Preparation of Amylose-Carboxymethyl Cellulose Conjugated Supramolecular Networks by Phosphorylase-Catalyzed Enzymatic Polymerization

: Enzymatic polymerization has been noted as a powerful method to precisely synthesize polymers with complicated structures, such as polysaccharides, which are not commonly prepared by conventional polymerization. Phosphorylase is one of the enzymes which have been used to practically synthesize well-deﬁned polysaccharides. The phosphorylase-catalyzed enzymatic polymerization is conducted using α - D -glucose 1-phosphate as a monomer, and maltooligosaccharide as a primer, respectively, to obtain amylose. Amylose is known to form supramolecules owing to its helical conformation, that is, inclusion complex and double helix, in which the formation is depended on whether a guest molecule is present or not. In this paper, we would like to report the preparation of amylose-carboxymethyl cellulose (CMC) conjugated supramolecular networks, by the phosphorylase-catalyzed enzymatic polymerization, using maltoheptaose primer-grafted CMC. When the enzymatic polymerization was carried out using the graft copolymer, either in the presence or in the absence of a guest polymer poly ( ε -caprolactone) (PCL), the enzymatically elongated amylose chains from the primers on the CMC main-chain formed double helixes or inclusion complexes, depending on the amounts of PCL, which acted as cross-linking points for the construction of network structures. Accordingly, the reaction mixtures totally turned into hydrogels, regardless of the structures of supramolecular cross-linking points. The amylose-grafted additionally, formed ﬁlms with the hierarchically constructed nanostructure.


Introduction
Enzyme-catalyzed reactions have been employed to precisely synthesize biological and bio-related polymeric molecules with regular structures, which are identified as a term of enzymatic polymerization because they strictly progress with controlled regio-, stereo-, and conformational arrangements [1][2][3][4]. Polysaccharides are the representative molecules, which have been beneficially constructed according to such advantages of enzymatic catalysis [5][6][7]. They are comprising very complicated structures which are comprised of a variety of monosaccharide units, linked through specific types of glycosidic linkages, with highly controlled regio-and stereoarrangements. For example, amylose with a well-defined structure is comprised of α(1→4)-linked glucose (G) repeating units, and synthesized by the phosphorylase-catalyzed enzymatic polymerization of the α-D-glucose 1-phospahte (G-1-P) monomer [6][7][8][9][10]. The polymerization is precisely initiated at the non-reducing end of a maltooligosaccharide primer, and G residues are consecutively transferred from the monomers to the non-reducing propagating chain end. Therefore, the molecular weights

Results and Discussion
Prior to demonstrating the phosphorylase-catalyzed enzymatic polymerization, G7-grafted CMC was prepared by condensation of carboxyates in CMC (molecular weight (MW) = 2.5 × 10 6 , degree of carboxymethylation (DC) = 0.7), with an amine-functionalized G7 in the presence of the condensing agent, comprising 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in NaOH aq. at room temperature according to our previous literature procedure [32]. The structure of the product, which was isolated by dialysis against water and a subsequent precipitation procedure into methanol, was confirmed by the 1 H NMR measurement in D2O. The degree of substitution (DS) value of the G7 primers, with total repeating units of CMC, was estimated by the integrated ratio of signals assignable to anomeric protons in the G7 graft-chains to a signal ascribable to anomeric protons in the CMC main-chain to be 3.8%.
The phosphorylase-catalyzed enzymatic polymerization of G-1-P, using the resulting G7-grafted CMC, was then carried out in different PCL/G7 feed ratios (0, 3, 5, and 10) in aqueous acetate buffer (Figure 1a). The consistent G7 concentration (= 3.0 µmol/0.4 mL acetate buffer) and G-1-P/G7 feed ratio (=100) were employed in all runs. Regardless of the PCL/G7 feed ratios, the reaction mixtures totally turned into the hydrogels after maintaining for 24 h at 45 °C (Figure 1b). On the other hand, the phosphorylase-catalyzed enzymatic polymerization, using G7-grafted PGA with similar MW and DS values (1.5 − 2.5 × 10 6 and 3.0%, respectively), under the same conditions of run 3, did not result in hydrogelation. These results strongly suggested that CMC was a better component than PGA for conjugation with amylose to construct the amylosic supramolecular network.
The resulting hydrogels were taken out of the vessels, washed with water and acetone several times for purification (the removal of un-included PCL, phosphorylase, unreacted G-1-P, and buffer salt), and lyophilized. The monomer conversions, amylose molecular weights (Mn), amylose contents, and water contents, were estimated by weights of the products (Table 1). By increasing the

Results and Discussion
Prior to demonstrating the phosphorylase-catalyzed enzymatic polymerization, G 7 -grafted CMC was prepared by condensation of carboxyates in CMC (molecular weight (MW) = 2.5 × 10 6 , degree of carboxymethylation (DC) = 0.7), with an amine-functionalized G 7 in the presence of the condensing agent, comprising 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in NaOH aq. at room temperature according to our previous literature procedure [32]. The structure of the product, which was isolated by dialysis against water and a subsequent precipitation procedure into methanol, was confirmed by the 1 H NMR measurement in D 2 O. The degree of substitution (DS) value of the G 7 primers, with total repeating units of CMC, was estimated by the integrated ratio of signals assignable to anomeric protons in the G 7 graft-chains to a signal ascribable to anomeric protons in the CMC main-chain to be 3.8%.
The phosphorylase-catalyzed enzymatic polymerization of G-1-P, using the resulting G 7 -grafted CMC, was then carried out in different PCL/G 7 feed ratios (0, 3, 5, and 10) in aqueous acetate buffer ( Figure 1a). The consistent G 7 concentration (= 3.0 µmol/0.4 mL acetate buffer) and G-1-P/G 7 feed ratio (=100) were employed in all runs. Regardless of the PCL/G 7 feed ratios, the reaction mixtures totally turned into the hydrogels after maintaining for 24 h at 45 • C (Figure 1b). On the other hand, the phosphorylase-catalyzed enzymatic polymerization, using G 7 -grafted PGA with similar MW and DS values (1.5 − 2.5 × 10 6 and 3.0%, respectively), under the same conditions of run 3, did not result in hydrogelation. These results strongly suggested that CMC was a better component than PGA for conjugation with amylose to construct the amylosic supramolecular network.
The resulting hydrogels were taken out of the vessels, washed with water and acetone several times for purification (the removal of un-included PCL, phosphorylase, unreacted G-1-P, and buffer salt), and lyophilized. The monomer conversions, amylose molecular weights (M n ), amylose contents, and water contents, were estimated by weights of the products (Table 1). By increasing the PCL/G 7 feed ratios, the monomer conversions, molecular weights, and water contents decreased at the same tendency as had appeared in the previous study using G 7 -grafted PGA [29]. As already discussed in the previous report [29], inclusion complexes were predominantly formed in the presence of larger amounts of PCL, which resulted in the prevention of further progress of the enzymatic polymerization to produce the lower molecular weight amylose. Accordingly, the smaller networks were constructed from inclusion complexes with the shorter amylose graft chains as cross-linking points than those obtained from double helixes, resulting in lower water contents of the hydrogels. The formation of either the double helix or inclusion complex, as the amylose assembling structure in the products, was evaluated by the powder X-ray diffraction (XRD) measurement of the lyophilized samples ( Figure 2). Because of the absence of PCL, the XRD profile of the sample of run 1 shows the diffraction peaks at around 17 • and 22 • , ascribed to the double helix (Figure 2a,e). When the PCL/G 7 feed ratio of 3 was employed, the diffraction peaks at 13 • and 20 • , ascribable to the inclusion complex which are mildly detectable, in addition to those due to the double helix (Figure 2b,f). With increasing the PCL/G 7 feed ratios (=5 and 10), the XRD profiles mostly observed the diffraction peaks assignable to the inclusion complex (Figure 2c,d). The weight ratios of the inclusion complex to the double helix in the lyophilized samples were precisely estimated from weights of the recovered (un-included) PCL present in the acetone fractions. Based on a theoretical PCL/amylose unit ratio (0.16) in the inclusion complex with 6 1 helix [14], the weight ratios of two amylosic assemblies (inclusion complex and double helix) could be calculated, which increased with increasing the PCL/G 7 feed ratios. These data are in good agreement with the abovementioned XRD results.
The amylosic assembling structures in the supramolecular networks, depending on reaction times, were then evaluated. The phosphorylase-catalyzed enzymatic polymerization, using G 7 -grafted CMC, was conducted under the conditions of run 2 with different reaction times of 0.5, 1, 3, and 5 h. The M n values of the amylose graft chains, which were evaluated by weights of the lyophilized samples, increased in accordance with prolonged reaction times of 8000, 11,000, 16,000, and 17,000. The XRD profile of the lyophilized product with shorter reaction time (0.5 h) sorely observed the diffraction peaks ascribable to the inclusion complex (Figure 3a). With prolonged reaction times, the diffraction peaks caused by the double helix gradually appear (Figure 3b-d). These results indicated that the short amylose graft chains included PCL in the cavities at an early stage of the enzymatic polymerization, according to the vine-twining polymerization manner (Figure 4a). At following stages, the formation of the double helixes gradually occurred from the longer amylose chains (Figure 4b). The products obtained with different reaction times were further characterized by dynamic viscoelastic measurement. The frequency dependences of storage and loss moduli (G and G", respectively) of all the products before lyophilization observe signature of the typical viscoelastic material with the predominance of the G' values on the whole frequency range (Figure 4). Furthermore, the profiles in Figure 4 have been similar to those of the hydrogel previously obtained by the phosphorylase-catalyzed enzymatic polymerization using G 7 -grafted PGA under similar conditions with a reaction time of 48 h [29]. This suggests, in enzymatic polymerization using G 7 -grafted CMC, even with shorter reaction times (0.5-5 h), the hydrogels with comparable property to that using G 7 -grafted PGA with longer reaction time (48 h), are produced. These results also indicated that CMC was better than PGA as the component for conjugation with amylose to construct the amylosic supramolecular network. previously obtained by the phosphorylase-catalyzed enzymatic polymerization using G7-grafted PGA under similar conditions with a reaction time of 48 h [29]. This suggests, in enzymatic polymerization using G7-grafted CMC, even with shorter reaction times (0.5-5 h), the hydrogels with comparable property to that using G7-grafted PGA with longer reaction time (48 h), are produced. These results also indicated that CMC was better than PGA as the component for conjugation with amylose to construct the amylosic supramolecular network.  previously obtained by the phosphorylase-catalyzed enzymatic polymerization using G7-grafted PGA under similar conditions with a reaction time of 48 h [29]. This suggests, in enzymatic polymerization using G7-grafted CMC, even with shorter reaction times (0.5-5 h), the hydrogels with comparable property to that using G7-grafted PGA with longer reaction time (48 h), are produced. These results also indicated that CMC was better than PGA as the component for conjugation with amylose to construct the amylosic supramolecular network.    The macroscopic morphologies of the lyophilized samples of runs 1-4 were then evaluated by the SEM measurement ( Figure 5). With increasing the PCL/G7 feed ratios, smaller network morphologies are observed in the SEM images. In the presence of larger amounts of PCL, the inclusion complexes were mostly formed at the early stage, according to the vine-twining polymerization manner, which prevented further progress of the enzymatic polymerization to obtain the smaller networks with short amylose chains upon molecular level, resulting in the hierarchical fabrication of the smaller macroscopic networks (Figure 6a). Meanwhile, in the absence of PCL or the presence of smaller amounts of PCL longer amylose chains were smoothly produced, without prevention by inclusion complexes in the enzymatic polymerization, to form the larger networks at a molecular level (Figure 6b). Therefore, the larger macroscopic networks were hierarchically constructed under these conditions.   The macroscopic morphologies of the lyophilized samples of runs 1-4 were then evaluated by the SEM measurement ( Figure 5). With increasing the PCL/G 7 feed ratios, smaller network morphologies are observed in the SEM images. In the presence of larger amounts of PCL, the inclusion complexes were mostly formed at the early stage, according to the vine-twining polymerization manner, which prevented further progress of the enzymatic polymerization to obtain the smaller networks with short amylose chains upon molecular level, resulting in the hierarchical fabrication of the smaller macroscopic networks (Figure 6a). Meanwhile, in the absence of PCL or the presence of smaller amounts of PCL longer amylose chains were smoothly produced, without prevention by inclusion complexes in the enzymatic polymerization, to form the larger networks at a molecular level (Figure 6b). Therefore, the larger macroscopic networks were hierarchically constructed under these conditions. Catalysts 2019, 9, x FOR PEER REVIEW 6 of 9 Figure 3. XRD profiles of products by lyophilization of run 2 with reaction times of (a-d) 0.5, 1, 3, and 5. The macroscopic morphologies of the lyophilized samples of runs 1-4 were then evaluated by the SEM measurement ( Figure 5). With increasing the PCL/G7 feed ratios, smaller network morphologies are observed in the SEM images. In the presence of larger amounts of PCL, the inclusion complexes were mostly formed at the early stage, according to the vine-twining polymerization manner, which prevented further progress of the enzymatic polymerization to obtain the smaller networks with short amylose chains upon molecular level, resulting in the hierarchical fabrication of the smaller macroscopic networks (Figure 6a). Meanwhile, in the absence of PCL or the presence of smaller amounts of PCL longer amylose chains were smoothly produced, without prevention by inclusion complexes in the enzymatic polymerization, to form the larger networks at a molecular level (Figure 6b). Therefore, the larger macroscopic networks were hierarchically constructed under these conditions.     The macroscopic morphologies of the lyophilized samples of runs 1-4 were then evaluated by the SEM measurement ( Figure 5). With increasing the PCL/G7 feed ratios, smaller network morphologies are observed in the SEM images. In the presence of larger amounts of PCL, the inclusion complexes were mostly formed at the early stage, according to the vine-twining polymerization manner, which prevented further progress of the enzymatic polymerization to obtain the smaller networks with short amylose chains upon molecular level, resulting in the hierarchical fabrication of the smaller macroscopic networks (Figure 6a). Meanwhile, in the absence of PCL or the presence of smaller amounts of PCL longer amylose chains were smoothly produced, without prevention by inclusion complexes in the enzymatic polymerization, to form the larger networks at a molecular level (Figure 6b). Therefore, the larger macroscopic networks were hierarchically constructed under these conditions.

Phosphorylase-Catalyzed Enzymatic Polymerization of G-1-P Using G 7 -Grafted PGA in the Presence of PCL
A typical experimental procedure was as follows (run 4, PCL/G 7 = 10). G 7 -grafted CMC (0.019 g, 0.079 unit mmol, G 7 ; 3.0 µmmol) was first dissolved in an aqueous sodium acetate buffer solution (0.2 mol/L, pH 6.2, 0.4 mL). After G-1-P (disodium salt, 0.91 g, 0.3 mmol, 100 equiv. with primer) and a solution of PCL (7.0 mg, 10.0 µmmol) in acetone (0.06 mL) was mixed to the solution, the mixture was then maintained in the presence of thermostable phosphorylase (24.4 units) for 24 h at 45 • C. For purification, the obtained hydrogel was soaked and washed with water and acetone, in turn, several times (418 mg). The purified material was lyophilized to obtain the product (61.9 mg). The un-included PCL was recovered (16.8 mg) by evaporating the acetone fraction.

Conclusions
In this study, we have found that the phosphorylase-catalyzed enzymatic polymerization of G-1-P using G 7 -grafted CMC is a useful method to produce amylosic supramolecular networks conjugated with other polymeric components, which efficiently form hydrogels. The formation of double helix cross-linking points from the amylose graft chains in the absence of the guest polymer, PCL, gave rise to the construction of the larger network. By increasing the amounts of PCL, inclusion complex cross-linking points were predominantly formed according to the vine-twining polymerization manner, resulting in the formation of the smaller networks. The present supramolecular materials, which are consisting of bio-based and biodegradable polymers, that is, amylose, CMC, and PCL, have the potential to be practically employed as new soft materials in tissue engineering and biomedical applications in the future.