Supporting Information
Preparation of Hybrid Triple-Stimuli Responsive Nanogels Based on
Poly(L-Histidine)
Panayiotis Bilalis,*a Spyridon Varlas,a Aikaterini Kiafa,a Athanassios Velentzas,b
Dimitrios Stravopodis,b Hermis Iatrou*a
a
University of Athens, Department of Chemistry, Panepistimiopolis, Zografou,
15771, Athens, Greece
b
University of Athens, Department of Cell Biology & Biophysics, Faculty of Biology,
Panepistimiopolis, Zografou, 15784, Athens, Greece
*Corresponding authors: pbilalis@chem.uoa.gr (P. Bilalis), iatrou@chem.uoa.gr (H.
Iatrou); Tel.: +30-210-7274768; Fax: +30-210-7274186.
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Synthesis of N(im)-trityl-L-histidine N-carboxy anhydride (Trt-His-NCA):
A. Synthesis of Trt-His-NCA·HCl salt. In a round-bottom flask, 10.0 g (20.1 mmol) of
BOC-His(Trt)-OH were added and dried overnight under high vacuum. Then 75 mL
THF were distilled in the flask, giving a clear yellowish solution. Under continuous
argon flow, the reaction flask was placed in an ice-bath and 1.76 mL (24.1 mmol) of
thionyl chloride diluted in 20 mL of THF were added dropwise. After 2h the solution
was poured into excess amount of diethyl ether with precipitation of the Trt-HisNCA·HCl salt as the major product. The resulting solid was filtered and then dried in
vacuo. Recrystallization was conducted to the solid mixture, containing the HCl salt,
the initial substrate and free anhydride by distilling 150 mL of ethyl acetate (EtAc)
under high vacuum. The suspension then was removed from the high vacuum line and
placed into a water bath at 40 °C for 1 hour, resulting in complete dissolution of the
products. The solution was then cooled to 0 °C, yielding to the Trt-His-NCA·HCl salt
as a precipitate, which was filtered and dried overnight in vacuo.
B. Synthesis of Trt-His-NCA. Subsequently, 100 mL of EtAc were distilled into the
flask of the HCl salt of the NCA, which was then filled with argon and placed in an icebath. At 0 °C, 2.25 mL (16.0 mmol) of stoichiometric amount of distilled triethylamine
dissolved in 10 mL of the same solvent were added dropwise under vigorous stirring to
the reaction solution. The resulting salt of triethylamine hydrochloride was filtered off
and the filtrate was poured into 1 L of hexane in order to precipitate and recrystallize
the desired Trt-His-NCA monomer. A second recrystallization occurred with a mixture
of EtAc–hexane (1:5) and the white solid precipitate of Trt-His-NCA (6.1 g, 14.3 mmol,
71.1%) was isolated by filtration, dried overnight and stored in a glove box. The
successful synthesis of Trt-His-NCA was confirmed by
1
H-NMR and FT-IR
spectroscopy (Figure S1). 1H-NMR (300 MHz, CDCl3, δ, ppm): 2.80–3.20 (2H, –CH2–
), 4.50–4.65 (1H, –CH– of the NCA ring), 6.70 (1H, –NH– of the NCA ring), 7.00–
7.45 (16H, ArH of trityl group and N–CH=C of the imidazole ring), 7.65–7.85 (1H, N–
CH=N of the imidazole ring). FT-IR (KBr): 1848, 1780 cm-1 (v C=O, s), 751 cm-1, 706
cm-1 (v =C–H out of plane bend, s).
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Figure S1. FT-IR spectra of: a) the initial substance BOC-His(Trt)-OH and b) the
resulting Trt-His-NCA.
Scheme S2. Conversion reaction of mPEO-OH to mPEO-NH2.
Figure S3. FT-IR spectra of: a) the initial substance (Z-Cys-OH)2 and b) the resulting
L-Cys-NCA.
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Figure S4. 1H-NMR spectrum of the L-Cys-NCA in DMSO-d6.
Figure S5. FT-IR spectra a) of the final deprotected nanogel 35% and b) after
treatment of the sample with 10 mM GSH for 30 min. The characteristic band at 2566
cm-1 reveals the successful reduction of the disulfide bonds to sulfhydryl functional
groups (-SH).
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Figure S6. TEM microphotograph of the synthesized nanogel 12% (the scale bar
represents 100 nm).
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Table S7. Dependence of the nanogel dimensions containing 7% cystine as a function
of pH, temperature and GSH concentration obtained by DLS.
Sample: Nanogel 7%
Temperature
(oC)
25
37
41
Temperature
(oC)
25
37
41
Temperature
25
37
41
(oC)
CGSH (mM)
pH=7.4
Diameter, Dh (nm)
Poly. Index
KCounts
absence
absence
0.2
absence
0.2
64.3
73.0
69.5
95.8
95.4
0.399
0.425
0.481
0.388
0.471
178.7
160.1
180.5
165.3
188.6
CGSH (mM)
pH=6.5
Diameter, Dh (nm)
Poly. Index
KCounts
absence
absence
0.2
10.0
absence
0.2
10.0
76.3
89.0
91.2
388.9
92.2
90.8
535.3
0.545
0.551
0.582
0.894
0.426
0.578
0.944
138.4
135.2
110.8
82.0
139.1
103.9
74.0
CGSH (mM)
pH=5.0
Diameter, Dh (nm)
Poly. Index
KCounts
absence
absence
10.0
absence
10.0
108.8
143.1
210.0
241.5
550.9
0.601
0.483
0.690
0.535
0.796
148.9
153.1
85.8
149.3
65.1
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Table S8. Dependence of the nanogel dimensions containing 12% cystine as a function
of pH, temperature and GSH concentration obtained by DLS.
Sample: Nanogel 12%
Temperature
(oC)
25
37
41
Temperature
(oC)
25
37
41
Temperature
25
37
41
(oC)
CGSH (mM)
pH=7.4
Diameter, Dh (nm)
Poly. Index
KCounts
absence
absence
0.2
absence
0.2
62.2
69.7
65.1
75.4
70.5
0.380
0.370
0.600
0.379
0.559
175.0
170.6
129.1
169.3
135.7
CGSH (mM)
pH=6.5
Diameter, Dh (nm)
Poly. Index
KCounts
absence
absence
0.2
10.0
absence
0.2
10.0
67.8
75.7
73.5
358.6
87.8
97.9
227.3
0.501
0.506
0.611
0.812
0.494
0.573
0.822
134.1
135.2
110.6
57.9
144.3
122.3
50.6
CGSH (mM)
pH=5.0
Diameter, Dh (nm)
Poly. Index
KCounts
absence
absence
10.0
absence
10.0
100.7
130.3
417.9
228.0
514.8
0.497
0.490
0.846
0.590
0.854
144.6
143.6
84.4
128.9
78.8
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Table S9. Dependence of the nanogel dimensions containing 35% cystine as a function
of pH, temperature and GSH concentration obtained by DLS.
Sample: Nanogel 35%
Temperature
(oC)
25
37
41
Temperature
(oC)
25
37
41
Temperature
25
37
41
(oC)
CGSH (mM)
pH=7.4
Diameter, Dh (nm)
Poly. Index
KCounts
absence
absence
0.2
absence
0.2
46.4
56.5
53.1
60.1
65.5
0.398
0.440
0.567
0.563
0.633
159.7
136.0
140.3
174.2
170.1
CGSH (mM)
pH=6.5
Diameter, Dh (nm)
Poly. Index
KCounts
absence
absence
0.2
10.0
absence
0.2
10.0
59.8
61.1
59.7
102.6
71.8
69.8
171.9
0.573
0.542
0.619
0.712
0.495
0.553
0.755
210.3
194.1
231.8
100.7
200.5
195.4
98.3
CGSH (mM)
pH=5.0
Diameter, Dh (nm)
Poly. Index
KCounts
absence
absence
10.0
absence
10.0
67.3
86.2
397.4
84.0
303.7
0.547
0.462
0.844
0.513
0.781
153.4
132.7
80.9
143.8
85.6
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Figure S10. DLS diagrams showing the dimensions dependence of the nanogel 7%
on GSH concentration, at different pH and temperature values.
Figure S11. DLS diagrams showing the dimensions dependence of the nanogel 12%
on GSH concentration, at different pH and temperature values.
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Figure S12. DLS diagrams showing the dimensions dependence of the nanogel 35%
on GSH concentration, at different pH and temperature values.
Figure S13. Polymerization of the polypeptide-based disulfide cross-linked nanogels
after: A) 4 days and B) 8 days of reaction.
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