2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liu et al. Sci China Chem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
chain can form hydrogels or colloidal particles by adding
HRP and H2O2 [23–27].
NCA monomer was added into the solution. After complete
reaction, the solution was precipitated in excess amount of
cold diethyl ether (67.3% yield). The obtained product was
further washed twice with diethyl ether and dry overnight
under vacuum.
Herein, we prepared a novel class of nonionic peptide
amphiphile (PA) by sequential ring-opening polymerization
(ROP) of γ-benzyl-L-glutamate N-carboxyanhydride (BLG-
NCA) and L-tyrosine N-carboxyanhydride (Tyr-NCA) using
hexamethyldisilazane (HMDS) as an initiator, followed by
aminolysis with ethanolamine. These PA samples can readily
form a clear hydrogel with a critical gelation concentration as
low as 0.5 wt%. The hydrogel-to-nanogel transition is ob-
served under physiological conditions in the presence of
horseradish peroxidase (HRP) and hydrogen peroxide
(H2O2).
2.4 Aminolysis of BLG-b-Tyr oligomers
The BLG-b-Tyr oligomer and ethanolamine (EA) (8
equivalent of the benzyloxycarbonyl groups) were dissolved
in dry DMF in a reaction flask with agitator, then 2-hydro-
xypyridine (2-HP) (5 equivalent of the benzyloxycarbonyl
units) was added to as a catalyst. The aminolysis reaction
proceeded under a N2 atmosphere at 50 °C for 48 h. After
that, most of the solution DMF was removed using rotary
evaporator to give a crude concentrated product solution.
The crude product was precipitated in excess diethyl ether.
By re-dissolving the product into deionized water and dia-
lysis (1000 Da molecular weight cutoff) against deionized
water for 48 h, the light yellow spongy powder oligo(L-
glutamate-g-EA)-b-oligo(L-tyrosine) (G-EA)-b-Tyr oligo-
mers was obtained after lyophilization (58.5% yield).
2 Experimental
2.1 Materials and methods
Hexane and tetrahydrofuran (THF) were purified by passing
through activated alumina columns prior to use. HRP, hex-
amethyldisilazane (HMDS, 99%) and ethanolamine (EA)
were purchased from Aladdin (USA). Hydrogen peroxide
(H2O2), diethyl ether and 2-hydroxypyridine (2-HP) were
purchased from Sinopharm Chemical Reagent Co., Ltd.
(China). N,N-Dimethylformamide (DMF) was stored over
calcium hydride (CaH2) and purified by vacuum distillation
with CaH2. Benzyl-L-glutamic acid and L-Tyrosine acid
were purchased from GL Biochem (China) Ltd.
2.5 Preparation of hydrogel
Hydrogel solutions were prepared by dissolving freeze-dried
the oligopeptide samples in deionized water at the desired
concentrations. The homogeneous solutions were obtained
by sonication and blow heating. The formation of the gel was
determined via inverting tube method.
2.2 Synthesis of γ-benzyl-L-glutamate N-carboxyanhy-
dride (BLG-NCA) and L-tyrosine N-carboxyanhydride
(Tyr-NCA)
2.6 Enzymatic reaction
γ-Benzyl-L-glutamate BLG (20 g, 84.3 mmol) was sus-
pended in anhydrous THF (200 mL) at 50 °C and stirred
under N2. Triphosgene (8.88 g, 30 mmol) was then added.
After the reaction solution became clear, the reaction solu-
tion was cooled and then precipitated in n-hexane (500 mL).
The mixture was filtered and recrystallization in THF/hex-
ane. 14.6 g BLG-NCA was obtained. Yield: 68.7%. Tyr-
NCA was synthesized by reacting L-tyrosine with tri-
phosgene in dry THF in N2 atmosphere in a similar way.
Yield: 76.4%.
All samples were prepared in vials by enzymatic oxidation
using HRP and H2O2 at room temperature. In a typical pro-
cedure, (G-EA)4-Tyr2 was dissolved in phosphate buffered
solution (PBS, pH 7.4), freshly prepared PBS solution of
HRP (30 U mL–1 stock solution) and H2O2 were added and
the mixture was gently vortexed, followed by standing
overnight at room temperature. In all cases, the mole ratio of
H2O2 to the phenol group was fixed at 0.5 and the final
concentration is 1 wt%. The experiments for (G-EA)6-Tyr3
and (G-EA)8-Tyr4 were performed in a similar way.
2.3 Synthesis of oligo(γ-benzyl-L-glutamate)-b-oligo(L-
tyrosine) (BLG-b-Tyr) oligomers
2.7 Characterizations
1H NMR spectra were recorded on Bruker AV500 FT-NMR
spectrometer (Germany). FTIR spectra were performed by
using a Bruker HQL005 FTIR spectrometer (Germany) by
using the transmission method (TR). MALDI-TOF mass
spectrometry analysis was performed on a Bruker Microflex-
LRF mass spectrometer. TEM experiments were conducted
on FEI TECNAI 20 (USA). 3 mL of the oligopeptide solu-
BLG-NCA was dissolved in 40 mL dry DMF in a reaction
flask, followed by addition of HMDS in DMF solution at
room temperature. The polymerization was monitored by
observing the disappearance of characteristic peaks from
NCA (1790 and 1850 cm−1) using an FTIR spectrometer at
room temperature. After the peaks disappeared, the Tyr-