Synthesis and characterization of enzymatically biodegradable peg and peptide-based hydrogels prepared by click chemistry

Article abstract of DOI:10.1021/bm1002637

Herein we describe the synthesis and rheological characterization of a series of enzymatically sensitive PEG and peptide-based hydrogels by the Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction. The hydrogels were synthesized by a combination of alkyne-f

Full text of DOI:10.1021/bm1002637

1608
Biomacromolecules 2010, 11, 1608–1614
Synthesis and Characterization of Enzymatically Biodegradable
PEG and Peptide-Based Hydrogels Prepared by Click Chemistry
Maarten van Dijk,†,‡ Cornelus F. van Nostrum,‡ Wim E. Hennink,*,‡ Dirk T. S. Rijkers,† and
Rob M. J. Liskamp*,†
Divisions of Medicinal Chemistry and Chemical Biology and Pharmaceutics, Utrecht Institute for
Pharmaceutical Sciences, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University,
P.O. Box 80082, 3508 TB Utrecht, The Netherlands
Received March 11, 2010; Revised Manuscript Received April 29, 2010
Herein we describe the synthesis and rheological characterization of a series of enzymatically sensitive PEG and
peptide-based hydrogels by the Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction. The hydrogels were synthesized
by a combination of alkyne-functionalized star-shaped PEG molecules (two 4-armed PEGs with Mw 10 and 20
kDa, respectively, and one 8-armed PEG of 20 kDa) and the protease-sensitive bis-azido peptide, NR-(azido)-D-
alanyl-phenylalanyl-lysyl-(2-azidoethyl)-amide (6) in the presence of CuSO4 and sodium ascorbate in aqueous
solution. The swelling ratio and the storage modulus (G′) of the hydrogels could be tailored by several parameters,
for example, the initial solid content of the hydrogel, the molecular weight of the PEG derivative, and by the
architecture of the PEG molecule (4- versus 8-armed PEG derivative). The peptide sequence, D-Ala-Phe-Lys,
was sensitive toward the proteases plasmin and trypsin to render the hydrogels biodegradable.
azides and terminal alkynes was independently discovered in
2002 by the groups of Meldal17,18 and Sharpless19 and is
compatible with most functional groups present in proteins and
peptides, abolishing the need for protection groups. Furthermore,
the Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction, also
known as the most prominent example of “click chemistry”,20,21
can be performed in aqueous solution under mild reaction
conditions in terms of pH and temperature, it has a high reaction
rate, and generally results in a high yield of the desired product.
These properties render the Cu(I)-catalyzed 1,3-dipolar cycload-
dition reaction highly suitable for the synthesis of hydrogels22,23
based on unprotected bis-azido peptides and PEG-derivatized
alkynes.
Previously, we have shown that the Cu(I)-catalyzed 1,3-
dipolar cycloaddition reaction is an effective tool for the
synthesis of biodegradable peptide-based polymers.24,25 In the
present study we aimed to design a modular approach for the
synthesis of tailorable biodegradable hydrogels. The Cu(I)-
catalyzed 1,3-dipolar cycloaddition reaction between a trypsin-
and plasmin-sensitive bis-azido peptide and star-shaped alkyne-
derivatized PEG moieties resulted in a series of hydrogels in
which the chain length, chain architecture, and alkyne density
of the PEG derivatives were found to be important factors in
the design of the macroscopic properties of the hydrogels.
Introduction
Hydrogels are three-dimensional, hydrophilic polymeric
networks capable of absorbing large amounts of water.1 There
has been a lively interest in hydrogels, because they can be used
for a wide range of applications including drug delivery systems
for the entrapment and release of pharmaceutically active
proteins and as scaffolds for tissue engineering and repair.2,3
Two major classes of hydrogels can be distinguished based on
the nature of cross-linking that form the three-dimensional
hydrophilic network.4 In chemically cross-linked hydrogels,
covalent bonds form the network, while in physically cross-
linked hydrogels, noncovalent interactions like hydrogen bond-
ing, stereocomplex formation, hydrophobic, and ionic interac-
tions are responsible for network formation.5 To render chemically
cross-linked hydrogels biodegradable, hydrolysis sensitive moi-
eties, like ester bonds, have to be incorporated either in the cross-
linking entity or in the (polymeric) backbone of the hydrogel.
A special class of biodegradable hydrogels comprise the
enzymatically degradable hydrogels, which allow the degrada-
tion and a subsequent release of the entrapped bioactive
compound to be controlled by cell-secreted and cell-activated
enzymes.6,7 A frequently used approach to obtain enzymatically
degradable hydrogels is the incorporation of a short peptide
sequence that can act as a substrate, which is specifically
recognized and cleaved by endogenous proteases, like trypsin,
plasmin, or a matrix metalloproteinase, as schematically shown
in Figure 1.7-12
Experimental Section
Materials and Methods. Chemicals were obtained from commercial
sources and used without further purification. The star-shaped 4- and
8-armed poly(ethylene glycol) derivatives (PEG4-10k (7), PEG4-20k
(8), and PEG8-20k (11)) were purchased, with a free hydroxyl moiety,
from JenKem Technology U.S.A. Reactions were carried out at room
temperature unless stated otherwise. Column chromatography was
performed with Silica-P Flash silica gel (Silicycle). Retention factor
values (Rf) were determined with thin layer chromatography (TLC) by
using Merck silica gel 60 F-254 plates. Spots were visualized by UV-
quenching, ninhydrin, TDM/Cl2,26 KMnO4, or PPh3/ninhydrin.271H
NMR spectra (300 MHz) were recorded on a Varian Mercury plus
An attractive approach toward the synthesis of enzymatically
degradable hydrogels that contain protease-sensitive peptide
sequences is via the Cu(I)-catalyzed cycloaddition.13-16 The
Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction between
* To whom correspondence should be addressed. Phone: +31 30 253
7396/7307 (R.M.J.L.); +31 30 253 6964 (W.E.H.). Fax: +31 30 253 6655
(R.M.J.L.); +31 30 251 7839 (W.E.H.). E-mail: r.m.j.liskamp@uu.nl
(R.M.J.L.); w.e.hennink@uu.nl (W.E.H.).
† Division of Medicinal Chemistry and Chemical Biology.
‡ Division of Pharmaceutics.
10.1021/bm1002637
 2010 American Chemical Society
Published on Web 05/24/2010

Enzymatically Biodegradable Hydrogels by Click Chemistry
Biomacromolecules, Vol. 11, No. 6, 2010 1609
Figure 1. Schematic overview of the synthesis of enzymatically degradable PEG-peptide based hydrogels that were synthesized via the Cu(I)-
catalyzed azide-alkyne cycloaddition reaction.
spectrometer and chemical shift values (δ) are given in ppm relative
to TMS. 13C NMR spectra (75 MHz) were recorded using the attached
proton test (APT) pulse sequence and chemical shift values are given
in ppm relative to CDCl3 (77.0 ppm) or DMSO-d6 (39.5 ppm). Fourier
transform infrared spectra (FTIR) were measured on a Bio-Rad FTS-
25 spectrophotometer. The retention time values (Rt) and purities of
the synthetic peptide intermediates were evaluated by analytical RP-
HPLC on a Shimadzu automated HPLC system equipped with a
UV-vis detector operating at λ ) 214 and 254 nm and by using an
Alltech Prosphere C18 column (250 × 4.6 mm, 5 µm particle size,
300 Å pore size) at a flow rate of 1 mL/min using a linear gradient of
100% buffer A (0.1% TFA in H2O/CH3CN 95:5 v/v) to 100% buffer
B (0.1% TFA in CH3CN/H2O 95:5 v/v) in 20 or 40 min.
TFA. The obtained residue was dissolved in CH2Cl2 (300 mL) and
Fmoc-Lys(Boc)-OH (9.4 g, 20 mmol), BOP (8.8 g, 20 mmol), and
DIPEA (11 mL, 63 mmol) were added, and the reaction mixture was
stirred for 16 h at room temperature. Then, the solvent was removed
in vacuo and the residue was dissolved in EtOAc (300 mL), and this
solution was subsequently washed with 1 N KHSO4 (3 × 100 mL),
H2O (1 × 100 mL), 5% NaHCO3 (3 × 100 mL), H2O (1 × 100 mL),
and brine (3 × 100 mL). The EtOAc solution was dried (Na2SO4),
filtered, and concentrated in vacuo. The residue was purified by column
chromatography (CH2Cl2/MeOH 96:4 v/v) to give Fmoc-Lys(Boc)-(2-
azidoethyl)-amide 3 as a white solid in 97% yield (10.4 g). Rf ) 0.78
1
(CHCl3/MeOH/AcOH 95:20:3 v/v/v). FTIR (KBr) ν: 2110 cm-1. H
NMR (CDCl3, 300 MHz) δ: 1.35-1.50 (m, 4H, γCH2/δCH2 Lys), 1.43
(s, 9H, (CH3)3), 1.65 (m, 1H, ꢀCH2 Lys (1H)), 1.86 (m, 1H, ꢀCH2 Lys
(1H)), 3.10 (m, 2H, εCH2 Lys), 3.41 (m, 4H, CH2CH2N3), 4.13 (m,
2H, RCH Lys (1H)/CH Fmoc (1H)), 4.40 (m, 2H, CH2 Fmoc), 4.64
(broad s, 1H, NH urethane), 5.52 (broad s, 1H, NH urethane), 6.57
(broad s, 1H, NH amide), 7.26-7.78 (m, 8H, arom H Fmoc). 13C NMR
(CDCl3, 75 MHz) δ: 22.5, 28.4, 29.5, 31.9, 38.9, 39.7, 47.1, 50.6, 54.8,
67.0, 79.2, 119.9, 125.0, 127.0, 127.7, 141.2, 143.7, 156.2, 172.1, 180.1.
Fmoc-Phe-Lys(Boc)-(2-azidoethyl)-amide (4). Compound 3 (10.4 g,
19.3 mmol) was dissolved in piperidine/THF 1:4 v/v (150 mL) and
the reaction mixture was stirred for 2 h at room temperature before the
volatiles were removed by evaporation. The residue was coevaporated
with toluene (3×) and CHCl3 (3×) and subsequently purified by column
chromatography (CH2Cl2/MeOH 9:1 v/v) to give the intermediate
H-Lys(Boc)-(2-azidoethyl)-amide as a colorless oil in 79% yield (4.8
Synthesis of Nr-(Azido)-D-alanyl-phenylalanyl-lysyl-(2-azido-
ethyl)-amide (6). tert-Butyl 2-Azidoethylcarbamate (2). To a cooled
(0 °C) solution of 2-bromoethylamine hydrobromide 1 (16.8 g, 82.7
mmol) and Boc2O (19.8 g, 91 mmol) in CH2Cl2 (100 mL), Et3N (12.6
mL, 91 mmol) was added dropwise, and the obtained reaction mixture
was stirred for 1 h at 0 °C, followed by 16 h at room temperature.
Then, the reaction mixture was diluted with CH2Cl2 (100 mL) and this
solution was subsequently washed with 1 N KHSO4 (3 × 100 mL)
and brine (3 × 100 mL), dried (Na2SO4), filtered, and concentrated in
vacuo. The resulting N-(tert-butyloxycarbonyl)-2-bromoethylamine was
obtained as a white solid in 87% yield (17.7 g). Rf ) 0.16 (CHCl3/
MeOH/AcOH 95:20:3 v/v/v). 1H NMR (CDCl3, 300 MHz) δ: 1.45 (s,
9H, (CH3)3), 3.45 (m, 2H, CH2Br), 3.52 (m, 2H, NHCH2), 5.06 (broad
s, 1H, NH). 13C NMR (CDCl3, 75 MHz) δ: 28.3, 32.6, 42.3, 79.7, 155.5.
In the next step, a mixture of N-(tert-butyloxycarbonyl)-2-bromoethyl-
amine (17.7 g, 79 mmol) and NaN3 (10.3 g, 158 mmol) was dissolved
in dry DMF (300 mL) and stirred for 16 h at room temperature. Then,
the solvent was removed in vacuo and the residue was dissolved in
CH2Cl2 (330 mL), and this solution was subsequently washed with 1
N KHSO4 (3 × 100 mL) and brine (3 × 100 mL), dried (Na2SO4),
filtered, and concentrated in vacuo. The residue was coevaporated with
toluene (3×) and CHCl3 (3×) to remove any residual DMF, to give
tert-butyl 2-azidoethylcarbamate (2) as a white solid in 87% yield (12.9
g). Rf ) 0.95 (CHCl3/MeOH/AcOH 95:20:3 v/v/v). FTIR (KBr) ν: 2110
1
g). Rf ) 0.25 (DCM/MeOH 9:1 v/v). H NMR (CDCl3, 300 MHz) δ:
1.37-1.61 (m, 5H, ꢀCH2 (1H)/γCH2/δCH2 Lys), 1.44 (s, 9H, (CH3)3),
1.87 (m, 1H, ꢀCH2 Lys (1H)), 3.12 (m, 2H, εCH2 Lys), 3.34-3.48
(m, 5H, RCH Lys (1H)/CH2CH2N3), 4.61 (broad s, 1H, NH urethane),
7.70 (broad s, 1H, NH amide). 13C NMR (CDCl3, 75 MHz) δ: 22.8,
28.4, 29.9, 34.4, 38.6, 40.1, 50.8, 55.0, 79.1, 156.0, 175.3. In the next
step, H-Lys(Boc)-(2-azidoethyl)-amide (4.8 g, 15.2 mmol), BOP (7.1
g, 15.2 mmol), and Fmoc-Phe-OH (5.9 g, 15.2 mmol) were dissolved
in CH2Cl2 (250 mL), and to this solution, DIPEA (7.9 mL, 45 mmol)
was added dropwise. The reaction mixture was stirred for 16 h at room
temperature and the desired compound was removed by filtration and
subsequently dissolved in a large volume of EtOAc (400 mL). This
solution was washed with 1 N KHSO4 (4 × 100 mL), H2O (1 × 100
mL), 5% NaHCO3 (3 × 100 mL), H2O (1 × 100 mL), and brine (3 ×
100 mL), and subsequently dried (Na2SO4), filtered, and evaporated to
dryness to give Fmoc-Phe-Lys(Boc)-(2-azidoethyl)-amide 4 as an off-
white solid in 80% yield (8.3 g). Rf ) 0.85 (CHCl3/MeOH/AcOH 95:
1
cm-1. H NMR (CDCl3, 300 MHz) δ: 1.45 (s, 9H, (CH3)3), 3.31 (m,
2H, CH2), 3.42 (m, 2H, CH2), 4.98 (broad s, 1H, NH). 13C NMR
(CDCl3, 75 MHz) δ: 28.2, 40.0, 51.1, 79.6, 155.7.
Fmoc-Lys(Boc)-(2-azidoethyl)-amide (3). Azide 2 (3.9 g, 20 mmol)
was dissolved in TFA/CH2Cl2 1:1 v/v (80 mL), and the reaction mixture
was stirred for 1 h at room temperature. After this period of stirring,
the solvents were removed by evaporation and the residue was
coevaporated with toluene (3×) and CHCl3 (3×) to remove any residual
1
20:3 v/v/v). FTIR (KBr) ν: 2110 cm-1. H NMR (CDCl3, 300 MHz)

1610 Biomacromolecules, Vol. 11, No. 6, 2010
Table 1. Characterization of the Hydrogels
Dijk et al.
a
swelling
solid
Mc
entry
hydrogel
G′ (kPA)
ratio (Wt/W0)
content (%)
(g/mol)
1
2
3
4
5
6
7
8
2% PEG4-10k
5% PEG4-10k
7% PEG4-10k
10% PEG4-10k
15% PEG4-10k
2% PEG4-20k
5% PEG4-20k
7% PEG4-20k
10% PEG4-20k
15% PEG4-20k
2% PEG8-20k
5% PEG8-20k
7% PEG8-20k
10% PEG8-20k
15% PEG8-20k
1.4 ( 0.3
12.7 ( 0.8
17.7 ( 0.8
24.9 ( 2.3
37.7 ( 4.2
1.1 ( 0.1
4.6 ( 0.4
7.3 ( 0.6
15.5 ( 0.6
25.5 ( 0.6
1.6 ( 0.2
14.4 ( 0.3
23.7 ( 0.1
39.8 ( 1.0
53.3 ( 3.2
n.d.
0.96 ( 0.06
n.d.
1.50 ( 0.07
1.87 ( 0.04
n.d.
1.79 ( 0.04
n.d.
2.28 ( 0.10
2.92 ( 0.08
n.d.
0.83 ( 0.05
n.d.
1.21 ( 0.03
1.64 ( 0.04
n.d.
5.2
n.d.
6.7
8.0
n.d.
2.8
n.d.
4.4
5.1
n.d.
6.0
n.d.
8.2
9.3
34900 ( 4400
9600 ( 600
9700 ( 400
9900 ( 1100
44400 ( 3700
26600 ( 2300
23500 ( 1800
15900 ( 600
14600 ( 300
30500 ( 3400
8500 ( 200
7300 ( 100
6200 ( 200
7000 ( 400
n.d.
9
10
11
12
13
14
15
a Theoretical value for Mc: PEG4-10k, 5500 g/mol; PEG4-20k, 10500 g/mol; PEG8-20k, 5500 g/mol.
δ: 1.23-1.54 (m, 4H, γCH2/δCH2 Lys), 1.41 (s, 9H, (CH3)3), 1.59 (m,
1H, ꢀCH2 Lys (1H)), 1.83 (m, 1H, ꢀCH2 Lys (1H)), 3.00 (m, 4H, ꢀCH2
Phe/εCH2 Lys), 3.33 (m, 4H, CH2CH2N3), 4.15 (m, 1H, RCH Lys),
4.28 (m, 1H, CH Fmoc), 4.40 (m, 3H, RCH Phe (1H)/CH2 Fmoc (2H)),
4.73 (broad s, 1H, NH urethane), 5.64 (broad s, 1H, NH urethane),
6.80 (broad s, 2H, NH amide), 7.16-7.76 (m, 13H, arom H Fmoc (8H)/
arom H Phe (5H)). 13C NMR (CDCl3, 75 MHz) δ: 22.5, 28.4, 29.3,
31.4, 38.1, 39.0, 47.0, 50.4, 53.1, 56.3, 67.3, 120.0, 124.9, 127.0, 127,7,
128.7, 129.2, 136.1, 141.2, 143.6, 156.2, 171.3, 171.5.
amide), 7.17-7.77 (m, 6H, arom H Phe (5H)/NH amide (1H)). 13C
NMR (CDCl3, 75 MHz) δ: 17.0, 22.7, 28.4, 29.5, 32.2, 38.5, 39.0,
40.1, 53.0, 54.1, 58.2, 79.0, 127.0, 128.5, 129.3, 131.1, 136.1, 156.1,
170.6, 170.9, 171.8. In the final step, N3-D-Ala-Phe-Lys(Boc)-(2-
azidoethyl)-amide (4.2 g, 7.5 mmol) was dissolved in TFA/CH2Cl2 1:1
v/v (60 mL) and the reaction mixture was stirred for 1 h at room
temperature. Then, the solvents were removed by evaporation and the
residue was coevaporated with toluene (3×) and chloroform (3×) to
remove any residual TFA, and finally lyophilized from H2O to yield
quantitatively NR-(azido)-D-alanyl-phenylalanyl-lysyl-(2-azidoethyl)-
amide 6 as a white solid (3.7 g). Rt ) 18.47 min (C18). FTIR (KBr) ν:
2110 cm-1. 1H NMR (CDCl3, 300 MHz) δ: 1.33-1.47 (m, 2H, γCH2
Lys), 1.38 (d (J ) 7.2 Hz), 3H, ꢀCH3 Ala), 1.61-1.70 (m, 3H, ꢀCH2
(1H)/δCH2 Lys), 1.82 (m, 1H, ꢀCH2 Lys (1H)), 2.93-3.00 (m, 3H,
ꢀCH2 Phe (1H)/εCH2 Lys (2H)), 3.11-3.18 (dd (Jax ) 6.6, Jab ) 13.8
Hz), 1H, ꢀCH2 Phe (1H)), 3.36 (m, 4H, CH2CH2N3), 3.93 (m, 1H,
RCH Ala), 4.35 (m, 1H, RCH Lys), 4.62 (m, 1H, RCH Phe), 7.19-7.33
(m, 6H, arom H Phe (5H)/NH amide (1H)), 7.52 (d, 1H, NH amide),
7.91 (d, 1H, NH amide), 7.98 (broad s, 3H, NH3). 13C NMR (CDCl3,
75 MHz) δ: 16.7, 21.9, 26.4, 31.0, 37.7, 38.9, 39.3, 50.2, 53.0, 54.7,
58.1, 127.0, 128.5, 129.1, 136.0, 171.3, 171.7, 172.1. ESI-LCMS: Calcd
for C20H30N10O3, 458.25; found m/z [M + H]+, 459.20, [M + Na]+
481.24, [2 M + H]+ 917.40.
N3-D-Ala-OH (5). N3-D-Ala-OH was synthesized according to a
procedure described by Lundquist and Pelletier,28 which was based on
the method developed by Wong and co-workers.29,30 Azide 5 was
obtained as a yellowish oil in quantitative yield. Rf ) 0.59 (CHCl3/
MeOH/AcOH 95:20:3 v/v/v). FTIR (KBr) ν: 2110 cm-1 1H NMR
.
(CDCl3, 300 MHz) δ: 1.55 (d, 3H, ꢀCH3), 4.03 (m, 1H, RCH). 13C
NMR (CDCl3, 75 MHz) δ: 16.6, 57.0, 177.4.
NR-(Azido)-D-alanyl-phenylalanyl-lysyl-(2-azidoethyl)-amide (6). Pro-
tected dipeptide 4 (8.3 g, 12.2 mmol) was dissolved in piperidine/THF
1:4 v/v (200 mL), and the reaction mixture was stirred for 2 h at room
temperature before the volatiles were removed by evaporation. The
residue was coevaporated with toluene (3×) and CHCl3 (3×) and
subsequently purified by column chromatography (CH2Cl2/MeOH 9:1
v/v) to give the desired H-Phe-Lys(Boc)-(2-azidoethyl)-amide as a
yellow oil in 75% yield (4.2 g). Rf ) 0.42 (CH2Cl2/MeOH 95:5 v/v).
1H NMR (CDCl3, 300 MHz) δ: 1.25-1.68 (m, 5H, ꢀCH2 (1H)/γCH2/
δCH2 Lys), 1.44 (s, 9H, (CH3)3), 1.83 (m, 1H, ꢀCH2 (1H) Lys),
2.71-2.79 (dd (Jax ) 9.1, Jab ) 13.8 Hz), 1H, ꢀCH2 Phe (1H)), 3.09
(m, 2H, εCH2 Lys), 3.21-3.27 (dd (Jax ) 4.0, Jab ) 13.6 Hz), 1H,
ꢀCH2 Phe (1H)), 3.42 (m, 4H, CH2CH2N3), 3.65 (m, 1H, RCH Phe),
4.34 (m, 1H, RCH Lys) 4.59 (broad s, 1H, NH urethane), 6.73 (broad
s, 1H, NH amide), 7.20-7.36 (m, 5H, arom H Phe), 7.75 (broad d,
NH amide). 13C NMR (CDCl3, 75 MHz) δ: 22.6, 28.3, 29.4, 31.7, 38.9,
40.0, 40.7, 50.4, 52.5, 56.1, 78.9, 126.8, 128.6, 129.2, 137.4, 156.0,
171.9, 175.0. In the next step, N3-D-Ala-OH (1.2 g, 9 mmol), H-Phe-
Lys(Boc)-(2-azidoethyl)-amide (4.2 g, 9 mmol), and BOP (4.3 g, 9
mmol) were dissolved in CH2Cl2 (250 mL), and to this solution, DIPEA
(5 mL, 27 mmol) was added dropwise. The reaction mixture was stirred
for 16 h at room temperature. Then, the solvent was removed in vacuo
and the residue was dissolved in EtOAc (250 mL). This solution was
subsequently washed with 1 N KHSO4 (4 × 100 mL), H2O (1 × 100
mL), 5% NaHCO3 (3 × 100 mL), H2O (1 × 100 mL), and brine (3 ×
100 mL), dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by column chromatography (CH2Cl2/MeOH 97:3
v/v) to give the protected tripeptide, N3-D-Ala-Phe-Lys(Boc)-(2-
azidoethyl)-amide, as a white solid in 84% yield (4.2 g). 1H NMR
(CDCl3, 300 MHz) δ: 1.26-1.64 (m, 7H, ꢀCH2 (1H)/γCH2/δCH2 Lys/
ꢀCH3 Ala), 1.44 (s, 9H, (CH3)3), 1.84 (m, 1H, ꢀCH2 Lys (1H)), 3.08
(m, 4H, ꢀCH2 Phe/εCH2 Lys), 3.41 (m, 4H, CH2CH2N3), 4.00 (m, 1H,
RCH Ala), 4.41 (m, 1H, RCH Lys), 4.76 (m, 2H, RCH Phe (1H)/NH
urethane (1H)), 6.80 (broad s, 1H, NH amide), 6.89 (broad s, 1H, NH
Functionalization of Star-Shaped PEG Derivatives with Al-
kyne Moieties. In a typical procedure, PEG derivative 7 (PEG4-10k,
10.2 g, 1.02 mmol) was dissolved in dry THF (150 mL), and to this
solution, NaH (420 mg as a 60%-suspension in mineral oil, 10.5 mmol,
2.5 equiv per arm) was added and the reaction mixture was stirred for
15 min at room temperature. Then, propargyl bromide (600 µL as an
80% solution in toluene, 5.4 mmol, 1.3 equiv per arm) was added, and
the resulting reaction mixture was stirred for 16 h at room temperature.
The solvent was removed in vacuo, and the residue was dissolved in
H2O (150 mL) and dialyzed against demi-H2O (MWCO 3500 Da). After
lyophilization, the alkyne-functionalized PEG derivative 9 was obtained
1
as a white solid in 88% yield (8.8 g). H NMR (CDCl3, 300 MHz) δ:
2.44 (broad s, 4H, (∼O-CH2-C≡CH)4), 3.64 (broad s, ∼904H, (∼O-
(CH2-CH2∼)57)4), 3.88 (m, 8H, C-(CH2-O∼)4), 4.20 (broad s, 8H, (∼O-
CH2-C≡CH)4).
Preparation of the Hydrogels. In a typical gelation experiment (10%
gel, 10 kDa 4-arm PEG, entry 4, Table 1), a stock solution was prepared
that contained PEG derivative 9 (1.35 g, 0.13 mmol ≈ 0.52 mmol alkyne),
NR-(azido)-D-alanyl-phenylalanyl-lysyl-(2-azidoethyl)-amide 6 (120 mg,
0.26 mmol ≈ 0.52 mmol azide), and sodium ascorbate (135 mg, 0.68
mmol), and this mixture was dissolved in H2O (4.5 mL), which resulted
in an alkyne/azide molar ratio of 1. A sample of this stock solution (220
µL) was transferred into a syringe and diluted with H2O (270 µL).
Subsequently, an aliquot (63 µL) of an aqueous solution of CuSO4 (100
mg (0.40 mmol) CuSO4 ·5H2O in 10 mL of H2O) was added, and the
reaction mixture was vortexed. A gel was formed within several minutes.

Enzymatically Biodegradable Hydrogels by Click Chemistry
Biomacromolecules, Vol. 11, No. 6, 2010 1611
Scheme 1. Synthesis of the Bis-azido Peptide, NR-(Azido)-D-alanyl-phenylalanyl-lysyl-(2-azidoethyl)-amide (6)
it can be hydrolyzed by trypsin as well as plasmin.31,32 The
Rheological Characterization. Rheological characterization of the
hydrogels was performed on an AR G-2 Rheometer (TA Instruments)
equipped with a 2° steel cone geometry with a diameter of 40 mm and
a solvent trap. After the addition of the CuSO4 solution to the aqueous
mixture of PEG/peptide/sodium ascorbate, an aliquot (600 µL) of the
reaction mixture was immediately placed between the plates of the
rheometer. Rheological gel characteristics were monitored by oscillatory
time sweep experiments. During the time sweep experiments, the G′
(shear storage modulus) and G′′ (loss modulus) were measured in the
oscillation mode with a controlled strain of 0.1% at a frequency of 1
Hz at 20 °C for a period of 20 min.
Swelling Behavior of the Hydrogels. In a typical swelling experi-
ment the gels (dried with a tissue) were weighed (W0), placed into 100
mM PBS buffer (10 mL, pH 7.4, containing 0.3 mg/mL NaN3), and
incubated at 37 °C. To determine the swelling ratio (defined as Wt/
W0), the gels were removed from the buffer solution at regular time
intervals, dried with a tissue, and weighed (Wt). The buffer solution
was refreshed every 24 h.
Degradation Experiments. The gels were incubated at room
temperature in 0.1 M EDTA solution for 24 h, followed by several
washings with PBS buffer mainly to remove residual copper ions, which
resulted in colorless/opaque hydrogels. Subsequently, the gels were
incubated at 37 °C in 100 mM PBS buffer (5 mL, pH 7.4, 0.3 mg/mL
NaN3), and after 24 h, the gels were dried with a tissue, weighed (W0),
and subsequently incubated in 100 mM PBS buffer (5 mL, pH 7.4, 0.3
mg/mL NaN3) containing either plasmin (2.6 µM) or trypsin (0.8 µM).
At regular time intervals, the gels were removed from the buffer
solution, dried with a tissue, and weighed (Wt). The enzyme-containing
buffer solution was refreshed every 24 h. To study the degradation of
the peptide, bis-azido peptide 6 (3 mg) was dissolved in PBS buffer (1
mL, pH 7.4), and to this solution an aqueous solution of either trypsin
(at a final concentration of 0.8 µM) or plasmin (at a final concentration
of 2.6 µM) was added. The peptide/enzyme mixtures were incubated
at 37 °C. At regular time intervals a sample (100 µL) was drawn and
quenched with ice-cold 1 M NaOAc buffer (200 µL, pH 3.8) and stored
at 4 °C prior to HPLC analysis.
synthesis started with the protection of the amino group with a
Boc functionality and a subsequent substitution of the bromine
with an azide moiety. Azide 2 was obtained in 76% overall
yield based on 2-bromoethylamine hydrobromide 1 as starting
compound. After Boc-removal by treatment with TFA, the
resulting amine was coupled to Fmoc-Lys(Boc)-OH in the
presence of BOP/DIPEA as coupling reagents to give compound
3 in 97% yield over two steps. In the next step, the Fmoc group
was removed by piperidine in THF and after flash chromatog-
raphy the intermediate amine was directly used in the coupling
reaction with Fmoc-Phe-OH in the presence of BOP/DIPEA.
The fully protected dipeptide 4 was obtained as an off-white
solid in 80% yield. Then, treatment with piperidine/THF resulted
in the removal of the Fmoc group and the R-amine was coupled
to N3-D-Ala-OH (5) with BOP/DIPEA to give the protected
tripeptide in 63% yield over two steps. Azide 5 was prepared
by a Cu(II)-catalyzed diazotransfer with triflic azide based on
a procedure as described by Lundquist and Pelletier28 according
to the method of Wong and co-workers.29,30 Finally, the
protected tripeptide was treated with TFA to remove the Boc
group and the bis-azido tripeptide 6 was obtained as a white
solid in quantitative yield. The purity of this bis-azido building
block 6 was analyzed by TLC and HPLC analysis and the
1
identity was characterized by H, 13C, FTIR, and ESI-LCMS.
The functionalization of the star-shaped PEG derivatives 7,
8, and 11 was performed as depicted in Scheme 2. The PEG
alcohols were treated with NaH and subsequently reacted with
propargyl bromide. The alkynes 9, 10, and 12 were purified by
1
dialysis and the degree of substitution was determined by H
NMR spectroscopy, which showed close to quantitative sub-
stitution based on the intensities of the methylene protons of
the PEG chains and the propargyl moieties.
Preparation and Characterization of the Hydrogels. The
hydrogels were prepared by mixing an equimolar amount of
bis-azido peptide 6 with a PEG-alkyne derivative (9, 10, or 12)
in H2O in the presence of sodium ascorbate. To initiate gel
formation an aqueous solution of CuSO4 was added. In general
terms, gel formation was rapid and complete within 5 min at
room temperature, as judged by rheology measurements since
the storage modulus (G′) did not increase after this time point
Results and Discussion
Synthesis and Characterization of the Hydrogel Build-
ing Blocks. The synthesis of bis-azido peptide 6, NR-(azido)-
D-alanyl-phenylalanyl-lysyl-(2-azidoethyl)-amide, was per-
formed as shown in Scheme 1. This tripeptide was chosen since

1612 Biomacromolecules, Vol. 11, No. 6, 2010
Dijk et al.
Scheme 2. Synthesis of the Alkyne-Functionalized Star-Shaped PEG Derivatives, PEG4-10k (9), PEG4-10k (10), and PEG8-20k (12)
(Figure 2). The dried hydrogels were analyzed by FTIR and
the characteristic signal of the azide moiety (at ν 2110 cm-1
was absent, indicating that the azides were quantitatively
transformed into triazoles by reacting with alkynes in the
presence of Cu(I) as the catalyst.
PEG4-10k have the same length and consequently both hydro-
gels have the same average molecular weight between adjacent
cross-links (Mc), the storage modulus is the highest in case of
PEG8-20k due to an increased cross-link density (entries 4, 9,
and 14, Table 1).33
)
The rate of hydrogel formation was followed by rheological
measurements. It turned out that in less than 5 min after the
addition of CuSO4, a fully elastic hydrogel was formed
characterized by a storage modulus (G′) of 25 kPa and tan δ <
0.01, as shown in Figure 2. Furthermore, increasing the solid
content of the hydrogels resulted not only in an increased rate
of hydrogel formation due to an increased concentration of the
mutually reactive azide/alkyne functionalities, but also in an
increased storage modulus of the hydrogel because a higher solid
content resulted in a higher cross-link density, as shown in
Figure 3 and Table 1.
The storage modulus of the hydrogel was also dependent on
the molecular weight and the number of arms of the star-shaped
PEG-alkyne derivatives. Within a series of equal solid content
(e.g., 10%), increasing the molecular weight from PEG4-10k to
PEG4-20k resulted in a decrease of the storage modulus from
24.9 ( 2.3 to 15.5 ( 0.6 kPa (entries 4 and 9, Table 1). This
difference can be explained by the fact that the molar concentra-
tion of the alkyne groups in PEG4-20k is half as in PEG4-10k
and the lower concentration of reactive groups will result in a
lower cross-link density and, thus, lower storage modulus. On
the other hand, at equal polymer concentration, increasing the
number of arms from PEG4-20k to PEG8-20k increased the
storage modulus from 15.5 ( 0.6 to 39.8 ( 1.0 kPa (entries 9
and 14, Table 1). Although the PEG-arms of PEG8-20k and
The average molecular weight between adjacent cross-links
(Mc) was calculated from the plateau modulus (G0), using eq 1,
which was derived from the rubber elasticity theory,34-36 and
have been listed in Table 1.
G0 ) FRT/Mc
(1)
In this equation, F represents the concentration of the polymer
solution (g/m3), R represents the gas constant, and T represents
the absolute temperature. The theoretical Mc values represent
two times the sum of the molecular weight of each PEG-arm
and the bis-azido peptide (PEG4-10k, 5.5 kDa; PEG4-20k, 10.5
kDa; PEG8-20k, 5.5 kDa). The calculated Mc values were found
to be higher than the theoretical values, which can be explained
by imperfections in the hydrogel networks, like loop formation
and the presence of loose ends.37 It should further be mentioned
that eq 1 is only valid for ideal networks. The most pronounced
differences in calculated and theoretical Mc values were found
in hydrogels with the lowest solid content, an indication that at
Figure 3. Kinetics of hydrogel formation as function of solid content.
Reaction conditions: PEG4-10k 9, bis-azido peptide 6 (molar ratio 1:1),
sodium ascorbate, and CuSO4 in H2O at 20 °C. Solid content: 2%
(solid line), 5% (round dot), 7% (square dot), 10% (dash-dot), and
15% (long-dash dot-dot).
Figure 2. Kinetics of hydrogel formation (10% PEG4-10k, entry 4,
Table 1) in H2O (G′ (solid line) and tan δ (dotted line)).

Enzymatically Biodegradable Hydrogels by Click Chemistry
Biomacromolecules, Vol. 11, No. 6, 2010 1613
Figure 4. Swelling ratios (Wt/W0) of hydrogels based on PEG4-10k
(a), and PEG4-20k (b) in PBS buffer (pH 7.4 at 37 °C). Solid content:
5% (filled square), 10% (filled diamond), and 15% (filled triangle).
The data are shown as average ( SD and n ) 3.
Figure 5. The swelling ratios (Wt/W0) of hydrogels based on PEG4-
10k in the presence of trypsin at 0.8 µM (a), and PEG4-10k (b) in the
presence plasmin at 2.6 µM of PBS buffer (pH 7.4 at 37 °C). Solid
content: 5% (filled square), 10% (filled diamond), and 15% (filled
triangle). The data are shown as average ( SD and n ) 3.
a low alkyne/azide concentration the intramolecular loop
formation was preferred over the intermolecular cross-link
reaction. Another indication of loop-formation rather than the
presence of loose ends was found on basis of FTIR analysis,
because the characteristic signal of the azide moiety (ν 2110
cm-1) was clearly absent.
and plasmin.28 Plasmin plays an important role in fibrinolysis
and wound healing, however, it has also a critical role in tumor
cell invasiveness and metastasis39,40 and is present at signifi-
cantly elevated concentrations in tumor tissue.41 The elevated
concentration of plasmin (and other proteolytic enzymes) at the
site of the tumor has been used for triggered drug release from
enzymatically degradable dendrimers.42 Therefore, the tripeptide
D-Ala-Phe-Lys was converted into a bis-azido derivative, azido-
D-alanyl-phenylalanyl-lysyl-(2-azidoethyl)-amide 6, which was
suitable for a Cu(I)-catalyzed 1,3-dipolar cycloaddition with
alkyne-functionalized star-shaped PEG derivatives for the
formation of enzymatically degradable peptide-hydrogels. The
bis-azido peptide 6 was digested in a PBS buffer (100 mM, pH
7.4 at 37 °C) by trypsin (at 0.8 µM) and plasmin (at 2.6 µM)
with half-life times t1/2 of approximately 60 min and 60 h,
respectively, an indication that the N- and C-terminal modifica-
tion were of minimal influence on the hydrolysis rate.
A series of three hydrogels based on PEG4-10k with a solid
content of, respectively, 5, 10, and 15%, were prepared and
incubated with either trypsin (at 0.8 µM) or plasmin (at 2.6
µM) in a PBS buffer (100 mM, pH 7.4 at 37 °C). In the case of
incubation with trypsin, the swelling ratio increased during the
first 20 h to obtain a maximum, after which the swelling ratio
rapidly decreased until the hydrogels were completely degraded
after 40-80 h (Figure 5a). In line with the expectations, because
the hydrogels with the lowest solid content and thus with the
lowest cross-linking density, were degraded most rapidly.
Another series of hydrogels (PEG4-10k, solid content 5, 10,
and 15%, respectively) was incubated with plasmin. Contrary
to our expectations, plasmin was not able to degrade the
The swelling behavior (expressed as swelling ratio: Wt/W0)
of the hydrogels was evaluated by incubating the gels in PBS
buffer (100 mM, pH 7.4 at 37 °C) during a particular time
period. In the case of the hydrogel prepared with PEG4-10k,
the maximal swelling ratio was reached after 20 h of incubation
(Figure 4a, Table 1). Interestingly, the hydrogel with 5% solid
content did not increase in weight, while the hydrogels with 10
and 15% solid content swelled to 1.5 and 1.8 times their initial
weight, respectively (Figure 4a). Apparently, the hydrogel with
5% solid content is dimensionally stable, which could be
explained by the fact that the PEG-chains were already stretched,
which made the hydrogel unable to absorb an additional amount
of water. The swelling behavior of hydrogels based on PEG4-
20k is shown in Figure 4b and Table 1. From the literature it is
known that the degree of equilibrium swelling of a polymeric
hydrogel is inversely proportional to the mechanical strength.33,38
The swelling ratios of the hydrogels based on PEG4-20k were
found to be in agreement with the literature, because the highest
swelling ratios were found with these hydrogels presently
studied even in case of the gel with 5% solid content was found
to absorb almost 80% of its intitial water content (Figure 4b
and Table 1, entry 7).
Enzymatic Degradation of the Hydrogels. The tripeptide
D-alanyl-phenylalanyl-lysine (D-Ala-Phe-Lys) is known from the
literature as a selective substrate for the serine proteases trypsin

1614 Biomacromolecules, Vol. 11, No. 6, 2010
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star-shaped PEG derivatives. The storage modulus of the
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1
Supporting Information Available. Copies of H NMR
spectra of compounds 6, 9, 10, and 12, 13C NMR spectrum and
HPLC and LCMS analysis data of compound 6. This material
is available free of charge via the Internet at http://pubs.acs.org.
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