reaction time.
ε-PL-Analogous Click Polypeptides. When the above
synthesized Z-protected click polypeptides were treated with
CF3COOH/HBr for 2 h at 0 °C under a nitrogen atmosphere,
the ε-PL-analogous polypeptides with abundant and regularly
arranged R-amino groups on the side chains could be obtained
successfully, as illustrated in Scheme 2. As an example, after the
sample solubilitya Mn (104 Da)b PDIb Tg (°C)c Tm (°C)c Td (°C)d
7
F, S
2.85
2.26
3.55
5.30
6.69
3.60
9.35
2.85
3.32
4.03
2.30
2.34
4.12
3.35
4.11
3.86
106.7
103.2
99.2
236.9
232.3
239.5
229.7
267.2
328.0
266.2
344.1
8
F, S
9
F, S
10
11a
11b
12a
12b
F, S
96.8
F, S, W
F, S, W
F, S, W
F, S, W
27.5
41.0
28.8
41.0
a F = DMF, S = DMSO, W = water. b By SEC-MALLS analyses in DMF.
c By DSC (second heating scan) in nitrogen atmospheres. d Thermal
decomposition temperature (Td) was recorded by TGA at a temperature
of 5.0% decomposition.
ppm in 1H NMR (Figure 1A, t) as well as 122.9 and 144.7 ppm in
13C NMR spectra (Supporting Information Figure S6A). It is
noteworthy that because the click polymerization was taken place
under high monomer concentration with microwave-assisting,
the reaction time was compressed within 30 min,31,32 and the
molecular weights of click polypeptides 7-10 were all about
several ten of thousands of Da with polydispersities from 2.3 to
4.0, which was determined by SEC-MALLS analyses. (See
Table 1 and Supporting Information Figure S7.)
1
deprotection procedure, the signals in H and 13C NMR of Z
groups on polypeptide 7 disappeared completely, the signals of
triazole groups still remained (Figure 1B, t, and Figure S6B of the
Supporting Information), and the infrared adsorption of benzyl
groups in 740 and 698 cm-1 vanished, too (Figure S8 of the
Supporting Information). These results clearly indicated the
complete deprotection of click polypeptides with the main chains
keeping invariant under our treating conditions.
The main properties of the deprotected click polypeptides,
including pKa value and cell cytotoxicity, were investigated and
compared with that of the biosynthesized ε-PL from literatures
reported to evaluate whether the chemically synthesized click
polypeptides possess the similar biofunction as ε-PL. The pKa of
DCP7 determined by conventional potentiometric titration was
∼7.5, which was very close to that of ε-PL (pKa = 7.6) (Figure S9
of the Supporting Information). The results showed that the
R-amino groups on the DCP7 have the similar dissociation
property as ε-PL. Unlike the normal PLL, which bared strong
cationic character (pKa = 9 to 10), the lower values of pKa of the
click polypeptides would be very important for the selective
removal of the endotoxin from a protein containing biological
reagent because many acidic proteins possess negative charge
under physiological conditions.
As a kind of cationic polymer, DCP7 could also have some
toxicity because the cationic property would have the possibility
to destroy the cell membrane. The cytotoxicity of DCP7 was
evaluated against L929 mouse fibroblasts in vitro by MTT assay.
Chemically synthesized PLL and biosynthesized ε-PL were used
as the negative and positive controls, respectively. As shown in
Figure 3, the DCP7 exhibited close cytotoxicity to that of ε-PL
but had much lower cytotoxicity than that of PLL in all three
different concentrations. However, considering the negative
effect to the cytotoxicity results, which might come from the
harmful copper ions remaining in DCP7 even in trace amount,
we also synthesized another DCP sample by microwave-assisted
thermal click polymerizations (120 °C, 75 min) without Cu (I)
catalyst. (The sample is called thermal DCP7 for short.) This
time, thermal DCP7 showed much better cell compatibility than
All click polypeptides 7-10 were amorphous polymers with
high Tg values (∼100 °C) (Table 1). These Tg values were not
only much higher than that of poly (γ-benzyl-L-glutamate)
(PBLG) (∼19 °C),10 but also higher than that of nylon66
(45.0 °C), nylon610 (50.0 °C), and even nylon46 (75.0 °C). It
is obvious that the rigid triazole structure in the polymer main
chain plays the important role of the increase in Tg, as previous
reports revealed.27,35,36 The TGA analyses (Figure 2 and
Table 1) of click polypeptides 7-10 showed that all click
polypeptides were relatively stable with the thermal decomposi-
tion temperatures (Td values) at ∼230 °C. About 30% weight
loss occurred from 230 to 270 °C, which was caused by the
thermal decomposition of the amino protecting groups of click
polypeptide. After that, each TGA curve presented a platform
from 270 to 350 °C. Finally, the main chain of the polymers
started to break and decompose by the elevation of the tem-
perature to >350 °C. All of the click polypeptides 7-10 indicated
good solubility in strong polar solvents such as DMSO and DMF.
PEG-Peptide Click Copolymers. To confirm further the
reliability of the A-A, B-B strategy click polymerization with
microwave technique, we synthesized the PEG-containing alternat-
ing copolymers 11a, 11b, 12a, and 12b by the click polyaddition of
dialkyne-terminated PEG 6a (Mw, 1000 Da) or 6b (Mw, 2000 Da)
and diazide monomer 4 or 5. Unlike amino acid monomers,
dialkyne-terminated PEG did not have one identical molecular
weight, which made it difficult to obtain the exactly equivalent molar
ratio between the dialkyne-terminated PEG and the diazide amino
acid. To avoid the confused results of the final copolymers, we
designed the molar quantity of the dialkyne-terminated PEG 6a or
6b to be a little excess to that of the diazide monomer 4 or 5 to apply
the alkyne terminals to both ends of the PEG-containing multiblock
copolymers, which was confirmed by 1H and 13C NMR (Figures S4
and S5 of the Supporting Information).
All of these PEG-containing alternating copolymers showed
good solubility in DMSO, DMF, and water. They were
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Figure 5. Change of endotoxin concentration before (Origin) and after treatment with polycation-immobilized epoxy-containing cross-linked
polystyrene (PS-EO) microspheres. We treated 1 mL of endotoxin solution (10 EU/mL or 100 EU/mL) with about equal total amino content of
polycation immobilized microspheres (DCP7- immobilized PS-EO microspheres: 17.5 μmol/g ꢁ 10 mg; ε-PL-immobilized PS-EO microspheres: 13.0
μmol/g ꢁ 11 mg; PLL-immobilized PS-EO microspheres: 14.5 μmol/g ꢁ 11 mg).
Scheme 2. Structure Imitation of Poly(ε-lysine) (b) with De-Protected Click Polypeptides (a)
those other systems even in the lower PLL concentration
(0.25 mg mL-1). At the same time, the cells incubated in the
solution of 0.5 mg mL-1 DCP7 (Figure 4; B1, B2) as well as
thermal DCP7 (C1, C2) showed good growing status during
48 h, which was quite similar to that of ε-PL (A1, A2) and blank
(Blank1, Blank2), indicating the high compatibilities of the
polymer systems.
As an application example, DCP7, ε-PL (as positive control) and
PLL (as negative control) were immobilized onto the epoxy-
containing cross-linked polystyrene (PS-EO) microspheres (see
Figures S10 and S11 of the Supporting Information for the
characterization of PS-EO microspheres) for the evaluation of the
endotoxin-selective removal properties. The results showed that
after treatment with the polycation (DCP7, ε-PL, or PLL)-immo-
bilized microspheres, the endotoxin concentration remained in the
solution decreased sharply from the initial 10 EU/mL to <2 EU/mL
(Figure 5A). The PS-EO microspheres themselves also showed
some endotoxin absorbing capacity for its hydrophobic properties. It
is noteworthy that DCP7-immobilized microspheres exhibited the
Figure 6. Protein recoveries after treatment with different polycations
immobilized epoxy-containing cross-linked polystyrene (PS-EO) micro-
spheres. (A) PS-EO-ε-PL, (B) PS-EO-DCP7, (C) PS-EO-PLL, and
(D) PS-EO. The concentrations of cytochrome c, γ-globulin, hemoglo-
highest endotoxin removal ability. More than 96% of the endotoxin
was removed by the DCP7-immobilized microspheres with an
endotoxin adsorption capacity of 953 EU/g. When the initial
concentration of endotoxin increased to 100 EU/mL, the endotoxin
adsorption capacity of all of the adsorbents increased, but the
difference between them enlarged (Figure 5B). At this time, the
endotoxin adsorption capacity of DCP7 increased to 9250 EU/g. It
has been mentioned that the hydrophobic adsorbent can only
adsorb the single endotoxin chain through hydrophobic interaction
however, the cationic adsorbent can also adsorb the supramolecular
aggregates (micelle or vesicles) formed by endotoxin molecules that
have anionic groups on their surface.
bin, and BSA were 0.278, 0.456, 0.496, and 0.49 mg/mL, respectively.
ε-PL (Figure 3). Because there are only little difference in the
main chain structures between DCP7 and thermal DCP7 (the
triazole groups in DCP7 are all 1,4-triazole structures, whereas
thermal DCP7 has a little amount of 1,5-triazole structure), the
cytotoxicity value of thermal DCP7 could be considered to be the
real index of DCP7 without any interference from the copper
ions. More intuitional results could be obtained from the optical
images of L929 fibroblast cells after incubation for 24 and 48 h
(Figure 4). In the PLL (D1, D2) system, most of the cells died
within 24 h, and the remaining living cells were smaller than
Protein recovery is another important character for the
endotoxin-adsorbing material because protein is the most
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The characteristic results above bring the great possibility for
the chemosynthesized deprotected click polypeptides (DCPs) to
imitate ε-PL in its main properties and to be applied in the
endotoxin selective removal area.
’ CONCLUSIONS
We developed a novel strategy for the elaboration of click
polypeptides and PEG-containing alternating copolymers with
triazole units in the main chain and potential amino groups on
the side chains by microwave-assisted click polymerizations using
aspartic (or glutamic)-acid based dialkyne and diazide mono-
mers. It is really an encouragement that the combination of the
A-A, B-B strategy click polymerization with microwave tech-
nique could provide us a convenient way to synthesize copoly-
mers baring R-amino groups on the side chains with regular
arrangement. It is not only a chemically available approach to
synthesize the ε-PL-analogous polymers but also the new
designation to cultivate the new series of the cationic polymers,
which would have the great potential to be used as functional
gene vector with PEG as shadowing system as well as NCA
initiator to get amphiphilic graft polymers.
Figure 7. Endotoxin adsorption and hemoglobin recovery properties of
different polycations immobilized epoxy-containing cross-linked poly-
styrene (PS-EO) microspheres. The concentrations of hemoglobin and
endotoxin were 0.721 mg/mL and 4230 EU/mL, respectively.
commonly involved component during the actual endotoxin removal
process, like endotoxin removal from bacterial-possessed protein
solution and blood. To determine the protein recovery, we mixed
the polycation (ε-PL, DCP7, or PLL)-immobilized PS-EO
microspheres with different protein solutions and checked the
changes of the protein concentrations. As shown in Figure 6,
without polycations immobilization, the blank PS-EO micro-
spheres (D) showed little effect on the protein concentrations
with high protein recoveries (all higher than 90%). After
immobilized with polycations, the protein recoveries of the
microspheres decreased in all protein solutions except for BSA.
Among them, PLL- immobilized microsphere (C) had the lowest
protein recoveries, whereas DCP7-immobilized microsphere (B)
had significantly higher protein recoveries that were very close to
that of the blank microspheres and ε-PL-immobilized micro-
sphere (A), indicating the excellent low protein adsorption
property for its low pKa. It is noteworthy that the protein
adsorption behaviors of the polycations-immobilized micro-
spheres were not coincident with the protein charges. For
example, as the acidic protein with a pI (isoelectric point) of
4.9, BSA should be mostly adsorbed by the polycation immobi-
lized microspheres, theoretically. However, the results showed
that the BSA recoveries of the three kinds of polycation-
immobilized microspheres were all the highest compared with
other proteins. The abnormal phenomenon is assumed to be
caused by the low amino contents of the adsorbents and the
surface property of cross-linked PS-EO microspheres.
To confirm further the selective removal properties of DCP7
immobilized microspheres, the removal of endotoxin from a
endotoxin mixed protein solution was investigated using hemo-
globin as an example. As shown in Figure 7, after being treated by
polycation (DCP7, ε-PL, or PLL)-immobilized microspheres,
the endotoxin concentration that remained in the mixture
solution decreased vastly from the initial 4230 to ∼1000 EU/
mL (Figure 7). Again, DCP7 exhibited the best adsorption ability
with an endotoxin adsorption capacity of 3.4 ꢁ 105 EU/g. At the
same time, >90% of the hemoglobin still remained. The protein
recovery results of all of the adsorbents remained consistent with
the results obtained by protein recovery experiments from pure
hemoglobin solution (Figure 7, top right corner). It is obvious
that trace amount of the endotoxin in the protein solution could
be removed easily and completely without much loss of the
protein content.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra of
S
b
compounds 2-5, 6a, 11a, and12a, the 13C NMR and ATR-FTIR
spectra of click polypeptide 7 before and after deprotection, the
pH-V curve and d(pH)/dV-V curve of deprotected click
polypeptide 7, SEC-MALLS curves of click polypeptides 7-10
and PEG-containing alternate copolymers 11a, 11b and 12a,
12b, the preparation of epoxy-containing cross-linked polystyr-
ene (PS-EO) microspheres, and the immobilization of polyca-
tions processes, the SEM and XPS characterizations of cross-
linked PS-EO microspheres. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: þ86-431-85262769. E-mail: ybhuang@ciac.jl.cn.
’ ACKNOWLEDGMENT
We would like to acknowledge the financial support from
National Natural Science Foundation of China (nos. 20774094,
51021003, 50733003, and 20874097), the Ministry of Science and
Technology of China (973 project no. 2009CB930102; 863 project
no. 2007AA03Z535), “100 Talents Program” of the Chinese
Academy of Sciences (no. KGCX2-YW-802), and Jilin Provincial
Science and Technology Department (no. 20082104, 20100588).
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