A Simplified Method of Synthesis to Obtain Zwitterionic Cellulose under Mild Conditions with Active Ionic Moieties

A simplified procedure to synthesize zwitterionic cellulose by means of N-protected aspartic anhydride under mild conditions was developed. The preparation of modified cellulose samples was carried out under heterogeneous, aqueous conditions by reacting NH4OH-activated cellulose with aspartic anhydrides N-protected with trifluoroacetyl (TFAc) and carbobenzyloxy (Cbz). Modified cellulose samples Cel-Asp-N-TFAc and Cel-Asp-N-Cbz were characterized by Fourier Transform Infrared (FTIR) and 13C solid state Nuclear Magnetic Resonance (NMR) spectroscopy. The functionalization degree of each cellulose sample was determined by the 13C NMR signal integration values corresponding to the cellulose C1 vs. the Cα of the aspartate residue and corroborated by elemental analysis. In agreement, both analytical methods averaged a grafting degree of 20% for Cel-Asp-N-TFAc and 16% for Cel-Asp-N-Cbz. Conveniently, Cel-Asp-N-TFAc was concomitantly partially N-deprotected (65%) as determined by the ninhydrin method. The zwitterion character of this sample was confirmed by a potentiometric titration curve and the availability of these amino acid residues on the cellulose was inspected by adsorption kinetics method with a 100 mg L−1 cotton blue dye solution. In addition, the synthesis reported in the present work involves environmentally related advantages over previous methodologies developed in our group concerning to zwitterionic cellulose preparation.


Nuclear Magnetic Resonance Analysis
The estimation of the grafting degree (GD) in cellulose was calculated from the spectra of the samples of cellulose modified with N-protected aspartic anhydride. The protecting groups were trifluoroacetyl (Cel-Asp-N-TFAc) and carbobenzyloxy (Cel-Asp-N-Cbz), as shown in Figures S1 and S2, respectively. For each spectrum, the integration value of the C1 of the anhydroglucose unit (AGU) of cellulose (~106 ppm) was compared with that of the alpha carbon (Cα) of the grafted aspartate residue, in each sample (52-53 ppm), as can be observed in both spectra ( Figure S1 and S2). Furthermore, to provide assurance that the integration values are trustworthy, all the signals have their integration values depicted. The integration values for each carbon in cellulose (110-60 ppm) should be similar, as should be those belonging to the aspartic acid graft [1]. Thus, the signals corresponding to the aspartic graft as beta carbon (Cβ) should have similar integration values to those of the neighboring Cα, in order to consider that the NMR experimental parameter chosen were appropriate. In the case of the spectrum of sample Cel-Asp-N-TFAc, the cellulose carbon integration value had an average of 57 and that for the aspartate Cα and Cβ was around 10.3. Carbon signal integration areas for Cel-Asp-N-Cbz were around 52.5 for cellulose and an averaged value of 7.7 for the aspartic carbons. Accordingly, the reference carbon area was assigned as 100% to that belonging to the C1 of the AGU of cellulose, so the ratio of this value vs the integration value Cα of the aspartate in each sample was used to calculate the degree of substitution.

Elemental Analysis (EA)
Elemental analysis was carried out with around 2 mg of each sample (Cel-Asp-N-TFAc and Cel-Asp-N-Cbz) in duplicated using a Fisons Instrument model EA1108 for Carbon, Hydrogen, Nitrogen and Sulfur (CHNS). The percentage of C, N and H of each sample was determined and averaged to calculate the GD in cellulose. Table S1 shows the resulting data. Here the experimental data from EA of cellulose [2] and modified cellulose are comparable with theoretical values according to the degree of substitution calculated in base of the relationship of AGU´s of cellulose vs aspartate residue. The Cel-Asp-N-TFAc sample shows a ratio of 5:1 anhydoglucose units per aspartate residues, calculated from the C and N content, corresponding to a functionalization (GD) of 20%. In the case of Cel-Asp-N-Cbz a 17% of GD was obtained and is related to the 6:1 ratio of AGU and aspartate residues. Table S1. Elemental analysis data from samples of Cel-Asp-N-TFAc and Cel-Asp-N-Cbz compared with their theoretical functionalization degrees.

Sample
Run

N-Deprotection Degree on Celluloses
Celluloses with N-deprotected aspartates react with a ninhydrin solution developing a blue-purple color due to the Schiff base formation [3]. The deprotection degree in each sample was measured using a UV/Vis spectrophotometer and calculated by reading the absorbance values at 575 nm, employing a calibration curve with different concentrations of amino acid valine (2, 4, 6, 8 and 10 mM) following a modified Moore-Steain methodology [4]. Five hundred µL of valine solution were placed in 5 mL test tubes and then reacted with 1500 µL of 0.5% ninhydrin solution, capped and heated in a water bath at 95 °C during 15 min. After this period, the tubes were cooled to room temperature with tap water. Each tube developed a blue-purple color with different intensities according to its valine concentration (5 to 10 mM). 100 µL of the colored solution were withdrawn from each tube and bought up to 1 mL with 900 µL of distilled water and transferred to a plastic cuvette (for visible spectroscopy). Every solution was measured at 575 nm using distilled water as blank. The absorbance values, as shown in Table S2, were used to construct a calibration curve ( Figure S3). Linear regression analysis was applied to fit the curve, using the equation y = mx−b, where y is the equipment response, m is the slope, c the concentration of the sample and b corresponded to the intercept [5]. Furthermore, squared correlation coefficient, R 2 , was determined by linear regression analysis in order to determine how well the model fits the experimental data. The closer the R 2 value to 1.0, the better the data points are described by linear regression [6]. In our case, R 2 was resulted in 0.9907 as can be seen in the calibration curve shown in Figure S2. Table S2. Absorbance values for valine solutions with different concentrations used for the calibration curve.

Concentration
(mM) Absorbance In the case of modified celluloses, 5 mg of each sample (Cel-Asp-N-TFAc and Cel-Asp-N-Cbz) were suspended in 500 µL of distilled water and reacted with 1500 µL of 0.5% ninhydrin solution, in a 5 mL screw-capped tubes. Placed in a water bath at 95 °C for 20 min, with periodic shaking during 3 sec using a vortex after every 5 min. Again, the tubes were cooled at room temperature with tap water, after which 100 µL of the supernatant were taken and transferred to a visible cuvette using 900 µL of distilled water. The grafting degree is calculated by the ratio of the CAsp/CAGU, which corresponded to 0.1299 ~ 0.13. In other words, the functionalization of aspartate N-deprotected (Cel-Asp-N-H) is 13%. According to 13 C NMR and EA of the Cel-Asp-N-TFAc sample, the total degree of substitution was 20% and the 13% of this grafted aspartate is N-H. It means that N-deprotection occurred in the same process of modification of cellulose reaching up to 65%.