Hydrophobization of surfaces on cellulose nanofibers by enzymatic grafting of partially 2-deoxygenated amylose.

Recently, attention has been paid to cellulose nanofibers, such as 2,2,6,6-tetramethylpiperidine-1-oxyl-oxidized cellulose nanofibers (TOCN), as new bio-based materials. In addition, hydrophobized surface on TOCNs can be expected to provide new applications. Based on our previous finding that partially 2-deoxygenated (P2D)-amylose, which was synthesized by GP-catalyzed enzymatic copolymerization of D-glucal with α-d-glucose 1-phosphate (Glc-1-P) as comonomers, was hydrophobic, in this study, hydrophobization of surfaces on TOCNs was investigated by the GP-catalyzed enzymatic grafting of P2D-amylose chains on TOCNs. After maltooligosaccharide primers were modified on TOCNs, the GP-catalyzed enzymatic copolymerization of D-glucal with Glc-1-P was performed for grafting of P2D-amylose chains. 1H NMR spectroscopic analysis confirmed the production of P2D-amylose-grafted TOCNs with different 2-deoxyglucose/Glc unit ratios. The powder X-ray diffraction profiles of the products indicated that the entire crystalline structures were strongly affected by the unit ratios and chain lengths of the grafted polysaccharides. The SEM images observed differences in nanofiber diameter in the reaction solutions and surface morphology after film formation, due to grafting of P2D-amylose chains from TOCNs. The water contact angle measurement of a cast film prepared from the product indicated its hydrophobicity.

Enzymatic Assembly of Chitosan-Based Network Polysaccharides and Their Encapsulation and Release of Fluorescent Dye.

We prepared network polysaccharide nanoscopic hydrogels by crosslinking water-soluble chitosan (WSCS) with a carboxylate-terminated maltooligosaccharide crosslinker via condensation. In this study, the enzymatic elongation of amylose chains on chitosan-based network polysaccharides by glucan phosphorylase (GP) catalysis was performed to obtain assembly materials. Maltoheptaose (Glc7) primers for GP-catalyzed enzymatic polymerization were first introduced into WSCS by reductive amination. Crosslinking of the product with the above-mentioned crosslinker by condensation was then performed to produce Glc7-modified network polysaccharides. The GP-catalyzed enzymatic polymerization of the α-d-glucose 1-phosphate monomer from the Glc7 primers on the network polysaccharides was conducted, where the elongated amylose chains formed double helices. Enzymatic disintegration of the resulting network polysaccharide assembly successfully occurred by α-amylase-catalyzed hydrolysis of the double helical amyloses. The encapsulation and release of a fluorescent dye, Rhodamine B, using the CS-based network polysaccharides were also achieved by means of the above two enzymatic approaches.

A Mini-Review: Fabrication of Polysaccharide Composite Materials Based on Self-Assembled Chitin Nanofibers.

This mini-review presents the fabrication methods for polysaccharide composite materials that employ self-assembled chitin nanofibers (ChNFs) as functional components. Chitin is one of the most abundant polysaccharides in nature. However, it is mostly not utilized because of its poor feasibility and processability. Self-assembled ChNFs are efficiently obtained by a regenerative bottom-up process from chitin ion gels using an ionic liquid, 1-allyl-3-methylimodazolium bromide. This is accomplished by immersing the gels in methanol. The resulting dispersion is subjected to filtration to isolate the regenerated materials, producing ChNF films with a morphology defined by highly entangled nanofibers. The bundles are disintegrated by electrostatic repulsion among the amino groups on the ChNFs in aqueous acetic acid to produce thinner fibers known as scaled-down ChNFs. The self-assembled and scaled-down ChNFs are combined with other chitin components to fabricate chitin-based composite materials. ChNF-based composite materials are fabricated through combination with other polysaccharides.

An overview on acylation methods of α-chitin.

This article overviews the acylation methods of α-chitin developed over the last four decades. The acylation of polysaccharides has been identified as a useful approach for conferring properties such as thermoplasticity. Owing to the poor solubility of α-chitin, its acylation using acid anhydrides and acyl chlorides has been traditionally investigated under heterogeneous conditions in strong acidic media. Although chitin chains depolymerize under acidic conditions, the resultant derivatives exhibit certain properties and functions. Solvents, such as LiCl/N,N-dimethyladcetamide, ionic liquids, and deep eutectic solvents, are suitable for α-chitin dissolution; therefore, acylation methods for α-chitin under homogeneous conditions have been developed using these solvents as reaction media. The functional materialization of the resultant derivatives was achieved by introducing appropriate substituents and controlling their ratios.

Synthesis of Mixed Chitin Esters via Acylation of Chitin in Deep Eutectic Solvents.

The development of efficient derivatization methods of chitin, such as acylation, has been identified to confer new properties and functions to chitin. In this study, we investigate the synthesis of mixed chitin esters via the acylation of chitin in deep eutectic solvents (DESs) comprising 1-allyl-3-methylimidazolum chloride and 1,1,3,3-tetramethylguanidine based on a previous study that reported the development of efficient acylation of chitin in the DES to obtain single chitin esters. A stearoyl group was selected as the first substituent, which was combined with several bulky acyl and long oleoyl groups as the second substituents. After dissolution of chitin in the DES (2 wt%), the acylation reactions were conducted using stearoyl and the desired acyl chlorides for 1 h + 24 h at 100 °C in the resulting solutions. The IR and 1H NMR spectra of the isolated products confirmed the structures of mixed chitin esters with two different substituents. The substituent ratios in the derivatives, which were estimated via the 1H NMR analysis, were changed according to the feed ratios of two acyl chlorides.

Fabrication of Nanostructured Supramolecules through Helical Inclusion of Amylose toward Hydrophobic Polyester Guests, Biomimetically through Vine-Twining Polymerization Process.

This review article presents the biomimetic helical inclusion of amylose toward hydrophobic polyesters as guests through a vine-twining polymerization process, which has been performed in the glucan phosphorylase (GP)-catalyzed enzymatic polymerization field to fabricate supramolecules and other nanostructured materials. Amylose, which is a representative abundant glucose polymer (polysaccharide) with left-handed helical conformation, is well known to include a number of hydrophobic guest molecules with suitable geometry and size in its cavity to construct helical inclusion complexes. Pure amylose is prepared through enzymatic polymerization of α-d-glucose 1-phosphate as a monomer using a maltooligosaccharide as a primer, catalyzed by GP. It is reported that the elongated amylosic chain at the nonreducing end in enzymatic polymerization twines around guest polymers with suitable structures and moderate hydrophobicity, which is dispersed in aqueous polymerization media, to form amylosic nanostructured inclusion complexes. As the image of this system is similar to how vines of a plant grow around a support rod, this polymerization has been named 'vine-twining polymerization'. In particular, the helical inclusion behavior of the enzymatically produced amylose toward hydrophobic polyesters depending on their structures, e.g., chain lengths and substituents, has been systematically investigated in the vine-twining polymerization field. Furthermore, amylosic supramolecular network materials, such as hydrogels, are fabricated through vine-twining polymerization by using copolymers, where hydrophobic polyester guests or maltooligosaccharide primers are covalently modified on hydrophilic main-chain polymers. The vine-twining polymerization using such copolymers in the appropriate systems induces the formation of amylosic nanostructured inclusion complexes among them, which act as cross-linking points, giving rise to supramolecular networks at the nanoscale. The resulting materials form supramolecular hydrogels, films, and microparticles.

Fabrication of all-chitin composite films.

The aim of this study is to propose a first concept for the procedure to prepare an all-chitin composite. The fabrication of all-chitin composite films was investigated for the first time via the mixing of low-crystalline matrix dispersions with high-crystalline fiber dispersions. Self-assembled chitin nanofiber (ChNF) films, prepared from a chitin ion gel, were treated with aqueous NaOH for deacetylation, followed by treatment with different types of aqueous acids via ultrasonication to produce dispersions. When the treatment was carried out with 1.0 mol/L aqueous acetic acid, we obtained a scaled-down ChNF (high-crystalline chitin fiber) dispersion, as previously reported. The crystallinity was reduced by treatment with 1.0 mol/L aqueous trifluoroacetic acid for 10 min at room temperature via ultrasonication and subsequent treatment for 24 h at 50 °C with stirring to produce a low-crystalline chitin matrix dispersion. The resulting two dispersions were mixed, and treated by suction filtration and drying to produce all-chitin composite films. The mechanical properties of the obtained composite films with appropriate weight ratios of the two components were superior to those of the high-crystalline scaled-down ChNF film. All-chitin complexes are expected to be used in the future as sustainable materials for a variety of applications.

Synthesis of Thermoplastic Mixed Chitin Esters.

Mixed chitin esters, that is, chitin benzoate stearates, exhibiting thermoplasticity, were synthesized by the acylation of chitin using benzoyl and stearoyl chlorides in the presence of pyridine and N,N-dimethyl-4-aminopyridine for 1 h + 24 h at 100 °C in an ionic liquid, 1-allyl-3-methylimidazolium bromide. IR and 1H NMR spectroscopic analyses confirmed the formation of the desired chitin benzoate stearates. Powder X-ray diffraction analysis of the products indicated that the crystalline structures of the chitin main-chains and stearoyl side-chains were strongly affected by the benzoyl/stearoyl substituent ratios. Introducing a small number of benzoyl groups, in addition to a large ratio of stearoyl groups, contributed to disrupting the intrinsic chitin crystals and enabling the chitin main chains and stearoyl side chains to form regularly controlled layered and parallel arrays, respectively. The resulting products exhibited meting points, associated with regular stearoyl packings, and formed melt-pressed films during the melt-pressing process. These results suggest that chitin benzoate stearates with appropriate benzoyl/stearoyl substituent ratios exhibit thermoplasticity.

Hydrogelation from Self-Assembled and Scaled-Down Chitin Nanofibers by the Modification of Highly Polar Substituents.

Chitin nanofibers (ChNFs) with a bundle structure were fabricated via regenerative self-assembly at the nanoscale from a chitin ion gel with an ionic liquid using methanol. Furthermore, the bundles were disentangled by partial deacetylation under alkaline conditions, followed by cationization and electrostatic repulsion in aqueous acetic acid to obtain thinner nanofibers called scaled-down ChNFs. This review presents a method for hydrogelation from self-assembled and scaled-down ChNFs by modifying the highly polar substituents on ChNFs. The modification was carried out by the reaction of amino groups on ChNFs, which were generated by partial deacetylation, with reactive substituent candidates such as poly(2-oxazoline)s with electrophilic living propagating ends and mono- and oligosaccharides with hemiacetallic reducing ends. The substituents contributed to the formation of network structures from ChNFs in highly polar dispersed media, such as water, to produce hydrogels. Moreover, after the modification of the maltooligosaccharide primers on ChNFs, glucan phosphorylase-catalyzed enzymatic polymerization was performed from the primer chain ends to elongate the amylosic graft chains on ChNFs. The amylosic graft chains formed double helices between ChNFs, which acted as physical crosslinking points to construct network structures, giving rise to hydrogels.

Vine-Twining Inclusion Behavior of Amylose towards Hydrophobic Polyester, Poly(ß-propiolactone), in Glucan Phosphorylase-Catalyzed Enzymatic Polymerization.

This study investigates inclusion behavior of amylose towards, poly(ß-propiolactone) (PPL), that is a hydrophobic polyester, via the vine-twining process in glucan phosphorylase (GP, isolated from thermophilic bacteria, Aquifex aeolicus VF5)-catalyzed enzymatic polymerization. As a result of poor dispersibility of PPL in sodium acetate buffer, the enzymatically produced amylose by GP catalysis incompletely included PPL in the buffer media under the general vine-twining polymerization conditions. Alternatively, we employed an ethyl acetate-sodium acetate buffer emulsion system with dispersing PPL as the media for vine-twining polymerization. Accordingly, the GP (from thermophilic bacteria)-catalyzed enzymatic polymerization of an α-d-glucose 1-phosphate monomer from a maltoheptaose primer was performed at 50 °C for 48 h in the prepared emulsion to efficiently form the inclusion complex. The powder X-ray diffraction profile of the precipitated product suggested that the amylose-PPL inclusion complex was mostly produced in the above system. The 1H NMR spectrum of the product also supported the inclusion complex structure, where a calculation based on an integrated ratio of signals indicated an almost perfect inclusion of PPL in the amylosic cavity. The prevention of crystallization of PPL in the product was suggested by IR analysis, because it was surrounded by the amylosic chains due to the inclusion complex structure.

Fabrication of Chitosan-Based Network Polysaccharide Nanogels.

In this study, we developed a method to fabricate chitosan-based network polysaccharides via the condensation between amino groups in water-soluble chitosan (WSCS) and a carboxylate-terminated maltooligosaccharide crosslinker. We previously reported on the fabrication of network-polysaccharide-based macroscopic hydrogels via the chemical crosslinking of water-soluble chitin (WSCh) with the crosslinker. Because the molecular weight of the WSCS was much smaller than that of the WSCh, in the present investigation, the chemical crosslinking of the WSCS with the crosslinker was observed at the nanoscale upon the condensation between amino and carboxylate groups in the presence of a condensing agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide, affording nano-sized chitosan-based network polysaccharides. The occurrence of the crosslinking via the formation of amido linkages was supported by the IR analysis and 1H NMR measurements after the dissolution via acid hydrolysis in DCl/D2O. The products formed nanogels, whose sizes depended on the amino/carboxylate feed ratio. The nanoscale morphology and size of the products were evaluated via scanning electron microscopy, dynamic light scattering analyses, and transition electron microscopy. In the present study, we successfully developed the method to fabricate nanogel materials based on network polysaccharide structures, which can practically be applied as new polysaccharide-based 3D bionanomaterials.

Synthesis of thermoplastic chitin hexanoate-graft-poly(ε-caprolactone).

Herein, we report that chitin hexanoate-graft-poly(ε-caprolactone) (ChHex-g-PCL) is thermoplastic, as confirmed by the formation of a melt-pressed film. Chitin hexanoates with degrees of substitution (DSs) of 1.4-1.8 and bearing free hydroxy groups were first prepared by the hexanoylation of chitin using adjusted feed equivalents of hexanoyl chloride in the presence of pyridine and N,N-dimethyl-4-aminopyridine in 1-allyl-3-methylimidazolium bromide, an ionic liquid. Surface-initiated ring-opening graft polymerization of ε-caprolactone from the hydroxy groups of the chitin hexanoates was conducted in the presence of tin(II) 2-ethylhexanoate as the catalyst at 100 °C to produce (ChHex-g-PCL)s. The feed equivalent of the catalyst, reaction time, and DS value were found to affect the molar substitution and degree of polymerization of the PCL graft chains. Longer PCL graft chains formed their crystalline structures and the (ChHex-g-PCL)s largely contained uncrystallized chitin chains. Accordingly, these (ChHex-g-PCL)s exhibited melting points associated with the PCL graft chains, leading to thermoplasticity.

Preparation of Composite Materials from Self-Assembled Chitin Nanofibers.

Although chitin is a representative abundant polysaccharide, it is mostly unutilized as a material source because of its poor solubility and processability. Certain specific properties, such as biodegradability, biocompatibility, and renewability, make nanofibrillation an efficient approach for providing chitin-based functional nanomaterials. The composition of nanochitins with other polymeric components has been efficiently conducted at the nanoscale to fabricate nanostructured composite materials. Disentanglement of chitin microfibrils in natural sources upon the top-down approach and regeneration from the chitin solutions/gels with appropriate media, such as hexafluoro-2-propanol, LiCl/N, N-dimethylacetamide, and ionic liquids, have, according to the self-assembling bottom-up process, been representatively conducted to fabricate nanochitins. Compared with the former approach, the latter one has emerged only in the last one-and-a-half decade. This short review article presents the preparation of composite materials from the self-assembled chitin nanofibers combined with other polymeric substrates through regenerative processes based on the bottom-up approach.

Synthesis of mixed chitin esters with long fatty and bulky acyl substituents in ionic liquid.

This study revealed that mixed chitin esters with long fatty and bulky acyl substituents were efficiently synthesized by acylation using acyl chlorides in the presence of pyridine and N,N-dimethyl-4-aminopyridine in an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), at 100 °C for 24 h. A stearoyl group was selected as the first substituent, which was combined with different long fatty and bulky acyl groups as the second substituents. In addition to IR analysis of the products, which suggested progress of the acylation, 1H NMR measurement was allowed for structural confirmation for high degrees of substitution (DSs) of the desired derivatives in CDCl3/CF3CO2H solvents. Crystalline structures and thermal property of the products were evaluated by powder X-ray diffraction and differential scanning calorimetry measurements, respectively. All the products showed film formability by casting from solutions in chloroform or chloroform/trifluoroacetic acid solvents. The occurrence of halogen exchange between acyl chlorides and AMIMBr in the present system was speculated to produce highly reactive acyl bromides in situ, which efficiently reacted with hydroxy groups in chitin to obtain high DS products.

Fabrication of highly flexible nanochitin film and its composite film with anionic polysaccharide.

This study investigated the fabrication of a nanochitin film via the aggregation of scaled-down chitin nanofibers (SD-ChNFs). A self-assembled ChNF film, which was prepared by regeneration from a chitin/ionic liquid ion gel using methanol, followed by filtration, was treated with aqueous NaOH for deacetylation and subsequently disintegrated by cationization and electrostatic repulsion in 1.0 mol/L aqueous acetic acid with ultrasonication to give a SD-ChNF dispersion. Isolation of the SD-ChNFs via filtration of the dispersion resulted in a highly flexible self-assembled ChNF film that bent and twisted easily. The film exhibited superior mechanical properties compared to the parent self-assembled ChNF film, where the flexibility was further enhanced by the compositing the SD-ChNFs with an anionic polysaccharide, namely ι-carrageenan, via multi-point ionic cross-linking. These enhanced mechanical properties and efficient compositing properties were attributed to the scaling down of the ChNFs.

Preparation of Amylose-Oligo[(R)-3-hydroxybutyrate] Inclusion Complex by Vine-Twining Polymerization.

In this study, we attempted to prepare an amylose-oligo[(R)-3-hydroxybutyrate] (ORHB) inclusion complex using a vine-twining polymerization approach. Our previous studies indicated that glucan phosphorylase (GP)-catalyzed enzymatic polymerization in the presence of appropriate hydrophobic guest polymers produces the corresponding amylose-polymer inclusion complexes, a process named vine-twining polymerization. When vine-twining polymerization was conducted in the presence of ORHB under general enzymatic polymerization conditions (45 °C), the enzymatically produced amylose did not undergo complexation with ORHB. However, using a maltotriose primer in the same polymerization system at 70 °C for 48 h to obtain water-soluble amylose, called single amylose, followed by cooling the system over 7 h to 45 °C, successfully induced the formation of the inclusion complex. Furthermore, enzymatic polymerization initiated from a longer primer under the same conditions induced the partial formation of the inclusion complex. The structures of the different products were analyzed by X-ray diffraction, 1H-NMR, and IR measurements. The mechanism of formation of the inclusion complexes discussed in the study is proposed based on the additional experimental results.

Preparation and gelation behaviors of poly(2-oxazoline)-grafted chitin nanofibers.

Based on our previous work on successful gelation of poly(2-methyl-2-oxazoline)-grafted chitin nanofibers (ChNFs) with high polar media, in this study, we investigated the preparation and gelation behaviors of the ChNFs having different poly(2-alkyl-2-oxazoline) graft chains, that is, poly(2-methyl-2-oxazoline), poly(2-isopropyl-2-oxazoline), and poly(2-butyl-2-oxazoline), with various disperse media. The grafting was carried out by reactions of living propagating ends of poly(2-alkyl-2-oxazoline)s with amino groups present on the self-assembled ChNFs, which were obtained from a chitin ion gel. The products formed gels in the reaction mixtures, which could be converted into hydrogels. All the products with the three poly(2-alkyl-2-oxazoline) graft chains formed gels with high polar media. Besides, gelation of the product with poly(2-butyl-2-oxazoline) was observed by immersing it in relatively non-polar media such as benzyl alcohol, ethyl acetate, and toluene. The formation process of network structures by the grafting of poly(2-alkyl-2-oxazoline)s on ChNFs is proposed to induce gelation of the products.

Evaluation of artificial crystalline structure from amylose analog polysaccharide without hydroxy groups at C-2 position.

In this study, we found that a new artificial crystalline structure was fabricated from an amylose analog polysaccharide without hydroxy groups at the C-2 position, i.e., 2-deoxyamylose. The polysaccharide with a well-defined structure was synthesized by facile thermostable α-glucan phosphorylase-catalyzed enzymatic polymerization. Powder X-ray diffraction (XRD) analysis of the product indicated the formation of a specific crystalline structure that was completely different from the well-known double helix of the natural polysaccharide, amylose. Molecular dynamics simulations showed that the isolated chains of 2-deoxyamylose spontaneously assembled to a novel double helix based on building blocks with controlled hydrophobicity arising from pyranose ring stacking. The simulation results corresponded with the XRD patterns.

Chemoenzymatic synthesis of carboxylate-terminated maltooligosaccharides and their use for cross-linking of chitin.

In this paper, we report chemoenzymatic synthesis of maltooligosaccharides having carboxylate groups at both ends (carboxylate-terminated maltooligosaccharides, GlcA-Glcn-GlcCOONa). The products were further used as cross-linker for water-soluble chitin (WSCh) to obtain network chitins. The synthesis of GlcA-Glcn-GlcCOONa was achieved by thermostable phosphorylase-catalyzed enzymatic α-glucuronylation using α-d-glucuronic acid 1-phosphate with a carboxylated maltooligosaccharide, which was prepared by chemical oxidation at the reducing end of maltoheptaose with sodium hypoiodite. The structures of GlcA-Glcn-GlcCOONa were evaluated by 1H NMR and MALDI-TOF mass spectra. The obtained GlcA-Glcn-GlcCOONa were used as cross-linker for WSCh by condensation in the presence of condensing agent. The reaction mixtures totally turned into hydrogel form in most cases. Morphologies of lyophilized samples (cryogels) from the hydrogels were evaluated by SEM measurement. The hydrogels could be converted into films by pressing. Furthermore, mechanical properties of the hydrogels and films were investigated by compression and tensile tests, respectively.

Preparation of chitin-based fluorescent hollow particles by Pickering emulsion polymerization using functional chitin nanofibers.

This study investigated the preparation of chitin-based fluorescent hollow particles by Pickering emulsion polymerization of styrene using bifunctional chitin nanofibers (ChNFs) as stabilizer, giving CNF-based composite particles, followed by solubilizing out inner polystyrene. In addition to the introduction of anionic maleyl groups on ChNFs to improve dispersibility in aqueous ammonia, polymerizable methacryl groups were substituted on ChNFs as second functionalization to provide ability in copolymerization with styrene for stabilization of hollow structures. Consequently, after the formation of styrene-in-water Pickering emulsion using the bifunctional ChNFs as stabilizer, radical polymerization was conducted in the presence of potassium persulfate as an initiator to produce the composite particles. The hollow particles were then fabricated by solubilizing out inner polystyrene with toluene, which stably dispersed in water. Encapsulation of a fluorescent dye, pyrene, into the cavity of the hollow particles was achieved by hydrophobic interaction with polystyrene present on the inner walls, which could be released by treatment of the resulting fluorescent hollow particles with surfactant, oleyl alcohol, in water. The same Pickering emulsion polymerization system was also performed in the presence of a pyrene derivative having a polymerizable group to obtain fluorescent composite/hollow particles with pyrene moieties covalently bound to polystyrene.