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Supplementary material
Synthesis, physicochemical characterization and biological
evaluation of chitosan sulfate as heparan sulfate mimics
Ernesto Doncel-Péreza, Inmaculada Aranazb, Agatha Bastidac, Julia Revueltac, Celia Camachob,
Niuris Acostab, Leoncio Garridod, Concepción Civerab, Eduardo García-Juncedac, Angeles Herasb,
Alfonso Fernández-Mayoralasc, *
a Laboratorio de Química Neuro-Regenerativa, Hospital Nacional de Parapléjicos, SESCAM,
Finca la Peraleda s/n, 45071 Toledo, Spain. b Departamento de Química Física II. Facultad de
Farmacia, Universidad Complutense de Madrid, Plaza de Ramón y Cajal, s/n, 28040 Madrid,
Spain. c Departamento de Química Bio-Orgánica, Instituto de Química Orgánica General (IQOG-
CSIC), CSIC, Juan de la Cierva 3, 28006 Madrid, Spain. d Departmento de Química-Física,
Instituto de Ciencia y Tecnología de Polímeros (ICTP-CSIC), CSIC, Juan de la Cierva 3, 28006
Madrid, Spain.
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Synthesis of N-succinyl Chitosan (SuccCh)
SuccCh was prepared according to a previous reported procedure slightly modified (Wang
et al., 2012). Briefly, chitosan (1 g) was dissolved in 3% (v/v) acetic acid in water (60 mL) and
then methanol (60 mL) was added. Afterwards, succinic anhydride (2 g) was added at room
temperature and the reaction was stirred overnight. After that, the mixture was
concentrated to one-half volume, then water (30 mL) was added and the pH value was
adjusted to 10 with 2N NaOH. The solution was dialyzed against water and lyophilized to
give SuccCh (1.25 g). The degree of N-succinylation was determined by NMR as the ratio of
the amines containing succinyl group to total amines (see Supplementary Fig. S1).
Sulfation
Sulfation reaction of SuccCh was carried out under homogeneous conditions using
chlorosulfonic acid, following the conditions described for sulfation of chitosan (Zhang et
al., 2010). SuccCh (0.5 g) was dissolved in formic acid (15 mL) within 1 h at room
temperature. Then DMF (78 mL) was added, followed by another 2 h of stirring. After that,
chlorosulfonic acid (0.88 mL) in DMF (5 mL) was dropped slowly into the chitosan solution
within 15 min and the mixture was kept at room temperature for 5 h. The resulting yellow
solution was poured into a saturated ethanolic solution of anhydrous sodium acetate (300
mL) and the mixture was kept overnight. The obtained precipitate was washed with
ethanol/water-mixture (4/1, v/v) and then dissolved in water (45 mL) and the pH value of
the solution was adjusted to 7.5. Subsequently, the solution was dialyzed against water and
lyophilized to give sulfated SuccChS (0.61 g).
This procedure was applied for the sulfation of chitosan under different conditions as
indicated in Table 1, to give ChS1-ChS4.
The total degree of sulfation was calculated from elemental analysis of the compounds
according to the following equation: Total DSS = (S%/32.06)/(N%/14.01)
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FIGURE S1. 1H NMR spectrum of compound SuccCh and determination of the degree of N-succinylation.[a]
[a] The degree of N-succinylation was determined as the ratio of the amines containing succinyl group to total amines according to the represented formula, where the values of c, d, d' and e have been calculated by integrating the selected signals of the proton spectrum.
5859606162
6364656667686970717273747576777879808182838485
FIGURE S2. 1H NMR and HSQC (1H-13C) of SuccChS8687
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FIGURE S3. 1H NMR, HSQC (1H-13C) and TOCSY of ChS1
1.71.81.92.02.12.22.32.42.52.62.72.82.93.03.13.23.33.43.53.63.73.83.94.04.14.24.34.44.54.64.74.84.95.0f2 (ppm)
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f1 (
pp
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Chit-O-sulf-1-ghsqcSTANDARD PROTON PARAMETERS
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CHIT51A4-tocsySTANDARD PROTON PARAMETERS
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FIGURE S4. 1H NMR and HSQC (1H-DEPT) of ChS2
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FIGURE S5. 1H NMR and HSQC (1H-DEPT) of ChS3
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Chit-O-sulf-3-ghsqcSTANDARD PROTON PARAMETERS
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FIGURE S6. 1H NMR and HSQC (1H-DEPT) of ChS4 125126
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Solid state NMR of chitosan derivatives.
N-15 solid state NMR measurements were performed on a Bruker Avance
spectrometer equipped with a wide bore superconductive magnet operating at 9.4 T (N-
15 Larmor frequency at 40.56 MHz). Powdered samples were packed in 4 mm zirconia
rotors. The CP/MAS NMR measurements with contact times of 3 ms, repetition rates of
2 s and high-power proton decoupling (75 kHz) were carried out at room temperature,
and spinning rates of 5 kHz. Generally, 45,000 scans were averaged unless specified
otherwise. The spectra were externally referenced to ammonium chloride (-338.1 ppm)
secondary to nitromethane (0.00 ppm).
FIGURE S7. N-15 CP/MAS NMR spectrum corresponding to a sample of SuccChS. The presence of amide (-253 ppm) and amine (-343 ppm) groups are observed.
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FIGURE S8. N-15 CP/MAS NMR spectrum corresponding to a sample of ChS1. The presence of amide (-256 ppm) and amine (-342 ppm) groups are observed.
References
Wang, T., Zhou, Y., Xie, W., Chen, L., Zheng, H., & Fan, L. (2012). Preparation and
anticoagulant activity of N-succinyl chitosan sulfates. International Journal of Biological
Macromolecules, 51, 808-814.
Zhang, K., Helm, J., Peschel, D., Gruner, M., Groth, T., & Fischer, S. (2010). NMR and
FT Raman characterisation of regioselectively sulfated chitosan regarding the
distribution of sulfate groups and the degree of substitution. Polymer, 51, 4698-4705.
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