Self-Assembly of Cellulose Oligomers into Nanoribbon Network

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Self-Assembly of Cellulose Oligomers into Nanoribbon Network Structures Based on Kinetic Control of Enzymatic Oligomerization Takeshi Serizawa, Yuka Fukaya, and Toshiki Sawada Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03653 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on October 31, 2017

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Self-Assembly of Cellulose Oligomers into Nanoribbon Network Structures Based on Kinetic Control of Enzymatic Oligomerization Takeshi Serizawa,* Yuka Fukaya, and Toshiki Sawada Department of Chemical Science and Engineering, School of Materials and Chemical Technology, Tokyo Institute of Technology, 2-12-1-H121 Ookayama, Meguro-ku, Tokyo 152-8550, Japan

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ABSTRACT: The ability to chemically synthesize desired molecules followed by their in situ self-assembly in reaction solution has attracted much attention as a simple and environmentally friendly method to produce self-assembled nanostructures. In this study, α-D-glucose 1-phosphate monomers and cellobiose primers were subjected to cellodextrin phosphorylase-catalyzed reverse phosphorolysis reactions in aqueous solution in order to synthesize cellulose oligomers, which were then in situ self-assembled into crystalline nanoribbon network structures. The average degree-of-polymerization (DP) values of the cellulose oligomers were estimated to be approximately 7-8 with a certain degree of DP distribution. The cellulose oligomers crystallized with the cellulose II allomorph appeared to align perpendicularly to the base plane of the nanoribbons in an anti-parallel manner. Detailed analyses of reaction time dependence suggested that the production of nanoribbon network structures was kinetically controlled by the amount of water-insoluble cellulose oligomers produced.

KEYWORDS: Self-Assembly, Crystallization, Enzymatic Reaction, Cellulose Oligomer, Nanoribbon, Network Structure, Hydrogel


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INTRODUCTION Self-assembly is a low-energy, simple bottom-up process that does not require any special equipment and is useful for the hierarchical production of complex and precise structures at a wide variety of scales.1-9 For molecular self-assembly in solution, self-assembling molecules are prepared as the first step, and then they are self-assembled into particular nanostructures in a separate flask. Meanwhile, the chemical synthesis of desired molecules followed by their in situ self-assembly in the same reaction solution has attracted much attention to more simply and efficiently produce self-assembled nanostructures.10-12 Chemical reactions of small/medium molecules13-17 as well as polymerization reactions18-22 have been used to induce the self-assembly of the products. For example, the production of dipeptide derivatives through enzymatic reactions at different enzyme concentrations induced the structurally diverse molecular and network assemblies.15 Furthermore, the production of amphiphilic block copolymers through precisely controlled living radical dispersion polymerizations induced the self-assembly of the products into various nanostructures (so-called “polymerization-induced self-assembly”).19-22 These previous studies clearly indicate that the reaction-based kinetic control of production amounts is potentially useful to control self-assembled nanostructures in non-equilibrium states. The in vitro enzymatic synthesis of biorelated polymers is an attractive approach that mimics biological syntheses proceeded under aqueous-based mild conditions,23-26 and it has been used to produce polypeptides27-30 and polysaccharides.31-36 Interestingly, some of the water-insoluble biorelated polymers were in situ self-assembled into unique nanostructures in the reaction solution through polymerization-induced self-assembly.29,34 For example, when cellulose oligomers were synthesized by cellodextrin phosphorylase (CDP)-catalyzed reverse phosphorolysis reactions using α-D-glucose 1-phosphate (αG1P) monomers and D-glucose primers in aqueous buffer solutions, the water-insoluble cellulose oligomers were in situ


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self-assembled into two-dimensional crystalline rectangular nanosheets, in which the oligomers crystallized with the cellulose II allomorph were aligned perpendicularly to the base plane of the nanosheets in an anti-parallel manner.37,38 The enzymatic synthesis of biorelated polymers is valuable to simply obtain non-natural nanostructures in a single step without any protection/deprotection processes. However, uncontrollable aggregation of the self-assembled products hampers further control of higher-order structures. Cellulose-based hydrogels have attracted growing interest for various applications in diverse fields due to their fascinating and versatile properties.39-44 Using the CDP-catalyzed synthetic system,26,32,34,37,38 we have successfully demonstrated two strategies for the in situ production of highly ordered crystalline nanoribbon network structures composed of cellulose oligomers or their derivatives, leading to the formation of novel cellulose-based hydrogels.45-48 One strategy is the use of αG1P monomers and D-glucose derivative primers with hydrophilic chains at the reducing end,46 instead of conventional D-glucose primers.37,38 The other strategy is the application of macromolecular crowding environments for reaction solutions using conventional reactants such as αG1P monomers and D-glucose primers.47,48 In both cases, the aggregation and subsequent precipitation of the colloidal precursors were suppressed so that there was strong growth of nanoribbon network structures. As the next step, we hypothesized that the kinetic control of enzymatic oligomerization, which modulates the rate of production of water-insoluble cellulose oligomers, would directly relate to the production and growth of colloidal precursors and therefore such control would be another strategy for nanostructure production of cellulose oligomers. In this study, we demonstrate a more straightforward and versatile system that produces nanoribbon network structures for the crystallization-driven in situ self-assembly of cellulose oligomers that are synthesized by CDP-catalyzed reverse phosphorolysis reactions using αG1P monomers and cellobiose primers (Figure 1). The kinetics of the production of


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water-insoluble cellulose oligomers differ considerably between cellobiose and conventional D-glucose

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primer systems under the same synthetic conditions, leading to different

mechanisms underlying the production of nanoribbons and nanosheets, respectively. These observations successfully proof that the kinetic control of production amounts is useful for the construction of highly ordered nanostructures through the self-assembly of solvent-insoluble biorelated molecules. Our findings open a new avenue for the bioprocess-based facile production of hydrogel materials.

EXPERIMENTAL SECTION Materials. αG1P disodium salt n-hydrate (the number of hydrated water was determined by 1H nuclear magnetic resonance (NMR) measurements) and 40% sodium deuteroxide (NaOD)/deuterium oxide (D2O) solution were purchased from Wako Pure Chemical Industries (Osaka, Japan). ProteoMassTM MALDI-MS Standard, 1% trifluoroacetic acid, acetonitrile, 2,5-dihydroxybenzoic acid, and D2O were purchased from Sigma-Aldrich (Tokyo, Japan). Dotite, collodion-coated copper EM grid, and EM stainer were purchased from Nisshin EM Corporation (Tokyo, Japan). Other reagents were purchased from Nacalai Tesque (Kyoto, Japan). A Milli-Q Advantage A-10 (Merck Millipore, Darmstadt, Germany) supplied ultrapure water with resistivity greater than 18.2 MΩ cm for all the experiments. Enzymatic Synthesis. The procedures for CDP preparation and CDP-catalyzed synthesis of cellulose oligomers were essentially followed from our previously reported protocols.38 CDP from Clostridium thermocellum YM4 was obtained from Escherichia coli BL21-Gold (DE3) harboring plasmid pET28a-CDP cultures after purification using a Ni-NTA column (GE Healthcare, Tokyo, Japan). Unless otherwise stated, αG1P monomers (200 mM) and cellobiose

primers

(50

mM)

were

incubated

with

CDP

(0.2

U

mL-1)

in

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution (0.5 M, pH 7.5)


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at 60 °C for 72 h. For reference experiments, D-glucose was used instead of cellobiose. To readily assess the gelation of the product solutions, the vials were inverted, and photographs were taken immediately. With the exception of products being assessed by SEM, the gelled or precipitated products were collapsed mechanically and were washed with ultrapure water by centrifugation (15,000 rpm)/redispersion cycles to remove 99.999% of the reaction mixture. The purified products were stored at 4 °C as water dispersions or as powders after lyophilization. For quantification of the product, a volume of the water dispersion was dried at 105 °C for 24 h, and the obtained residues were weighed. Chemical Structural Characterizations. For 1H NMR spectroscopy, the lyophilized products were dissolved in 4% NaOD/D2O at ≥2% (w/v). A Bruker DPX-300 spectrometer (Bruker Biospin, Yokohama, Japan, 300 MHz) was used to obtain the 1H NMR spectra at ambient temperature. Residual signals of water (δ = 4.79) as an internal standard were used to calibrate the spectra. For matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the products were dispersed in acetonitrile/water (1/1, v/v) with 2,5-dihydroxybenzoic acid (1.7 mg mL-1) and trifluoroacetic acid (0.02% (v/v)). The samples for measurement were prepared by deposition of the resulting dispersions onto a sample target plate. An AXIMA-performance mass spectrometer equipped with a nitrogen laser (λ = 337 nm) and pulsed ion extraction were used to obtain the spectra, which were calibrated with peptide standards (ProteoMassTM MALDI-MS Standard) at 757.3997 Da (Bradykinin fragment 1-7), 1,533.8582 Da (P14R), and 2,465.1989 Da (ACTH fragment 18-39). Crystal Structural Characterizations. A Rigaku (Tokyo, Japan) MiniFlex600 with Cu Kα radiation (λ = 1.54 Å) was used to obtain the wide-angle X-ray diffraction (WAXD) profiles of the lyophilized products in the 2θ range of 5-40 degrees with a step of 0.02 degrees at a scan speed of 2 degrees min-1 at ambient temperature. A JASCO (Tokyo, Japan)


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FT/IR-4100 spectrometer was used to obtain the attenuated total reflection-Fourier transform infrared (ATR-FTIR) absorption spectra of the lyophilized products with a cumulative number of 100 and a resolution of 2.0 cm-1 at ambient temperature. Morphological Characterizations. For scanning electron microscopy (SEM), the as-synthesized gelled products were purified by immersion of the vials in excess amounts of ultrapure water at 4 °C for at least 1 w. The ultrapure water that was used for purification was exchanged every day. Then, the water solvent of the gelled products was exchanged by sequentially immersing the products in 10, 20, 30, 40, 50, 60, 70, 80, 90% ethanol, ethanol, ethanol/tert-butyl alcohol (1/1, v/v), and tert-butyl alcohol. The resulting organogels with a tert-butyl alcohol solvent were rapidly frozen using liquid nitrogen. The frozen organogels were fractured using a razor blade and then lyophilized. A field-emission scanning electron microscope (S-4700, Hitachi High-Technologies, Tokyo, Japan) was used to obtain the SEM images at an accelerating voltage of 6 kV. For atomic force microscopy (AFM), the products dispersed in ultrapure water (0.001% (w/v)) were spin-cast on mica substrates at 600 rpm for 30 min. A Shimadzu (Kyoto, Japan) SPM-9600 was used to obtain the AFM images in tapping mode at ambient temperature in an air atmosphere. Quantification of Phosphate Ions. The quantification of phosphate ions eliminated from αG1P monomers in the product solutions was performed by following a previously reported protocol.38

In

brief,

the

product

solutions

were

10-fold

diluted

with

3-morpholinopropane-1-sulfonic acid (MOPS) buffer solution (50 mM, pH 7.5), and the resulting solutions were centrifuged at 15000 rpm at 4 °C for at least 5 min. The supernatants were adequately diluted with the MOPS buffer solution, and the molybdenum reagent and sodium ascorbate were added to the diluted solutions. The absorbance at 850 nm was measured in the presence of sodium dodecyl sulfate, followed by quantification of phosphate ions by using a standard curve.


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RESULTS AND DISCUSSION Enzymatic Oligomerization of αG1P Monomers from Cellobiose Primers. αG1P monomers (200 mM) were oligomerized from cellobiose primers (50 mM) by CDP-catalyzed reverse phosphorolysis reactions at CDP concentrations of 0.1, 0.2, and 0.4 U mL-1 in HEPES buffer solutions (0.5 M, pH 7.5) at 60 °C for 72 h, according to our previously reported protocols (except for CDP concentration).38,45-50 Note that 0.2 U mL-1 of CDP is the standard concentration in our previous studies. The transparent solutions became turbid after the reaction at all CDP concentrations (Figure 2), indicating the presence of colorless and water-insoluble products (that is, the production of water-insoluble cellulose oligomers with certain degree-of-polymerization (DP) values51). Significantly, owing to their being in hydrogel states, none of the product solutions flowed when the glass vials were turned upside down, suggesting the production of network structures. The conversions of αG1P monomers were estimated both from the total amounts of the water-insoluble products collected by centrifugation processes and the average DP values estimated by 1H NMR measurements (see below). The monomer conversions were 45 ± 4, 55 ± 3, and 63 ± 4% at CDP concentrations of 0.1, 0.2, and 0.4 U mL-1, respectively. This suggests that the monomer conversions tended to increase with increasing CDP concentration. Cellobiose was used as a primer for CDP-catalyzed synthesis of cellulose oligomers in a previous study, resulting in the production of precipitates composed of platelets with nanometer-scale thickness.52 However, the reaction conditions were significantly different; in particular, the concentrations of the reactants and CDP in the previous paper were lower than those in this study, suggesting that the selection of adequate reaction conditions is an essential factor for the production of network structures in hydrogel states. To the best of our knowledge, the production of network structures composed of


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cellulose oligomers that have been enzymatically synthesized from cellobiose primers has not been reported. When D-glucose was used as a primer instead of cellobiose for the production of cellulose oligomers under the same synthetic conditions, the product solutions were in sol states and contained aggregates of the products.37,38 This observation suggests that the selection of adequate primers is another essential factor for the production of network structures, even though cellobiose is transiently produced as an intermediate product for D-glucose primers. The conversion of αG1P monomers for D-glucose primers was estimated to be approximately 35% and was slightly greater than that for the enzymatic synthesis using cellobiose primers at a CDP concentration of 0.1 U mL-1, at which the network structures were produced as previously mentioned. These observations suggest that the monomer conversion did not directly correlate with the production of network structures. In other words, the production of network structures is characteristic of the enzymatic synthesis using cellobiose primers. Therefore, the structural differences in the products and the kinetic differences in the production of cellulose oligomers between the two primers will be investigated in the following sections. Chemical and Crystal Structural Characterizations of the Enzymatically Synthesized Products. The chemical structures of the products that were enzymatically synthesized by using cellobiose primers at different CDP concentrations were characterized by 1H NMR and MALDI-TOF-MS measurements (Figure 3). 1H NMR spectra of the products showed the representative proton signals assignable to cellulose oligomers in the range of 2.8-5.2 ppm (Figure 3a),53 indicating the successful propagation of αG1P monomers from cellobiose primers through CDP-catalyzed reverse phosphorolysis reactions for cellulose oligomer production. The average DP values of the products were estimated to be 7.1 ± 0.1, 7.4 ± 0.2, and 7.5 ± 0.1, respectively, from the integral ratios for H1α (δ = 5.1), H1β (δ = 4.5), and H1’,1”


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(δ = 4.3) protons. These values are acceptable for water-insoluble cellulose oligomers, because it is known that the water-solubility of cellulose oligomers is poor when the DP values are more than 7.51 In addition, the average DP values tended to increase slightly with increasing CDP concentration. This observation might be acceptable, when considering the possibility that the propagation reaction proceeded to a slightly greater extent at greater CDP concentrations before solidification of the products for the reaction termination, which corresponded to the isolation of the products from CDP. The MALDI-TOF-MS measurements of the products showed a series of peaks assignable to the sodium and potassium ion adducts of cellulose oligomers (Figure 3b). The peak-to-peak mass difference of 162 Da corresponded to the mass of a single glucosyl repeating unit, confirming cellulose oligomer production. The DP values obtained from MALDI-TOF-MS were close to those obtained from 1H NMR spectra, suggesting reliability of the DP values. The MALDI-TOF-MS measurements also revealed that the cellulose oligomers had a certain degree of DP distribution, mainly DP values of 6 to 8. The aforementioned average DP values were significantly smaller than those obtained from D-glucose

primers (approximately 10) under the same synthetic conditions.38 Therefore, these

products seemed to be more readily solidified to be isolated from CDP than those obtained from D-glucose primers, resulting in reaction termination at smaller DP values for the cellobiose primer system. Note that the average DP values of enzymatically synthesized cellulose oligomers in the previous hydrogels ranged 8-9,47,48 which were slightly greater than those for the present cellobiose primers, indicating that the DP values do not directly predominate the production of network structures. All these observations possibly suggest that the two synthetic systems using different primers (that is, cellobiose versus D-glucose) showed different kinetic mechanisms for cellulose oligomer production.


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The crystal structures of water-insoluble cellulose oligomers in the hydrogels prepared at different CDP concentrations were characterized by WAXD and ATR-FTIR absorption measurements (Figure 4). WAXD profiles showed three diffraction peaks at 2θ = 12.3, 19.9, and 22.1 degree for d-spacings of 0.72, 0.45, and 0.40 nm for all the products (Figure 4a), which were assignable to 1 1 0 , 1 1 0 , and 0 2 0 of the cellulose II allomorph, respectively.37 Furthermore, the ATR-FTIR absorption spectra showed two sharp peaks, at approximately 3440 and 3490 cm-1, for all the products (Figure 4b), and these were also assignable to stretching vibration bands of the OH groups of cellulose molecules in the same allomorph.54 Other peaks at 1372 cm-1, 1418 cm-1, and 2891 cm-1 were assignable to vibration bands of the C-H deformation, CH2 scissoring at C(6), and C-H stretch of cellulose molecules in the same allomorph, respectively.55 This allomorph was the same as that for the cellulose oligomers in rectangular nanosheets enzymatically produced by using D-glucose primers.38 It was therefore found that the cellulose oligomers enzymatically synthesized by using cellobiose primers were crystallized in an anti-parallel manner, irrespective of the CDP concentration. Morphological characterizations of the hydrogels’ network structures. The morphologies of the network structures in the hydrogels prepared at different CDP concentrations were observed by SEM after freeze fracturing and subsequent drying of the corresponding organogels prepared with a tert-butyl alcohol solvent (Figure 5). Significantly, the hydrogels were composed of well-formed nanoribbon network structures in all cases, indicating that the enzymatically synthesized cellulose oligomers were in situ self-assembled into highly ordered nanostructures in the reaction solutions. In addition, branched nanoribbons were not observed, suggesting that the physical contact of nanoribbons was the driving force for network formation. The widths and lengths of the nanoribbons were estimated to be several hundred nanometers and more than several micrometers, respectively, irrespective of the


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CDP concentration. Furthermore, AFM observations of the mechanically crushed nanoribbons revealed the uniform thicknesses to be 4.4 ± 0.3 nm, 4.7 ± 0.9 nm, and 4.5 ± 0.2 nm at CDP concentrations of 0.1, 0.2, and 0.4 U mL-1, respectively, which were comparable with the chain length (~4.6 nm) of cellononaose (DP = 9) in the cellulose II allomorph.37 The thicknesses of the nanoribbons were slightly greater than the chain lengths of the cellulose oligomers with the aforementioned average DP values. However, considering that the cellulose oligomers had a certain level of DP distribution and that the nanoribbons should be hydrated in an air atmosphere, those oligomers were likely to align perpendicularly to the base plane of the nanoribbons in an anti-parallel manner, as illustrated in Figure 1. It was therefore found that the thickness of the nanoribbons was defined by the DP values and crystal allomorph of cellulose oligomers at sub-ten nanometer scales. The widths of nanoribbons and the perpendicular alignment of cellulose oligomers were similar to those of rectangular nanosheets enzymatically produced by using D-glucose primers.38 In addition, it is known that the driving forces for crystal growth into rectangular nanosheets against the direction of the long and short axes, corresponding to the length and width directions of the nanosheets, are based on intermolecular hydrophobic and hydrogen-bonding interactions, respectively.37,56 Therefore, the findings suggest that the present nanoribbons were produced by promoting the hydrophobic interactions against the direction of the long axes. Furthermore, the anti-parallel molecular orientation perpendicular to the base plane of nanoribbons and the morphologies of nanoribbons in this study were quite similar to those of enzymatically synthesized oligo(ethylene glycol)-bearing46 or alkylated45 cellulose oligomer derivatives and of cellulose oligomers enzymatically synthesized in macromolecular crowding environments.47 This similarity suggests that cellulose oligomers and their derivatives have great potential for producing nanoribbon


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network structures through their crystallization-driven self-assembly, leading to the formation of unique cellulose-based hydrogels. Kinetic Mechanisms for Nanoribbon Production. The aforementioned observations revealed that the conversions of αG1P monomers did not directly correlate with the production of nanoribbon network structures and that the nanoribbons for cellobiose primers were composed of cellulose oligomers with smaller DP values than those in rectangular nanosheets for D-glucose primers,38 even though cellobiose is transiently produced as an intermediate product when D-glucose primers are used. These differences in the two synthetic systems using different primers might correlate with the kinetics of the production of water-insoluble cellulose oligomers; therefore, time-dependent differences in the production of cellulose oligomers were analyzed for the two synthetic systems. Figure 6 shows photographs of the reaction time dependence of the product solutions for the enzymatic reactions using the present cellobiose or conventional D-glucose primers. The product solutions for cellobiose primers were transparent and in sol states up to a reaction time of 3 h, and then transformed into turbid hydrogels after a reaction time of 6 h (Figure 6a), suggesting that water-insoluble cellulose oligomers that were sufficient for aggregate production were not synthesized until a reaction time of 3 h. Meanwhile, the product solutions for D-glucose primers became turbid after a reaction time of 3 h and produced aggregates, and the amount of the aggregates increased with the reaction time (Figure 6b). To analyze the progress of the CDP-catalyzed reverse phosphorolysis reactions in an alternative way, the amounts of phosphate ions eliminated from αG1P monomers in the product solutions were quantified (as another measure of monomer conversions). Then, the two monomer conversions estimated from the amounts of water-insoluble products collected by centrifugation processes and of phosphate ions were plotted against the reaction time (Figure 7).


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Significantly, the two profiles for cellobiose primers were clearly different (Figure 7a). In other words, the profile for the amount of water-insoluble products showed a latent period, while that for the amount of phosphate ions was saturated, suggesting that when using cellobiose primers, αG1P monomers were used not only for water-insoluble products but also for water-soluble ones. In fact, the production of phosphate ions when using cellobiose primers, up to a reaction time of 3 h, confirmed the progress of the enzymatic reactions even in the transparent product solutions. By contrast, the two profiles were identical when D-glucose

primers were used, and the two conversions increased gradually with the reaction

time (Figure 7b), indicating that water-soluble cellulose oligomers are rarely presented in the reaction solutions. In other words, αG1P monomers (when using D-glucose primers) were simply used for the production of water-insoluble cellulose oligomers irrespective of the reaction time. Note that the monomer conversions estimated from the water-insoluble products, for both cellobiose and D-glucose primers, were almost the same after reaction times of 6 h and 24 h, even though the states of the product solutions were considerably different (hydrogels and dispersions, respectively). This difference also confirmed that the kinetics of the production of water-insoluble cellulose oligomers were essential for the production of different morphologies. MALDI-TOF-MS measurements were conducted for the product solutions after a reaction time of 3 h (Figure 8). The sodium and potassium ion adducts of water-soluble cellulose oligomers with DP values of 4-6 were observed from the transparent product solutions when using cellobiose primers (Figure 8a). Considering the aforementioned production of phosphate ions at the same time (Figure 7a), it is likely that large amounts of cellobiose primers are propagated by using αG1P monomers, leading to the enzymatic synthesis of water-soluble cellulose oligomers at the initial reaction stage. The subsequent synthesis of large amounts of water-insoluble cellulose oligomers seemed to induce the production of


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nanoribbon network structures, as schematically illustrated in Figure 9a. Note that the catalytic efficiencies (kcat/Km) of the propagation reaction gradually decreased from cellobiose to cellotetraose, indicating that cellobiose primers are preferentially propagated at the initial reaction stage.37,57 On the other hand, the reaction time required for hydrogel production increased with increasing cellobiose concentration (Figure S1), suggesting that greater amounts of cellobiose primers were used for the propagation reaction at greater cellobiose concentrations, leading to the production of greater amounts of water-soluble cellulose oligomers. This observation also confirmed the preferential propagation of cellobiose primers at the initial reaction stage. Meanwhile, MALDI-TOF-MS signals for cellulose oligomers were rarely observed from the supernatants for D-glucose primers after a reaction time of 3 h, although those peaks were clearly present when precipitates were analyzed (Figure 8b). This observation is acceptable, because it is known that the small amounts of cellobiose synthesized from D-glucose were predominantly propagated by CDP to gradually synthesize water-insoluble cellulose oligomers and subsequently produce rectangular nanosheets as aggregates,38 as schematically illustrated in Figure 9b. Note that after a reaction time of 3 h the DP values for the products of D-glucose primers were obviously higher than those after 3 d. This observation might be acceptable when considering the possibility that the propagation reaction proceeded to a greater extent at the initial reaction stage before solidification of the products owing to retardation of crystallization by smaller concentrations of the products and/or by greater concentrations of αG1P monomers. As a consequence, it can be proposed that the kinetics of the production of water-insoluble cellulose oligomers, which depends on the much greater reactivity of cellobiose than D-glucose with CDP,37 controls the crystallization-driven self-assembly of enzymatically synthesized cellulose oligomers, followed by the production of well-formed nanoribbon network structures versus rectangular nanosheets. On the other


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hand, even though the enzymatic synthesis was demonstrated using cellobiose primers under the standard conditions after pre-incubation of CDP (0.32 U mL-1) with D-glucose primers (79 mM) at 60 °C for 1 h, the monomer conversion was not influenced by pre-incubation, confirming that CDP more preferentially recognizes cellobiose than D-glucose. When the aforementioned propagation mechanisms are interpreted from the viewpoint of conventional polymer synthesis, the cellobiose primer system is similar to the mechanism for living polymerizations, in which the propagation reaction proceeds from initiators side by side. By contrast, the D-glucose primer system is similar to the mechanism for free radical polymerizations of vinyl monomers, in which radical species produced from initiators preferentially propagate. Although the termination mechanisms differ considerably between enzymatic and conventional polymer syntheses, the DP distributions of water-insoluble cellulose oligomers for the cellobiose primer system (Figure 3b) were significantly smaller than those for the D-glucose primer system,38 which also reveals the similarity of the former system to living polymerizations. Therefore, this study successfully demonstrated that careful design of reaction mechanisms has strong potential for allowing the control of reaction-induced self-assembly of crystalline molecules.

CONCLUSIONS αG1P monomers were oligomerized from cellobiose primers by CDP-catalyzed reverse phosphorolysis reactions in aqueous solutions. The transparent reaction solutions were transformed into turbid and colorless hydrogels under appropriate reaction conditions, producing network structures composed of water-insoluble cellulose oligomers. 1H NMR spectra revealed the production of cellulose oligomers with average DP values of 7-8 at various CDP concentrations. MALDI-TOF-MS also confirmed the production of cellulose oligomers, which have the aforementioned DP values with a certain degree of DP


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distribution. WAXD profiles and ATR-FTIR absorption spectra revealed that the cellulose oligomers formed crystals with the anti-parallel cellulose II allomorph. SEM observations of the xerogels revealed that the hydrogels were composed of well-formed nanoribbon network structures, indicating that the cellulose oligomers were in situ self-assembled into highly ordered nanostructures in the reaction solutions. In fact, the nanoribbons had the widths of several hundred nanometers, lengths of more than several micrometers, and sub-ten nanometer uniform thicknesses. Analyses of reaction time-dependence suggested that the production of nanoribbon network structures was based on kinetic control of the amount of water-insoluble cellulose oligomers produced. It is generally difficult to control product nanostructures for precipitation reactions such as enzymatic syntheses of cellulose. This concept for the kinetic control of production amounts will be a versatile strategy for producing highly ordered hierarchical structures through reaction-induced self-assembly of solvent-insoluble molecules. Studies of the potential applicability of this strategy to other synthetic systems of self-assembling oligo-/polymers are now in progress.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX. Time-dependent photos of the product solutions at different cellobiose concentrations. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors wish to thank the Division of Materials Analysis Ookayama (Tokyo Tech) for the SEM observations and WAXD measurements. This study was partially supported by the Funding Program for Next Generation World-Leading Researchers (NEXT Program, GR022), the Grants-in-Aids for Scientific Research (26288056, 26620174, and 16K14075) from the Japan Society for the Promotion of Science, and the collaborative research with JX Nippon Oil & Energy.

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Figure 1. (a) Reaction scheme for the CDP-catalyzed synthesis of cellulose oligomers and (b) schematic illustration of nanoribbon network structures prepared by crystallization-driven in situ self-assembly of enzymatically synthesized cellulose oligomers.

Figure 2. Photographs of the product solutions prepared at different CDP concentrations.


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Figure 3. (a) 1H NMR spectra and (b) MALDI-TOF-MS spectra of the products prepared at different CDP concentrations.


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Figure 4. (a) WAXD profiles and (b) ATR-FTIR absorption spectra of the products prepared at different CDP concentrations.


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Figure 5. (a) SEM images of the xerogels prepared at different CDP concentrations and (b) AFM images of the mechanically crushed products prepared at different CDP concentrations.


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Figure 6. Photographs of the reaction time-dependence of the product solutions prepared by using (a) cellobiose and (b) D-glucose primers.

Figure 7. Reaction time-dependent monomer conversions of αG1P monomers for the products prepared by using (a) cellobiose and (b) D-glucose primers.


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Figure 8. MALDI TOF-MS of (a) the water-soluble products prepared by using cellobiose primers and (b) the supernatants and precipitated products prepared by using D-glucose primers after a reaction time of 3 h.


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Figure 9. Schematic illustration of the enzymatic synthesis of cellulose oligomers using (a) cellobiose and (b) D-glucose primers for the production of nanoribbon network structures and rectangular nanosheets, respectively.


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Table of Contents:


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Self-Assembly of Cellulose Oligomers into Nanoribbon Network

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