2.1 Material and methods
General Information: All starting materials and reagents were obtained from commercial sources, and were used as received unless otherwise noted. All solvents were freshly distilled before use.
Chitosan (molecular weight 100-300 kDa and a degree of deacetylation of about 85% determined on the 1H NMR spectrum), Boc-Gly-OH (>98%), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, >98%) 1-Hydroxybenzotriazole hydrate (HOBt, >99%), deuterium oxide (D2O, deuteration degree min. 99.9%) and CD3COOD (99.5%) were used for research.
1H NMR spectra were recorded with spectrometers operating at 600 MHz in D2O with addition of CD3COOD. The infrared analysis of materials was carried out using a PerkinElmer Frontier FT-IR spectrometer. The analyses were performed in transmittance mode in a wavenumber range 4000–516 cm-1, in ATR technique. Spectra were processed in SpectraGryph 1.2.15.
2.2 Results
The aim of the research was to obtain a "nitrogen-enriched" chitosan derivative. The initial plan was to substitute an amino acid containing two nitrogen atoms, such as L-lysine. The first choice was the method of azeotropic distillation of chitosan with L-lysine, in toluene, acc. the procedure described in section 1.2. Unfortunately, despite several repetitions of the reaction, it was not possible to obtain even traces of the product. Changing the amino acid to the smaller L-glycine also had no positive effect. Moreover, the obtained 1H NMR spectra were almost identical to those of the authors,[21] unfortunately the only change in relation to the spectrum of pure chitosan concerned the signal at 3.34 ppm, coming from traces of methanol after washing the product (see S1 in Supplementary Material).
Therefore, the azeotropic distillation method was abandoned in favor of amide bond coupling. The synthesis of L-lysine and chitosan was carried out using coupling reagents EDC and NHS, according to the procedure successfully used for lactobionic acid.[23] Unfortunately, the product was not obtained, probably due to the presence of two unprotected amino groups in the amino acid. These groups can react with the activated carboxyl group of the second molecule, condensing into oligopeptides before being introduced into the chitosan solution, thus preventing the reaction from occurring. For this reason, in the further part of the research, only amino acids with protected amino groups were used.
L-Glycine with a tert-butoxycarbonyl (Boc) group was selected as the first model substrate. Two reactions were carried out according to procedures of the group from the Szczecin University of Technology.[24] In order for the N-acyl form to be the dominant product, both EDC and NHS were used in the reaction. Syntheses were carried out in 1% acetic acid at pH 4.7 and 4.0. In both cases, only traces of the product were obtained – slightly more (3%) at higher pH. The presence of the product was confirmed in the 1H NMR spectrum, but due to the low yield, this method did not give hope for optimization.
The next step was an attempt to activate chitosan by HOBt, with simultaneous activation of the amino acid by carbodiimide EDC according to procedure described above for phenylalanine.[25] This time the synthesis was successful – chitosan substituted with Boc-L-glycine in 26% was obtained.
The highest degree of L-glycine substitution (26%) was obtained using a two-fold excess of coupling reagents (Table 1., No. 1). Attempting to change the solvent to MeOH, as well as eliminating and reducing the amount of water, resulted in a huge decrease in the reaction yield (Table 1, No. 2, 5, 6). We managed to reduce the amount of coupling reagents to one equivalent, which is a great success from the point of view of green chemistry and future application in industry (Table 1., No. 3).
Table 1. Reaction of chitosan and Boc-L-glycine – optimization
No.
|
Chitosan (mmol)
|
HOBt (mmol)
|
Boc-L-Gly (mmol)
|
EDC (mmol)
|
Solvent
|
DS*
|
1
|
1
|
2
|
1
|
2
|
H2O/EtOH (1:1)
|
26%
|
2
|
1
|
2
|
1
|
2
|
H2O/MeOH (1:1)
|
5%
|
3
|
1
|
1
|
1
|
1
|
H2O/EtOH (1:1)
|
21%
|
4
|
1
|
1
|
1
|
1
|
H2O/EtOH (1:1)
|
11.5%**
|
5
|
1
|
2
|
1
|
2
|
Anhydrous EtOH
|
<0.1%
|
6
|
1
|
2
|
1
|
2
|
H2O/EtOH (1:16)
|
3%
|
*The degree of substitution (DS) was calculated from the 1H NMR spectrum
**twice the concentration of reactants
After selecting the optimal reaction conditions, substitution with other L-amino acids were carried out. Syntheses with L-phenylalanine, L-valine and L-proline protected with a Boc group have been carried out. Compared to L-glycine, worse but quite satisfactory results were obtained (Table 2.).
Table 2. Reactions with another amino acids
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)
Reaction conditions: chitosan (1 mmol), HOBt (2 mmol), EDC (2 mmol), amino acid (1 mmol) H2O/EtOH (1:1, 16 ml);
*The degree of substitution (DS) was calculated from the 1H NMR spectrum
The structures of the products were confirmed by 1H NMR and FTIR spectra (see Fig. S2-S10 in Supplementary Material)