Rapid dissolution of chitin and chitosan with degree of deacetylation less than 80% in KOH/urea aqueous solution†

Yi Zhong‡ ^a, Xi Zhang‡ ^a, Qing Zhang ^a and Jie Cai *^ab
aHubei Engineering Centre of Natural Polymers-based Medical Materials, College of Chemistry & Molecular Sciences, Institute of Hepatobiliary Diseases, Zhongnan Hospital, Wuhan University, Wuhan 430072, China
^bResearch Institute of Shenzhen, Wuhan University, Shenzhen 518057, China. E-mail: caijie@whu.edu.cn

First published on 16th August 2023

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Introduction

Chitin and chitosan are linear 1,4-linked polysaccharides composed of 2-acetamido-2-deoxy-β-D-glucan and 2-amino-2-deoxy-β-D-glucan units, with different degrees of deacetylation (DD) that significantly influence their physical and chemical properties, in vivo degradation, and biological activity.^1–3 Although they exhibit excellent biodegradability, biocompatibility, and biosafety, making them suitable for applications as biomaterials, wearable flexible electronic devices, energy conversion and storage materials, and food packaging,^4–6 their utilization is considerably less investigated.⁷

To maximize the utilization of chitin and chitosan, it is vital to develop an effective and eco-friendly universal solvent with promising applications to facilitate research on their molecular-scale chain conformations and solution properties, enabling advanced applications. However, their backbones contain numerous hydroxyl, hydroxymethyl, acetamido, and amino groups forming strong inter- and intra-molecular hydrogen bonds, and are tightly packed into different crystalline forms,^8–12 inhibiting their melt-processing or dissolution in water and most common solvents. Despite similar chemical structures, chitin and chitosan exhibit significantly different solubilities due to distinct hydrogen-bond networks and aggregate structures. Traditionally, strong acids, inorganic-salt solutions, saturated CaCl₂·2H₂O/methanol, LiCl/dimethylacetamide, and hexafluoroisopropanol are used as chitin solvents,^13–17 while formic acid, acetic acid, dilute hydrochloric acid, and other acidic solutions are used as chitosan solvents.^18–20 However, these solvents exhibit numerous limitations, such as, low solubility, high viscosity, rapid degradation, toxicity, and poor regenerated-material mechanical properties. Although ionic liquids and deep eutectic solvents dissolve chitin and chitosan on heating,^21–26 they are expensive, difficult to produce and recycle, and hazardous to human health and the environment.^27,28

In our previous work, LiOH/urea and NaOH/urea aqueous solutions dissolve cellulose by a hydrogen bond-induced dynamic self-assembly at low temperatures;^29–31 they have also been used for chitin and chitosan dissolution and the regeneration and functionalization of chitin- and chitosan-based materials.^32–35 However, the dissolution involves repeated freezing–thawing cycles causing long production periods, high energy consumption, limited solubility, and difficulties in scaling up. Although KOH aqueous solutions have been used as deacetylation agents for chitin since 1924, they have not been used for chitin and chitosan dissolution.³⁶ Recently, a KOH/urea aqueous solution has been developed by us for chitin and chitosan dissolution within several minutes without any freezing–thawing cycles;^37–40 it produces chitin- and chitosan-based materials that are stronger than those produced from other solvents.^41–45 However, the dissolution mechanism of chitin and chitosan in alkali hydroxide/urea aqueous solutions remains unexplored. Therefore, a systematic investigation of the rapid dissolution of chitin and chitosan with degree of deacetylation less than 80% in KOH/urea aqueous solutions could elucidate the interactions occurring in the system, guiding the rational design of green solvents for chitin and chitosan and the construction of advanced chitin- and chitosan-based materials for a wide range of applications.

Results and discussion

The hydrogen-bond network and crystal structure of α-chitin are more stable than those of β-chitin; thus, shrimp shell-derived α-chitin was firstly used to evaluate the influence of deacetylation on its hierarchical structure.⁴⁶ The α-chitin has hierarchical structure (involved the twisted plywood structure, fibril plane, nanofibrils cluster, and hydrogen-bonded molecular chains) (Fig. 1a), and the deacetylation process generally shows the effect on the hydrogen-bond networks and crystalline structures of α-chitin, deacetylated α-chitin and chitosan (Fig. 1b, see the ESI Fig. S1†). The N-acetylated glucosamine units and glucosamine units were randomly distributed along the molecular chains during the heterogeneous deacetylation process.⁴⁷ Before analysis, purified-shrimp-shell α-chitin was produced by protein and mineral removal. It exhibited clear optically isotropic under polarized light (Fig. 1c). Subsequently, the microstructure of the α-chitin was investigated by scanning electron microscopy (SEM) and atomic force microscopy (AFM) (Fig. 1d–f). Chitin nanofibrils with diameters of 20–50 nm, arranged perpendicular to the shells in planar honeycomb-like arrays, were arranged around tunnel micro-cavities in planes that were stacked with small rotating angles, forming a characteristic twisted plywood-like structure; macropores and mesopores were distributed throughout the structure.^38,46 Deacetylation transformed the α-chitin to chitosan, without changing its appearance and polarized-light birefringence (Fig. 1g); it continued to exhibit twisted plywood-like structure (consisting of chitosan fibril planes, nanofibrils cluster, mesopores, and macropores) (Fig. 1h–j).

The molecular structures and hydrogen-bonding interactions of deacetylated α-chitin (CT-15, 15% of DD; CT-32, 32% of DD) and chitosan (CS-69, 69% of DD; CS-79, 79% of DD) were investigated using Fourier transform infrared spectroscopy (FT-IR) (Fig. 1k). Deacetylation affected the hydrogen bonding interactions between the hydroxyl, hydroxymethyl, acetamido, and amino groups of deacetylated α-chitin and chitosan; thus, deacetylated α-chitin (the CT-series) and chitosan (the CS-series) exhibited different FTIR spectra. CT-15 and CT-32 exhibited α-chitin characteristic absorption peaks at 3483 and 3446 cm⁻¹ (broad and intense) due to O–H stretching, 3267 and 3106 cm⁻¹ due to amide-I N–H, 1660 and 1625 cm⁻¹ for CO stretching (amide I), and 1575 and 1558 cm⁻¹ for N–H bending (amide II), suggesting the presence of inter-chain (N–H⋯OC, CO⋯H–O6, and O6–H⋯O6–H) and intra-chain (O3–H⋯O5′ and O3–H⋯O6′–H) hydrogen-bonding interactions between the deacetylated chitin chains.¹² CS-69 and CS-79 exhibited the characteristic absorption peaks of chitosan (at 3411 and 3307 cm⁻¹ due to O–H stretching vibrations, 1655 cm⁻¹ due to the amide I band, and 1595 cm⁻¹ due to N–H bending vibrations).⁴⁸ As the DD increased, the relative intensity of the bands at 1625 cm⁻¹ (for amide I) and at 1575 and 1558 cm⁻¹ (for amide II) decreased, with the development of a new peak at 1595 cm⁻¹ due to N–H bending; this indicated a reducing of the N–H⋯OC and CO⋯H–O6 hydrogen-bonding interactions and forming of the N–H⋯O6–H hydrogen-bonding interactions between the acetamido, amino, and hydroxymethyl moieties of adjacent chitosan chains. Solid-state cross-polarization/magic angle spinning ¹³C nuclear magnetic resonance (CP/MAS ¹³C NMR) was used to investigate the molecular structures further (Fig. 1l). The CT-15 and CT-32 spectra exhibited eight characteristic resonances of α-chitin.⁴⁹ Contrarily, CS-69 and CS-79 showed seven characteristic resonances of chitosan, with the C3 and C5 resonances merged into a single peak,⁵⁰ while the intensities of the CO and CH₃ resonances decreased significantly, and the C2 resonance shifted downfield by 2.3–2.6 ppm, confirming the deacetylation of α-chitin to chitosan. Furthermore, computing the integral area of the CH₃ carbon compared to that of the anomeric carbon, the DDs of CT-15, CT-32, CS-69, and CS-79 were evaluated to be 15, 32, 69, and 79%, respectively, in agreement with the potentiometric titration analysis (see the ESI Table S1†).

The aggregate structures of deacetylated α-chitin and chitosan were analyzed by X-ray diffraction (XRD) (Fig. 1m). CT-15 showed six characteristic diffraction peaks at approximately 9.2°, 12.7°, 19.2°, 20.8°, 23.2°, and 26.3°, corresponding to the (020), (021), (110), (120), (130), and (013) reflections of α-chitin crystals, respectively.⁵¹ On deacetylation, other than the (020) reflection, which exhibited a slight shift of approximately 0.1° towards larger diffraction angles, the diffraction peaks of CT-32 exhibited a negligible change. The slightly lower lattice spacing in CT-32 (9.57 Å) compared to that in CT-15 (9.59 Å) could be attributed to a steric-hindrance reduction between adjacent chitin chains on deacetylation. In addition, CS-69 exhibited two broad characteristic diffraction peaks at approximately 8.8° and 20.0° due to the (020) and superimposed (200)/(220) reflections of hydrated chitosan crystals, respectively.⁹ On deacetylation, the peak due to the (020) reflection in CS-79 exhibited a slight shift (by ∼0.2°) towards larger diffraction angles, indicating a reduction in the lattice spacing of the hydrated chitosan crystals (from 10.03 to 9.87 Å) due to the presence of more N–H⋯O6–H hydrogen-bonding interactions between adjacent chitosan chains at high DD values. Notably, the lattice spacings of CS-69 and CS-79 were larger than those of CT-15 and CT-32, possibly due to the presence of less inter-chain N–H⋯OC and CO⋯H–O6 hydrogen-bonding interactions in chitosan and the presence of water molecules within hydrated chitosan crystals.⁹ The degree of crystallinity of deacetylated α-chitin and chitosan decreased from 77 to 61% on increasing their DD values (see the ESI Table S1†). Although chitin and chitosan are deacetylated randomly, deacetylation significantly influenced the hydrogen-bond networks and crystalline structures of deacetylated α-chitin and chitosan, and could affect their swelling and dissolution in KOH/urea aqueous solutions.

Subsequently, the swelling behaviors of CT-32 and CS-79 were investigated in water, urea, KOH, and KOH/urea aqueous solutions at 25 °C. On water immersion (Fig. 2a), the CT-32 diffraction pattern was similar to that shown in Fig. 1m, due to the inter- and intra-sheet stability resulting from strong hydrogen-bonding interactions between the deacetylated α-chitin chains as shown in Fig. 1k. CT-32 exhibited two broad scattering peaks at 27.7° and 39.8° owing to background scattering due to air and absorbed water in the amorphous region of deacetylated α-chitin. Notably, the intensity and peak positions of the diffraction peaks changed negligibly on urea-aqueous-solution immersion compared to water immersion, indicating a negligible influence of urea on the deacetylated α-chitin crystals. However, on immersion in KOH and KOH/urea aqueous solutions at 25 °C, the diffraction-peak intensities decreased significantly, with similar peak positions as deacetylated α-chitin. This feature indicated that desolvated K⁺ ions and hydrated OH⁻ ions possibly penetrated into deacetylated α-chitin crystals from the outside to form deacetylated α-chitin-solvent intermediate complexes, weakening the inter-chain N–H⋯OC and CO⋯H–O6 hydrogen-bonding interactions, thereby causing a partial swelling of deacetylated α-chitin in KOH and KOH/urea aqueous solutions at 25 °C.³⁸ Furthermore, the chitosan aggregate structural changes differed from those of deacetylated α-chitin (Fig. 2b). On water and urea-aqueous-solution immersion at 25 °C, CS-79 exhibited two diffraction peaks and two scattering peaks corresponding to the (020) and superimposed (200)/(220) reflections of hydrated chitosan crystals, and background scattering due to air and absorbed water in the amorphous region of chitosan, respectively. Notably, the (020)-reflection peak of CS-79 shifted by approximately 1.7° and 2.0° towards lower diffraction angles on immersion in water and urea aqueous solutions, respectively, whereas the (200)/(220) reflection remained almost constant (at 20.0°). Thus, the lattice spacing between the (020) planes of the hydrated chitosan crystals of CS-79 (9.87 Å) increased to 12.03 Å in water and 12.89 Å in urea aqueous solutions. This anisotropic swelling of chitosan is comparable to that of β-chitin.⁵² Thus, water and urea molecules could intercalated into the (020) planes of hydrated chitosan crystals at ambient temperatures due to weak inter-chain (N–H⋯OC and CO⋯H–O6) and inter-sheet water-bridge hydrogen-bonding interactions mediated by water molecules, while strong intra-sheet N–H⋯O6–H hydrogen-bonding interactions between the amino and hydroxymethyl moieties of adjacent chitosan chains maintained pyranose-ring stacking.⁹ On immersion of CS-79 in a KOH aqueous solution at 25 °C, the diffraction intensity of the (200)/(220)-reflection peak decreased significantly with a slight shift (∼0.1°) towards larger diffraction angles, while the (020)-reflection peak completely disappeared; therefore, chitosan exhibited significant swelling in KOH aqueous solutions to form chitosan-solvent intermediate complexes, while retaining its pyranose-ring stacking. The (200)/(220)-reflection peak intensity of CS-79 reduced more significantly on immersion in KOH/urea aqueous solutions compared to only KOH aqueous solutions, indicating that urea molecules promoted the swelling of chitosan in the presence of KOH.

Deacetylated α-chitin and chitosan rapidly dissolved in KOH/urea aqueous solutions at low temperatures forming clear and viscous solutions (Fig. 2c; see the ESI Movies S1–S4†). Optical microscopy indicated that CT-15 and CT-32 exhibited moderate and significant swelling at −25 °C in the KOH/urea aqueous solution (Fig. 2d and e), with complete dissolution on stirring at −25 and −22 °C (see the ESI Movies S5 and S6†). Notably, CS-69 and CS-79 exhibited significant swelling on immersion in the KOH/urea aqueous solution at an ambient temperature, with fast dissolution on decreasing the temperature to 10 and 0 °C (Fig. 2f and g). Furthermore, under stirring, CS-69 and CS-79 exhibited complete dissolution at 15 and 5 °C (see the ESI Movies S7 and S8†) due to the presence of less inter-chain N–H⋯O6–H hydrogen-bonding interactions between the amino and hydroxymethyl moieties of adjacent chitosan chains at higher DD values. Notably, the optimal dissolution temperature of deacetylated α-chitin was higher than that of purified α-chitin (−30 °C),³⁸ while chitosan dissolved towards ambient temperatures (Fig. 2h). The same phenomenon was observed in the deacetylated β-chitin and chitosan. Thus, deacetylation significantly influenced the hydrogen-bond networks and crystalline structures of α-chitin and chitosan, facilitating the swelling and dissolution of deacetylated α-chitin and chitosan in KOH/urea aqueous solutions. However, chitosan with DD value higher than 80% obtained through repeated deacetylation of chitin was not soluble in the KOH/urea aqueous solution, even after undergoing freeze–thaw cycles. Further research is necessary to investigate the dissolution of chitosan with DD exceeding 80%.

In situ two-dimensional wide-angle X-ray diffraction (2D WAXD) (Fig. 3) was further used to investigate the aggregate structural changes of deacetylated α-chitin and chitosan in KOH/urea aqueous solutions during cooling process; the corresponding azimuthal-averaging XRD profiles were also analyzed. Significant changes were observed in the 2D WAXD patterns of deacetylated α-chitin and chitosan on decreasing the temperature. CT-15 exhibited the 2D WAXD pattern of a typical semi-crystalline polymer with an amorphous halo and several strong Bragg diffractions (Fig. 3a, left) corresponding to the characteristic (020), (021), (110), (120), (130), and (013) reflections of α-chitin crystals. After immersion in the KOH/urea aqueous solution, the Debye rings of CT-15 transformed into foggy arcs and eventually faded as the temperature decreased from −15 to −30 °C (Fig. 3a, right). Due to air and solvent scattering, a fuzzy ring was superimposed on the typical diffraction pattern of the α-chitin crystals. The intensity of the diffraction peaks decreased significantly on gradually reducing the temperature to −25 °C, while their positions remained similar to those exhibited by deacetylated α-chitin (Fig. 3b). This feature is different with the swelling behavior of α-chitin in NaOH and hydrazine aqueous solution,¹¹ most likely due to the desolvated K⁺ ions, hydrated OH⁻ ions and urea molecules could rapidly penetrated and dissolved deacetylated α-chitin crystals from the outside to form deacetylated α-chitin–KOH–urea complexes, making it difficult to determine the swelling and dissolution processes in real-time with decreasing temperature of the KOH/urea aqueous solution.³⁸ All these deacetylated-α-chitin-crystal diffraction peaks were absent at −30 °C, indicating a complete disruption of inter-chain N–H⋯OC, CO⋯H–O6, and O6–H⋯O6–H hydrogen-bonding interactions, causing the rapid swelling and completely dissolution of deacetylated α-chitin in KOH/urea aqueous solutions. Furthermore, the CT-15 solution exhibited similar 2D WAXD patterns at −30 °C and after cooling and subsequent thawing (−60 to 20 °C), indicating deacetylated α-chitin dissolution in the KOH/urea aqueous solution prior to freezing. Interestingly, CT-32 and CT-15 exhibited similar trends, with an exception (the diffraction peaks of CT-32 disappeared at a higher temperature (−25 °C) than those of CT-15) (Fig. 3c and d), possibly due to less inter-chain N–H⋯OC and CO⋯H–O6 hydrogen-bonding interactions at higher DD values, which facilitated the intercalation of desolvated K⁺ ions, hydrated OH⁻ ions, and hydrated urea molecules into the deacetylated α-chitin crystals, causing swelling and dissolution at higher temperatures during the cooling process.

Chitosan exhibited different 2D WAXD patterns (Fig. 3e and g, left), including an amorphous halo and two strong Bragg diffractions corresponding to the (020) and superimposed (200)/(220) reflections from hydrated chitosan crystals.⁹ On immersing CS-69 in the KOH/urea aqueous solution at 25 °C, the Debye ring corresponding to the superimposed (200)/(220) reflections of hydrated chitosan crystals transformed into a cloudy ring, whereas the (020) reflection disappeared (Fig. 3e, right). This indicated the formation of chitosan-solvent intermediate complexes with a retention of pyranose-ring stacking due to less inter-chain (N–H⋯OC and CO⋯H–O6) and weak inter-sheet water-bridge hydrogen-bonding interactions in the hydrated chitosan crystals, and more intra-sheet N–H⋯O6–H hydrogen-bonding interactions between the acetamido, amino, and hydroxymethyl moieties of adjacent chitosan chains.⁹ On decreasing the temperature from 20 to 10 °C, the Debye ring corresponding to the chitosan-solvent intermediate complexes disappeared, and only a fading ring due to scattering by the chitosan-solvent complex and solvents was observed. As indicated by the azimuthal-averaging XRD profiles (Fig. 3f), the (020)-reflection peak of the hydrated chitosan crystals almost disappeared on reducing the temperature, while the diffraction intensity of the superimposed (200)/(220)-reflection peak significantly decreased. At 15 °C, the superimposed (200)/(220)-reflection peak of the hydrated chitosan crystals disappeared, indicating a complete disruption of inter- and intra-chain (N–H⋯OC, CO⋯H–O6, O6–H⋯O6–H, and N–H⋯O6–H) and water-bridge hydrogen-bonding interactions between adjacent chitosan chains, enabling the rapid dissolution of chitosan in the KOH/urea aqueous solution. The 2D WAXD patterns of the CS-69 solution at 15 °C and on lowering the temperature further (to −60 °C) and thawing (to 20 °C) were identical, confirming chitosan dissolution in the KOH/urea aqueous solution before freezing. The CS-79 and CS-69 patterns exhibited similar characteristics (Fig. 3g and h), with an exception (all the hydrated-chitosan-crystal diffractions disappeared at 5 °C for CS-79), consistent with the optical-microscopy results (Fig. 2f and g), possibly due to more intra-sheet N–H⋯O6–H hydrogen-bonding interactions between adjacent chitosan chains at higher DD values, which caused a lower dissolution temperature in the KOH/urea aqueous solution. Notably, chitosan exhibited more facile swelling and dissolution in the KOH/urea aqueous solution than α-chitin and deacetylated α-chitin due to less inter-chain (N–H⋯OC and CO⋯H–O6) and weak inter-sheet water-bridge hydrogen-bonding interactions in the hydrated chitosan crystals, facilitating the intercalation of desolvated K⁺ ions, hydrated OH⁻ ions, and hydrated urea molecules.

To understand the dissolution mechanism of deacetylated α-chitin and chitosan, the effects of alkali-hydroxide species (KOH, NaOH, and LiOH) on their aggregate structures at various temperatures were investigated. According to previous publications, the swelling and dissolving ability of alkali-hydroxide aqueous solutions towards α-chitin follows the order KOH ≫ NaOH > LiOH.³⁸ Here, for deacetylated α-chitin, although the inter-chain N–H⋯OC and CO⋯H–O6 hydrogen-bonding interactions were weakened, the swelling and dissolving ability of alkali-hydroxide aqueous solutions followed the same order (KOH ≫ NaOH > LiOH (not shown)), indicating similar dissolution mechanisms for deacetylated α-chitin and α-chitin.³⁸ However, for chitosan, the dissolving ability of alkali-hydroxide aqueous solutions followed the order KOH ≫ LiOH > NaOH. In KOH aqueous solutions, the Debye ring of the hydrated chitosan crystals gradually disappeared as the temperature reduced from 20 to −20 °C (see the ESI Fig. S2†), indicating the facile intercalation of desolvated K⁺ ions and hydrated OH⁻ ions into the hydrated chitosan crystals, disrupting inter-chain (N–H⋯OC, CO⋯H–O6, O6–H⋯O6–H, and N–H⋯O6–H) and water-bridge hydrogen-bonding interactions between the acetamido, amino, hydroxyl, and hydroxymethyl moieties of adjacent chitosan chains at low temperatures. Therefore, the rapid swelling and dissolution of chitosan in the KOH/urea aqueous solution was mainly due to the dissolving ability of KOH, with urea molecules enhancing the dissolution. In contrast, NaOH and LiOH aqueous solutions exhibited poor chitosan complexation capabilities (see the ESI Fig. S3 and S4†). The (020)-reflection peak of the hydrated chitosan crystals disappeared after immersion in NaOH and LiOH aqueous solutions, whereas the superimposed (200)/(220)-reflection peak remained during the cooling process. This indicated that hydrated Na⁺ and Li⁺ ions, with larger hydration-ion sizes, intercalated into the [020] plane of the hydrated chitosan crystals to form chitosan-solvent intermediate complexes due to weak inter-sheet water-bridge hydrogen-bonding interactions; however, it was challenging to disrupt the strong intra-sheet N–H⋯O6–H hydrogen-bonding interactions between the amino and hydroxymethyl moieties of adjacent chitosan chains. Consequently, chitosan exhibited swelling without dissolution in NaOH and LiOH aqueous solutions during the cooling process. Therefore, the swelling and dissolution of deacetylated α-chitin and chitosan in alkali-hydroxide aqueous solutions exhibited ion specificity due to the hydrogen-bond networks and aggregate structures of deacetylated α-chitin and chitosan and the hydration structure of the alkali hydroxide species.

Furthermore, N-acetyl-D-glucosamine (GlcNAc) and D-glucosamine hydrochloride (GlcN·HCl) monosaccharides were chosen as model compounds for chitin and chitosan, respectively, to determine their molecular mechanisms of dissolution in KOH/urea aqueous solutions. As reported previously, strong ion-dipole and hydrogen-bonding interactions exist between desolvated K⁺ ions, hydrated OH⁻ ions, and GlcNAc, and urea molecules preferentially bind to the hydrophobic regions of GlcNAc.³⁸ Under alkaline conditions, GlcN·HCl was transformed to GlcN and underwent mutarotation, exhibiting two sets of chemical shifts corresponding to the α- and β-anomers of GlcN (Fig. 4a). ¹H and ¹³C NMR experiments with different KOH concentrations were used to examine the interactions between GlcN and KOH/D₂O (Fig. 4b–d; see the ESI Fig. S5†). In GlcN/D₂O, a strong proton signal at 4.74 ppm, attributed to HOD resonance due to rapid proton exchange between the active hydroxyl and N–H protons of GlcN and D₂O, shifted downfield to 5.16 ppm on increasing the concentration of KOH from 0 to 20 wt%. This is similar to the behavior of GlcNAc in KOH/D₂O.³⁸ Thus, the active protons of GlcN were significantly electron-deshielded, and GlcN exchanged active protons from its hydroxyl and N–H species with water molecules readily in the presence of KOH. Additionally, the GlcN-residue anomeric-proton characteristic peaks were observed at 5.18 and 5.19 ppm (H1) for the α-anomer and 4.55 and 4.57 ppm for the β-anomer in D₂O, with a slight downfield shift (0.03–0.06 ppm) in KOH/D₂O due to the pH-dependent mutarotation of the α- and β-anomers. Furthermore, chemical shifts (δ) in the range of 2.54–2.69 ppm corresponded to H2, and δ_H values in the range of 3.31–3.88 ppm were assigned to the overlapping resonance of the non-anomeric protons (H3–H6) of GlcN in D₂O. On increasing the KOH concentration from 4 to 20 wt%, all the δ_H values of GlcN shifted upfield by 0.12–0.25 ppm, indicating a greater electron shielding of all the GlcN backbone protons due to intensified hydrogen-bonding interactions between KOH and the amino groups, hydroxyl, and hydroxymethyl moieties of GlcN. Additionally, the δ_H values of GlcN shifted downfield by less than 0.02 ppm in urea/D₂O on increasing the urea concentration from 0 to 20 wt%, while the active protons shifted upfield by less than 0.05 ppm (see the ESI Fig. S6a†), owing to the facile replacement of the GlcN-solvation-shell water molecules by urea molecules, without a disruption of the GlcN-water molecule hydrogen-bonding network.^53–55 Furthermore, the carbon-nuclei resonances of GlcN in D₂O exhibited characteristic peaks corresponding to the α- and β-anomers of GlcN (Fig. 4d); no carbon resonance was observed for CO. As the KOH concentration increased from 0 to 20 wt%, these carbon resonances significantly shifted downfield (by 0.9–5.1 ppm), indicating a greater electron deshielding of the GlcN carbon nuclei due to enhanced hydrogen-bonding interactions between KOH and GlcN. In contrast, all the δ_C values of GlcN shifted downfield by 0.08–0.17 ppm in urea/D₂O on increasing the urea concentration from 0 to 20 wt%, while the δ_C of urea remained 162.7 ppm, regardless of the urea concentration (see the ESI Fig. S6b†). This indicated a preferential binding of the urea molecules with the hydrophobic regions of GlcN.

¹H and ¹³C NMR analyses of GlcN in LiOH/D₂O, NaOH/D₂O, and KOH/D₂O solutions with the same molar concentration were used to investigate the chitosan-alkali hydroxide interactions further (Fig. 4e–g; see the ESI Fig. S7†). Compared to the ¹H NMR spectrum of GlcN in KOH/D₂O, the δ_H values of the active protons exhibited downfield shifts by 0.16 ppm in LiOH/D₂O and 0.06 ppm in NaOH/D₂O, possibly due to the strong hydration capacity of the Li⁺ and Na⁺ ions. Furthermore, all the δ_H values of GlcN in KOH/D₂O exhibited slight upfield shifts (by less than 0.02 ppm) relative to LiOH/D₂O and NaOH/D₂O, indicating a significant electron shielding of these protons in KOH due to strong hydrogen bonding interactions between KOH and GlcN. Moreover, the active protons of GlcN in KOH/urea/D₂O appeared 0.07 ppm downfield compared to those in KOH/D₂O, while the δ_H values of GlcN changed negligibly, confirming a preferential binding of the urea molecules with the hydrophobic regions of GlcN. The GlcN-alkali hydroxide interactions also affected the electron density of the GlcN backbone carbons. At the same molar concentration, all the carbon resonances of GlcN exhibited a larger downfield shift in KOH (by 0.2–0.4 ppm) than in NaOH and LiOH (Fig. 4g), indicating that the GlcN carbon nuclei were electron deshielded in KOH to a significantly greater extent due to conformation transition of GlcN caused by KOH. Furthermore, the shapes and relative positions of all the GlcN carbon resonances in KOH/D₂O and KOH/urea/D₂O were similar, confirming a preferential binding of the urea molecules to the hydrophobic regions of GlcN through favorable hydrophobic interactions.

¹H and ¹³C NMR were used to analyze deacetylated α-chitin and chitosan in KOH/urea/D₂O to understand the dissolution mechanism at the molecular level (Fig. 5). The ¹H NMR spectra exhibited a prominent resonance peak at 4.83 ppm (Fig. 5a) due to HOD generated by rapid proton exchange between the active protons and D₂O; δ_H values in the range of 2.0–4.0 ppm could be attributed to backbone protons (H1–H6), while the acetyl proton peak appeared at 1.5 ppm. Considering the signal-strength variations with the DD values, the H1, H2, and H3 of deacetylated α-chitin and chitosan could be identified as the GlcNAc (A) and GlcN (D) residues.⁵⁶ The δ_H values of 3.93, 2.83, and 3.27 ppm could be assigned to H-1A, H-2A, and H-3A of the GlcNAc residues, respectively, while 3.86, 2.07, and 3.40 ppm could be assigned to H-1D, H-2D, and H-3D of the GlcN residues, respectively. The intensities of the GlcNAc protons (H-1A, H-2A and H-3A) decreased, and the GlcN protons (H-1D, H-2D and H-3D) shifted downfield on increasing the DD values from deacetylated α-chitin to chitosan, indicating a significant electron shielding of the deacetylated α-chitin chains. Furthermore, the ¹³C NMR spectra of the deacetylated α-chitin and chitosan solutions exhibited a prominent resonance signal at 162.3 ppm (Fig. 5b) corresponding to the CO of urea molecules. The δ_C values of 174.0, 101.1, and 22.1 ppm corresponded to the CO, C1, and CH₃ resonances, respectively, of deacetylated α-chitin and chitosan. The intensity of the CO and CH₃ resonances decreased on increasing the DD values of deacetylated α-chitin and chitosan. Additionally, δ_C values in the range of 50–85 ppm were attributed to the C2–C6 resonances of deacetylated α-chitin and chitosan, 56.2, 72.0, and 79.0 ppm were attributed to the C-2A, C-3A, and C-4A of the GlcNAc residues, respectively, and 57.2, 73.8, and 78.2 ppm were attributed to the C-2D, C-3D, and C-4D of the GlcN residues, respectively. Notably, the C-2A and C-3A of the GlcNAc residues exhibited a higher electron shielding than the GlcN-residue carbons, indicating stronger ion-dipole and hydrogen-bonding interactions between KOH and the acetamido and hydroxyl groups of the deacetylated α-chitin chains than the chitosan chains. Additionally, no new proton and carbon signals were observed in the ¹H and ¹³C NMR spectra, indicating no derivatization; thus, the KOH/urea aqueous solution was a non-derivative solvent for deacetylated α-chitin and chitosan at the molecular level.

The morphology of deacetylated α-chitin and chitosan in the KOH/urea aqueous solution were analyzed by AFM and transmission electron microscopy (TEM) (Fig. 6a–i). At a concentration of 1.0 μg mL⁻¹, the dissolved deacetylated α-chitin and chitosan chains self-assembled into dendritic structures composed of bundles of deacetylated α-chitin and chitosan chains with heights in the range of 1.05 ± 0.39 to 1.69 ± 0.42 nm (Fig. 6a–d; see the ESI Fig. S8†). At a concentration of 0.1 μg mL⁻¹, deacetylated α-chitin and chitosan exhibited the isolated semi-stiff chain conformation of structural polysaccharides (Fig. 6e–h) due to intra-chain O3–H⋯O5′ and O3–H⋯O6′–H hydrogen-bonding interactions along their chains. Statistical analysis indicated that the isolated deacetylated α-chitin and chitosan chains exhibited relative heights in the range of 0.47 ± 0.08 nm to 0.55 ± 0.09 nm (see the ESI Fig. S9†), within the thickness (0.48 nm) and width (0.94 nm) of pyranose rings,⁵⁷ confirming their dissolution in the KOH/urea aqueous solution at the molecular level. Notably, they exhibited facile lateral aggregation into twisted right-handed helical structures composed of nanofibrils in ethanol solution (Fig. 6i–l; see the ESI Fig. S10†), possibly due to chiral supramolecular inter-chain interactions (dominated by steric repulsion), van der Waals dispersion interactions, pyranose-ring hydrophobic stacking,⁵⁸ intrachain hydrogen bonds^59,60 and inter-chain hydrogen-bonding interactions between adjacent deacetylated α-chitin and chitosan chains. The right-handed twist of nanofibril bundles could be explained by the chirality transfer of β-D-glucan during self-assembly.⁶¹ The minimum diameter, maximum diameter, and period length of the twisted nanofibrils were labeled t, d, and p, respectively, and an assumed model was proposed for the twisted-structure repeating units (Fig. 6m). Although they exhibited different DD values, the twisted deacetylated α-chitin and chitosan nanofibers exhibited a similar set of t, d, and p parameters in the ranges of 7.0 ± 0.6 to 9.1 ± 0.9 nm, 13.2 ± 0.4 to 14.2 ± 0.5 nm, and 49.1 ± 4.8 to 53.6 ± 3.8 nm, respectively. Furthermore, the XRD and CP/MAS ¹³C NMR (Fig. 6n and o) confirmed that the self-assembled deacetylated α-chitin and chitosan nanofibrils were α-chitin and hydrated chitosan crystals, respectively.^49–51 Therefore, these results indicated twisted right-handed helical nanofibril formation by the lateral aggregation and recrystallization of deacetylated α-chitin and chitosan chains in a non-solvent. Moreover, the M_η and DD of self-assembled deacetylated α-chitin and chitosan nanofibrils exhibited slight changes (Table S1†), suggesting that the dissolution process did not result in significant degradation or deacetylation of the chitin and chitosan chains.

Based on the above results, a desolvation–intercalation dissolution mechanism was proposed for deacetylated α-chitin and chitosan in KOH/urea aqueous solutions (Fig. 7). Although deacetylation did not significantly influence the twisted plywood-like structure of α-chitin, it affected the hydrogen-bond networks and aggregate structures of deacetylated α-chitin and chitosan, causing significantly different swelling and dissolution behaviors in aqueous KOH/urea solutions. The dissolution of deacetylated α-chitin and chitosan involved the top-down disintegration of their hierarchical structure. Most important, desolvated K⁺ ions and hydrated OH⁻ ions intercalated into the deacetylated α-chitin crystals from the outside to form deacetylated α-chitin-solvent intermediate complexes due to less inter-chain N–H⋯OC and CO⋯H–O6 hydrogen-bonding interactions, causing partial swelling in the KOH/urea aqueous solution at ambient temperatures (Fig. 7a). On decreasing the temperature to −30 °C, the deacetylated α-chitin crystals expanded along the [020] and [110] directions, and the inter-chain N–H⋯OC, CO⋯H–O6, and O6–H⋯O6–H hydrogen-bonding interactions were disrupted completely by strong ion-dipole and hydrogen-bonding interactions between the K⁺ and OH⁻ ions and the acetamido, amino, hydroxyl, and hydroxymethyl moieties of the deacetylated α-chitin chains, causing rapid swelling and dissolution in the KOH/urea aqueous solution. For comparison, desolvated K⁺ ions and hydrated OH⁻ ions were intercalated into hydrated chitosan crystals. Chitosan exhibited rapid and highly anisotropic swelling along the [020] direction in the KOH/urea aqueous solution at ambient temperatures to form chitosan-solvent intermediate complexes with pyranose-ring stacking retention due to less inter-chain (N–H⋯OC and CO⋯H–O6) and weak inter-sheet water-bridge hydrogen-bonding interactions, and strong intra-sheet N–H⋯O6–H hydrogen-bonding interactions between adjacent chitosan chains (Fig. 7b). On decreasing the temperature, the swollen hydrated chitosan crystals expanded along the [200]/[220] directions, and the inter-chain N–H⋯OC, CO⋯H–O6, O6–H⋯O6–H, and N–H⋯O6–H hydrogen bonding, and water-bridge hydrogen-bonding interactions between adjacent chitosan chains were completely disrupted by ion-dipole and hydrogen-bonding interactions between the K⁺ and OH⁻ ions and the acetamido, amino, hydroxyl, and hydroxymethyl moieties of the chitosan chains, rapidly dissolving chitosan in the KOH/urea aqueous solution. Additionally, urea molecules did not cleave the hydrogen-bond networks of deacetylated α-chitin and chitosan; they exhibited preferential binding to the hydrophobic regions of their backbones, improving the solubility and stability of deacetylated α-chitin and chitosan solutions. Moreover, the isolated deacetylated α-chitin and chitosan chains exhibited a strong tendency for lateral aggregation and recrystallization to form twisted right-handed helical nanofibrils with α-chitin and hydrated chitosan crystals, driven by the entropy of hydrogen-bonding and hydrophobic interactions (Fig. 7c).

Conclusion

In this study, the rapid dissolution of chitin and chitosan with degree of deacetylation less than 80% in KOH/urea aqueous solution have been comprehensively investigated. The optimal dissolution temperature of α-chitin and chitosan exhibited a temperature-degree of deacetylation dependency. In addition, a new desolvation–intercalation dissolution mechanism for the dissolution of deacetylated α-chitin and chitosan in KOH/urea aqueous solutions was also proposed. The hydrogen-bond networks and aggregate structures of deacetylated α-chitin and chitosan significantly influenced the swelling and dissolution, driven by strong ion-dipole and hydrogen-bonding interactions between desolvated K⁺ ions, hydrated OH⁻ ions and the acetamide, amino, hydroxyl, and hydroxymethyl moieties of adjacent deacetylated α-chitin and chitosan chains. Moreover, urea molecules underwent preferential binding with the hydrophobic regions of deacetylated α-chitin and chitosan, improving the solubility and stability of their solutions. In addition, isolated deacetylated α-chitin and chitosan chains showed a strong tendency for lateral aggregation and recrystallization to form twisted right-handed helical nanofibril structures with α-chitin and hydrated chitosan crystals in non-solvents. This study provides a new molecular-level insight into the dissolution mechanism of chitin and chitosan, and the techniques and concepts described here could guide future research on biomacromolecules.

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

This research was funded by the National Natural Science Foundation of China (52173105, 21875170). The authors thank to the facility support of the Fundamental Research Funds for the Central Universities (2042023kfyq05), the Special Funds for Guiding Local Science and Technology Development of Central Government (XZ202202YD0021C), and the Basic Research Program of Central Government Funds for Shenzhen Science and Technology Development (2021Szvup099).

References

1. L. Bai, L. Liu, M. Esquivel, B. L. Tardy, S. Huan, X. Niu, S. Liu, G. Yang, Y. Fan and O. J. Rojas, Nanochitin: Chemistry, Structure, Assembly, and Applications, Chem. Rev., 2022, 122(13), 11604–11674 CrossRef CAS PubMed .
2. M. N. V. R. Kumar, R. A. A. Muzzarelli, C. Muzzarelli, H. Sashiwa and A. J. Domb, Chitosan Chemistry and Pharmaceutical Perspectives, Chem. Rev., 2004, 104(12), 6017–6084 CrossRef PubMed .
3. M. Rinaudo, Chitin and Chitosan: Properties and Applications, Prog. Polym. Sci., 2006, 31(7), 603–632 CrossRef CAS .
4. W. Suginta, P. Khunkaewla and A. Schulte, Electrochemical Biosensor Applications of Polysaccharides Chitin and Chitosan, Chem. Rev., 2013, 113(7), 5458–5479 CrossRef CAS PubMed .
5. A. Anitha, S. Sowmya, P. T. S. Kumar, S. Deepthi, K. P. Chennazhi, H. Ehrlich, M. Tsurkan and R. Jayakumar, Chitin and Chitosan in Selected Biomedical Applications, Prog. Polym. Sci., 2014, 39(9), 1644–1667 CrossRef CAS .
6. M. Wu, X. Zhang, Y. Zhao, C. Yang, S. Jing, Q. Wu, A. Brozena, J. T. Miller, N. J. Libretto and T. Wu, et al. A High-performance Hydroxide Exchange Membrane Enabled by Cu²⁺-crosslinked Chitosan, Nat. Nanotechnol., 2022, 17(6), 629–636 CrossRef CAS PubMed .
7. N. Yan and X. Chen, Don't Waste Seafood Waste, Nature, 2015, 524(7564), 155–157 CrossRef CAS PubMed .
8. T. Yui, K. Imada, K. Okuyama, Y. Obata, K. Suzuki and K. Ogawa, Molecular and Crystal Structure of the Anhydrous Form of Chitosan, Macromolecules, 1994, 27(26), 7601–7605 CrossRef CAS .
9. K. Okuyama, K. Noguchi, T. Miyazawa, T. Yui and K. Ogawa, Molecular and Crystal Structure of Hydrated Chitosan, Macromolecules, 1997, 30(19), 5849–5855 CrossRef CAS .
10. Y. Nishiyama, Y. Noishiki and M. Wada, X-ray Structure of Anhydrous β-Chitin at 1 Å Resolution, Macromolecules, 2011, 44(4), 950–957 CrossRef CAS .
11. D. Sawada, Y. Ogawa, Y. Nishiyama, E. Togawa, S. Kimura and P. Langan, Molecular Interactions in an α-Chitin/Hydrazine Complex: Dynamic Hydrogen Bonds and Improvement of Polymeric Crystallinity, Cryst. Growth Des., 2016, 16(6), 3345–3352 CrossRef CAS .
12. Y. Ogawa, C. M. Lee, Y. Nishiyama and S. H. Kim, Absence of Sum Frequency Generation in Support of Orthorhombic Symmetry of α-Chitin, Macromolecules, 2016, 49(18), 7025–7031 CrossRef CAS .
13. D. Gagnaire, J. Saint-Germain and M. Vincendon, NMR Studies of Chitin and Chitin Derivatives, Makromol. Chem., 1982, 183(3), 593–601 CrossRef CAS .
14. M. Vincendon, Regenerated Chitin from Phosphoric Acid Solutions, Carbohydr. Polym., 1997, 32(3), 233–237 CrossRef CAS .
15. H. Tamura, H. Nagahama and S. Tokura, Preparation of Chitin Hydrogel Under Mild Conditions, Cellulose, 2006, 13(4), 357–364 CrossRef CAS .
16. J. Jin, D. Lee, H. Im, Y. C. Han, E. G. Jeong, M. Rolandi, K. C. Choi and B. Bae, Chitin Nanofiber Transparent Paper for Flexible Green Electronics, Adv. Mater., 2016, 28(26), 5169–5175 CrossRef CAS PubMed .
17. Y. Zhong, J. Cai and L. Zhang, A Review of Chitin Solvents and Their Dissolution Mechanisms, Chin. J. Polym. Sci., 2020, 1–14 Search PubMed .
18. R. H. Chen and M. L. Tsaih, Effect of Temperature on the Intrinsic Viscosity and Conformation of Chitosans in Dilute HCl Solution, Int. J. Biol. Macromol., 1998, 23(2), 135–141 CrossRef CAS PubMed .
19. M. Rinaudo, G. Pavlov and J. Desbrières, Influence of Acetic Acid Concentration on the Solubilization of Chitosan, Polymer, 1999, 40(25), 7029–7032 CrossRef CAS .
20. Y. Dong, W. Qiu, Y. Ruan, Y. Wu, M. Wang and C. Xu, Influence of Molecular Weight on Critical Concentration of Chitosan/Formic Acid Liquid Crystalline Solution, Polym. J., 2001, 33(5), 387–389 CrossRef CAS .
21. H. Xie, S. Zhang and S. Li, Chitin and Chitosan Dissolved in Ionic Liquids as Reversible Sorbents of CO₂, Green Chem., 2006, 8(7), 630–633 RSC .
22. Y. Qin, X. Lu, N. Sun and R. D. Rogers, Dissolution or Extraction of Crustacean Shells Using Ionic Liquids to Obtain High Molecular Weight Purified Chitin and Direct Production of Chitin Films and Fibers, Green Chem., 2010, 12(6), 968–971 RSC .
23. S. S. Silva, J. F. Mano and R. L. Reis, Ionic Liquids in The Processing and Chemical Modification of Chitin and Chitosan for Biomedical Applications, Green Chem., 2017, 19(5), 1208–1220 RSC .
24. L. Zhang, J. Wang, M. Yang, N. Ke, B. Zhang, H. Wang, Q. Zheng and H. Xie, Dissolution, Materialization and Derivatization of Chitin and Chitosan in Ionic Liquids, Sci. China: Chem., 2019, 49(8), 1059–1072 Search PubMed .
25. M. Feng, X. Lu, J. Zhang, Y. Li, C. Shi, L. Lu and S. Zhang, Direct Conversion of Shrimp Shells to O-acylated Chitin with Antibacterial and Anti-tumor Effects by Natural Deep Eutectic Solvents, Green Chem., 2019, 21(1), 87–98 RSC .
26. J. Wang, C. Teng and L. Yan, Applications of Deep Eutectic Solvents in the Extraction, Dissolution, and Functional Materials of Chitin: Research Progress and Prospects, Green Chem., 2022, 24(2), 552–564 RSC .
27. M. Petkovic, K. R. Seddon, L. P. N. Rebelo and C. S. Pereira, Ionic, Liquids: A Pathway to Environmental Acceptability, Chem. Soc. Rev., 2011, 40(3), 1383–1403 RSC .
28. L. Lomba, M. P. Ribate, E. Sangüesa, J. Concha, M. Garralaga, D. Errazquin, C. B. García and B. Giner, Deep Eutectic Solvents: Are They Safe?, Appl. Sci., 2021, 11(21), 10061 CrossRef CAS .
29. J. Cai and L. Zhang, Rapid Dissolution of Cellulose in LiOH/Urea and NaOH/Urea Aqueous Solutions, Macromol. Biosci., 2005, 5(6), 539–548 CrossRef CAS PubMed .
30. J. Cai, L. Zhang, C. Chang, G. Cheng, X. Chen and B. Chu, Hydrogen-Bond-Induced Inclusion Complex in Aqueous Cellulose/LiOH/Urea Solution at Low Temperature, ChemPhysChem, 2007, 8(10), 1572–1579 CrossRef CAS PubMed .
31. J. Cai, L. Zhang, S. Liu, Y. Liu, X. Xu, X. Chen, B. Chu, X. Guo, J. Xu, H. Cheng, C. C. Han and S. Kuga, Dynamic Self-assembly Induced Rapid Dissolution of Cellulose at Low Temperatures, Macromolecules, 2008, 41(23), 9345–9351 CrossRef CAS .
32. X. Hu, Y. Du, Y. Tang, Q. Wang, T. Feng, J. Yang and J. F. Kennedy, Solubility and Property of Chitin in NaOH/urea Aqueous Solution, Carbohydr. Polym., 2007, 70(4), 451–458 CrossRef CAS .
33. B. Ding, J. Cai, J. Huang, L. Zhang, Y. Chen, X. Shi, Y. Du and S. Kuga, Facile Preparation of Robust and Biocompatible Chitin Aerogels, J. Mater. Chem. A, 2012, 22(12), 5801–5809 RSC .
34. B. Duan, Y. Huang, A. Lu and L. Zhang, Recent Advances in Chitin Based Materials Constructed via Physical Methods, Prog. Polym. Sci., 2018, 82, 1–33 CrossRef CAS .
35. M. Fan and Q. L. Hu, Chitosan-LiOH-urea Aqueous Solution-A Novel Water-based System for Chitosan Processing, Carbohydr. Res., 2009, 344(7), 944–947 CrossRef CAS PubMed .
36. C. K. S. Pillai, W. Paul and C. P. Sharma, Chitin and Chitosan Polymers: Chemistry, Solubility and Fiber Formation, Prog. Polym. Sci., 2009, 34(7), 641–678 CrossRef CAS .
37. J. Cai, J. Huang and L. Zhang, Solvent Composition for Dissolving Chitin, ZL 201310034088.4.
38. J. C. Huang, Y. Zhong, L. N. Zhang and J. Cai, Distinctive Viewpoint on the Rapid Dissolution Mechanism of Alpha-chitin in Aqueous Potassium Hydroxide-urea Solution at Low Temperatures, Macromolecules, 2020, 53(13), 5588–5598 CrossRef CAS .
39. J. Huang, Y. Zhong, P. Wei and J. Cai, Rapid Dissolution of β-chitin and Hierarchical Self-assembly of Chitin Chains in Aqueous KOH/urea Solution, Green Chem., 2021, 23(8), 3048–3060 RSC .
40. J. Cai, Y. Zhong, J. Huang and L. Zhang, Method for Continuously Preparing Chitin/Chitosan Solutions with Different Deacetylation Degrees, ZL 201711023349.7.
41. Q. Zhang, Y. Chen, P. Wei, Y. Zhong, C. Chen and J. Cai, Extremely Strong and Tough Chitosan Films Mediated by Unique Hydrated Chitosan Crystal Structures, Mater. Today, 2021, 51, 27–38 CrossRef CAS .
42. J. Huang, Y. Zhong, L. Zhang and J. Cai, Extremely Strong and Transparent Chitin Films: A High-Efficiency, Energy-Saving, and “Green” Route Using an Aqueous KOH/Urea Solution, Adv. Funct. Mater., 2017, 27(26), 1701100 CrossRef .
43. J. Huang, Y. Zhong, X. Zhang, H. Xu, C. Zhu and J. Cai, Continuous Pilot-Scale Wet-Spinning of Biocompatible Chitin/Chitosan Multifilaments from an Aqueous KOH/Urea Solution, Macromol. Rapid Commun., 2021, 42(16), 2100252 CrossRef CAS PubMed .
44. Y. Chen, Q. Zhang, Y. Zhong, P. Wei, X. Yu, J. Huang and J. Cai, Super-Strong and Super-stiff Chitosan Filaments with Highly Ordered Hierarchical Structure, Adv. Funct. Mater., 2021, 31(38), 2104368 CrossRef CAS .
45. D. Xu, J. Huang, D. Zhao, B. Ding, L. Zhang and J. Cai, High-Flexibility, High-Toughness Double-Cross-Linked Chitin Hydrogels by Sequential Chemical and Physical Cross-Linkings, Adv. Mater., 2016, 28(28), 5844–5849 CrossRef CAS PubMed .
46. D. Raabe, P. Romano, C. Sachs, A. Al-Sawalmih, H. G. Brokmeier, S. B. Yi, G. Servos and H. G. Hartwig, Discovery of a Honeycomb Structure in the Twisted Plywood Patterns of Fibrous Biological Nanocomposite Tissue, J. Cryst. Growth, 2005, 283(1–2), 1–7 CrossRef CAS .
47. S.-I. Aiba, Studies on chitosan: 3. Evidence for the presence of random and block copolymer structures in partially N-acetylated chitosans, Int. J. Biol. Macromol., 1991, 13(1), 40–44 CrossRef CAS PubMed .
48. J. Kumirska, M. Czerwicka, Z. Kaczynski, A. Bychowska, K. Brzozowski, J. Thoming and P. Stepnowski, Application of Spectroscopic Methods for Structural Analysis of Chitin and Chitosan, Mar. Drugs, 2010, 8(5), 1567–1636 CrossRef CAS PubMed .
49. S. F. Tanner, H. Chanzy, M. Vincendon, J. C. Roux and F. Gaill, High-resolution Solid-State C-13 Nuclear-Magnetic-Resonance Study of Chitin, Macromolecules, 1990, 23(15), 3576–3583 CrossRef CAS .
50. H. Saito, R. Tabeta and K. Ogawa, High-Resolution Solid-State C-13 Nmr-Study of Chitosan and its Salts with Acids - Conformational Characterization of Polymorphs and Helical Structures as Viewed from the Conformation-dependent C-13 Chemical-shifts, Macromolecules, 1987, 20(10), 2424–2430 CrossRef CAS .
51. Y. Ogawa, S. Kimura, M. Wada and S. Kuga, Crystal Analysis and High-resolution Imaging of Microfibrillar Alpha-chitin from Phaeocystis, J. Struct. Biol., 2010, 171(1), 111–116 CrossRef CAS PubMed .
52. Y. Noishiki, Y. Nishiyama, M. Wada, S. Okada and S. Kuga, Inclusion Complex of Beta-chitin and Aliphatic Amines, Biomacromolecules, 2003, 4(4), 944–949 CrossRef CAS PubMed .
53. A. K. Soper, E. W. Castner and A. Luzar, Impact of Urea on Water Structure: A Clue to its Properties as a Denaturant?, Biophys. Chem., 2003, 105(2–3), 649–666 CrossRef CAS PubMed .
54. H. Kokubo and B. M. Pettitt, Preferential Solvation in Urea Solutions at Different Concentrations: Properties from Simulation Studies, J. Phys. Chem. B, 2007, 111(19), 5233–5242 CrossRef CAS PubMed .
55. L. Hua, R. H. Zhou, D. Thirumalai and B. J. Berne, Urea Denaturation by Stronger Dispersion Interactions with Proteins than Water Implies a 2-stage Unfolding, Proc. Natl. Acad. Sci. U. S. A., 2008, 105(44), 16928–16933 CrossRef CAS .
56. M. R. Kasaai, Determination of the Degree of N-acetylation for Chitin and Chitosan by Various NMR Spectroscopy Techniques: A Review, Carbohydr. Polym., 2010, 79(4), 801–810 CrossRef CAS .
57. A. C. Neville, D. A. Parry and J. Woodhead-Galloway, The Chitin Crystallite in Arthropod Cuticle, J. Cell Sci., 1976, 21(1), 73–82 CrossRef CAS PubMed .
58. B. Lindman, B. Medronho, L. Alves, M. Norgren and L. Nordenskiöld, Hydrophobic interactions control the self-assembly of DNA and cellulose, Q. Rev. Biophys., 2021, 54, E3 CrossRef CAS PubMed .
59. L. Bu, M. E. Himmel and M. F. Crowley, The molecular origins of twist in cellulose I-beta, Carbohydr. Polym., 2015, 125, 146–152 CrossRef CAS PubMed .
60. S. K. Kannam, D. P. Oehme, M. S. Doblin, M. J. Gidley, A. Bacic and M. T. Downton, Hydrogen bonds and twist in cellulose microfibrils, Carbohydr. Polym., 2017, 175, 433–439 CrossRef CAS PubMed .
61. G. Fittolani, D. Vargová, P. H. Seeberger, Y. Ogawa and M. Delbianco, Bottom-Up Approach to Understand Chirality Transfer across Scales in Cellulose Assemblies, J. Am. Chem. Soc., 2022, 144(27), 12469–12475 CrossRef CAS PubMed .

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Footnotes

† Electronic supplementary information (ESI) available: The molecular structure of deacetylated α-chitin and chitosan, 2D WAXD patterns and azimuthal-averaging XRD profiles of the chitosan powder in KOH, NaOH, LiOH aqueous solutions, 1H NMR and 13C NMR spectra of GlcN in alkali/D2O, urea/D2O and alkali/urea/D2O solutions, statistical height histograms of bundles of deacetylated α-chitin chains and chitosan chains, and statistical height histograms of isolated deacetylated α-chitin chains and chitosan chains, TEM images of self-assembled deacetylated α-chitin nanofibrils and chitosan nanofibrils, table of the physical properties of deacetylated α-chitin and chitosan (PDF). Video showing the dissolution process of deacetylated α-chitin and chitosan in KOH/urea aqueous solutions (AVI). See DOI: https://doi.org/10.1039/d3gc02231e

‡ These authors contributed equally.

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