Article pubs.acs.org/Langmuir
Enzymatic Synthesis of Oligo(ethylene glycol)-Bearing Cellulose Oligomers for in Situ Formation of Hydrogels with Crystalline Nanoribbon Network Structures Takatoshi Nohara, Toshiki Sawada, Hiroshi Tanaka, and Takeshi Serizawa* Department of Chemical Science and Engineering, School of Materials and Chemical Technology, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan S Supporting Information *
ABSTRACT: Enzymatic synthesis of cellulose and its derivatives has gained considerable attention for use in the production of artificial crystalline nanocelluloses with unique structural and functional properties. However, the poor colloidal stability of the nanocelluloses during enzymatic synthesis in aqueous solutions limits their crystallization-based self-assembly to greater architectures. In this study, oligo(ethylene glycol) (OEG)bearing cellulose oligomers with different OEG chain lengths were systematically synthesized via cellodextrin phosphorylasecatalyzed oligomerization of α-D-glucose l-phosphate monomers against OEG-bearing β-D-glucose primers. The products were self-assembled into extremely well-grown crystalline nanoribbon network structures with the cellulose II allomorph, potentially due to OEG-derived colloidal stability of the nanoribbon’s precursors, followed by the in situ formation of physically cross-linked hydrogels. The monomer conversions, average degree of polymerization, and morphologies of the nanoribbons changed significantly, depending on the OEG chain length. Taken together, our findings open a new avenue for the enzymatic reaction-based facile production of novel cellulosic soft materials with regular nanostructures.
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properties of the nanocelluloses are still challenging.4,5,11 To widen the applications of nanocellulose-based materials, the preparation of nanocelluloses with the desired morphologies, colloidal stability, and functions are highly required. To obtain artificially designed and structurally controlled nanocelluloses, a one-step and environmentally friendly synthesis of cellulose via enzymatic reactions has been widely explored.24−27 Enzymatic synthesis is typically performed under aqueous or organic/aqueous mixture conditions using cellulase28−30 and cellodextrin phosphorylase (CDP)31−33 via glycosylation reactions. In the latter reaction in particular, when α-D-glucose l-phosphate (αG1P) monomers were oligomerized from β-D-glucose primers, sheet-like nanocelluloses with the cellulose II allomorph were produced in situ as colorless precipitates, in which the cellulose molecules with an average degree of polymerization (DP) of 9−10 were aligned perpendicularly to the base plane of the nanocelluloses, thereby exposing the terminal ends to the surfaces.32,33 The unique characteristic of the CDP-catalyzed reaction is that CDP has
INTRODUCTION Nanoscale control of molecularly assembled structures through covalent and noncovalent interactions has gained considerable attention due to the potential for fabricating new soft nanomaterials with unique physicochemical properties.1−3 In many biorelated nanomaterials, the remarkable properties of nature-based nanocelluloses, which are nanostructured cellulosic materials isolated from natural resources, have garnered renewed interest as fascinating building blocks for sustainable materials.4−9 Nanocelluloses are produced via top-down or bottom-up processes based on combinations of chemical, mechanical, and enzymatic treatments of raw materials4,9 or cultivating bacteria10 and are typically isolated as onedimensional colloidal fibrils with high aspect ratios and high crystallinity. Owing to their mechanical stiffness and chemical stability, their potential applications for composite materials, such as hydrogels and films, have been extensively investigated.5−8 However, nanocelluloses tend to aggregate during composite processes due to their limited dispersibility in various solvents,5,11 thus restricting their potential use as material components. To improve their colloidal stability, versatile chemical modifications of the surface hydroxyl groups have been investigated, including oxidation,12−14 silylation,15−17 esterification,18−20 and polymer grafting;21−23 however, surface modifications without dissolving cellulose molecules from the nanocellulose surfaces and undesired changes in the bulk © XXXX American Chemical Society
Special Issue: Tribute to Toyoki Kunitake, Pioneer in Molecular Assembly Received: April 29, 2016 Revised: June 14, 2016
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DOI: 10.1021/acs.langmuir.6b01635 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir poor substrate specificity against anomeric substitutes of β-Dglucosyl primers.24,27 Indeed, various primers, such as β-linked disaccharides,34,35 β-glycosyl antioxidants,36 and cellobiosylated dendrimers,37 were employed in the reaction to obtain terminus-functionalized cellulose oligomers as water-soluble products. In our previous work, sheet-like nanocelluloses with numerous azide groups on the surfaces were successfully synthesized via the oligomerization of αG1P monomers from βglucosyl azide primers, thus demonstrating the surfacefunctionalization of nanocelluloses through copper(I)-catalyzed Huisgen cycloaddition reactions.38 In principle, other functional primers can be applied to the CDP-catalyzed synthesis of cellulose derivatives for the facile production of novel artificial nanocelluloses. Owing to its highly hydrated and dynamic nature in aqueous phases, oligo(ethylene glycol) (OEG) chains have attract much attention as surface modifiers of nanoparticles, micelles, and vesicles to improve their colloidal stability and biorepellency.39−41 OEG chains have also been covalently introduced to biomolecules, such as proteins (or peptides),42−44 lipids,45,46 and carbohydrates,47,48 to increase their particle size, stability, and solubility. When OEG chains are covalently introduced to cellulose molecules during their enzyme-catalyzed synthesis, crystallization-driven self-assembly of the cellulose derivatives for nanocellulose formation should be affected. In this study, we demonstrated the CDP-catalyzed synthesis of cellulose derivatives using αG1P monomers and OEG-bearing β-Dglucose (Glc-EGn; n = 2, 4, 6, 8) primers with different OEG chain lengths (Figure 1a). The reaction mixtures were significantly transformed into hydrogels, regardless of the OEG chain length. Spectroscopic analysis revealed that the conversions of αG1P monomers into cellulose derivatives were altered depending on the OEG chain length and that the average DP tended to slightly increase with increasing OEG
chain length. Microscopic analysis also revealed that the hydrogels consisted of extremely well-grown nanoribbon networks with the cellulose II allomorph (Figure 1b), the morphologies of which were altered depending on the OEG chain length.
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EXPERIMENTAL SECTION
Materials. O-pentaaceyl-β-D-glucoside (>99%), diethylene glycol monomethyl ether (>99%), tetraethylene glycol monomethyl ether (>98%), hexaethylene glycol monomethyl ether (>96%), octaethylene glycol monomethyl ether (>96%), boron trifluoride diethyl etherate (>98%), and p-anisaldehyde (>99%) were purchased from Tokyo Chemical Industry Company. Dichloromethane (DCM, > 99%, dehydrated), ethyl acetate (EtOAc, > 99%), and silica gel 60N (40− 100 μm, spherical, neutral) were purchased from Kanto Chemical Company. αG1P disodium salt n-hydrate (>98%), molecular sieves 4A (MS-4A), and Dowex 50WX4 100−200 Mesh (H) cation-exchange resin were purchased from Wako Pure Chemical Industries. 0.2 mm TLC silica gel plate (60F-254) was purchased from Merck Millipore. All of the other reagents were purchased from Nacalai Tesque. Dry DCM was obtained using a Glass Contour solvent purification system. Ultrapure water with more than 18.2 MΩ·cm was obtained from MilliQ Advantage A-10 (Merck Millipore) and used throughout the study. Synthesis of Glc-EGn (n = 2, 4, 6, 8) Primers. Glc-EGn primers were synthesized by adopting previously reported methodologies.49 Briefly, a mixture of O-pentaaceyl-β-D-glucoside (1 equiv), oligo(ethylene glycol) monomethyl ether (1.2 equiv), and pulverized activated MS-4A in dry DCM was stirred at ambient temperature under argon atmosphere to remove any trace amount of water. The reaction solution was cooled to 0 °C and boron trifluoride diethyl etherate (5 equiv) was added portion-wise to the reaction solution. Following overnight stirring at ambient temperature, the reaction solution was quenched with a mixture of saturated aq. NaHCO3 and EtOAc. The mixture was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, and filtered to remove MgSO4. After removal of the solvent in vacuo, the resulting residue was purified by column chromatography on silica gel (20−50% acetone in hexane) to obtain O-tetraacetyl Glc-EGn as a colorless oil at 35−60% yield. A catalytic amount of sodium methoxide (0.1 equiv) was added to an ice−water cooled solution of the O-tetraacetyl Glc-EGn (1 equiv) in MeOH. The reaction mixture was stirred at ambient temperature for 5−8 h and then subjected to Dowex cation-exchange resin. After filtration through a pad of Celite, the filtrate was concentrated to obtain Glc-EGn primers as a colorless oil with 90−95% yield. All reactions were monitored using thin-layer chromatography performed on TLC silica gel plate and visualized using an ethanol solution of panisaldehyde and hydrogen sulfate. Glc-EGn primers were used for the CDP-catalyzed reaction without further purification. The detailed characterizations of Glc-EGn primers are further described in the Supporting Information. CDP-Catalyzed Synthesis of OEG-Bearing Cellulose. CDP preparation and CDP-catalyzed synthesis of cellulose derivatives were performed according to our previously reported protocols.38 Briefly, CDP from Clostridium thermocellum YM4 was produced in an Escherichia coli BL21-Gold (DE3) strain containing a plasmid including the cdp gene and purified using a Ni-NTA column (GE Healthcare) with fused His-Tag. αG1P monomers (200 mM) and GlcEGn primers (50 mM) were incubated with CDP (0.2 U mL−1) in 500 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.5) at 60 °C for 3 days. For scanning electron microscopy (SEM) observations, the hydrogels were soaked in ultrapure water at 4 °C for at least 1 week to remove water-soluble residues from the reaction. For other characterizations, the water-dispersed products were obtained by collapsing the hydrogels using water flow. The dispersions were washed with ultrapure water through centrifugation (15,000 rpm) and redispersion cycles to remove 99.999% of the reaction solution, and then heated at 100 °C for 10 min to inactivate the remaining CDP. The resultant products were stored at 4 °C prior to use for matrix-assisted laser desorption ionization time-of-flight
Figure 1. (a) Synthetic scheme of OEG-bearing cellulose oligomers via CDP-catalyzed oligomerizations of αG1P monomers from OEGbearing β-D-glucose primers. (b) Schematic illustration of the resulting hydrogels consisting of extremely well-grown crystalline nanoribbon networks with the cellulose II allomorph. B
DOI: 10.1021/acs.langmuir.6b01635 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir mass spectroscopy (MALDI-TOF-MS) measurements and atomic force microscopy (AFM) observations. For 1H nuclear magnetic resonance (NMR) spectra, attenuated total reflection-Fourier transform infrared (ATR-FTIR) absorption spectra, and wide-angle X-ray diffraction (WAXD), the dispersions were lyophilized prior to the measurements. Structural Analysis of OEG-Bearing Cellulose. The 1H NMR spectra of the lyophilized products in 4% NaOD/D2O (≥3% (w/v)) were recorded on a JEOL Model-ECP 400 at 400 MHz, and calibrated using residual solvent peaks (δ 4.79) as internal reference. The MALDI-TOF-MS spectra were recorded on a Shimadzu AXIMAperformance mass spectrometer equipped with a nitrogen laser (λ = 337 nm) and pulsed ion extraction, which was operated at an accelerating potential of 20 kV with a linear-positive ion mode. The mixture of 2,5-dihydroxybenzoic acid (10 mg mL−1, 1 μL), trifluoroacetic acid/acetonitrile solution (0.2% (v/v), 3 μL), and the dispersed products in ultrapure water (0.05% (w/v), 1 μL) were mounted onto a sample target plate. The ATR-FTIR absorption spectra of the lyophilized products were recorded on a JASCO FT/IR4100 spectrometer under ambient conditions, with the cumulative number of 100 and a resolution of 2.0 cm−1. The WAXD measurements of the lyophilized products were performed under ambient conditions on a Rigaku MiniFlex600 with Cu Kα radiation (λ = 0.154 nm). The SEM images were obtained on a JEOL fieldemission scanning electron microscope at an accelerating voltage of 5 kV. The water solvent of the hydrogels was exchanged by gradually immersing in 10, 20, 30, 40, 50, 60, 70, 80, 90, and 99.5% ethanol, 99.5% ethanol/tert-butyl alcohol (50:50), and 99% tert-butyl alcohol. The obtained organogels were rapidly frozen by liquid nitrogen, fractured using a razor blade, and lyophilized. The lyophilized samples were placed on substrates with conductive carbon tape and Dotite, and then coated with osmium before SEM observations. The AFM measurements were performed using a Shimadzu SPM-9600 in a tapping mode in air at ambient temperature. The samples were prepared by spin-cast of the dispersed products (0.001% (w/v)) on mica at 600 rpm for 30 min.
contrast, colorless hydrogels were produced from Glc-EGn primers (Figure 2a−d), indicating that the synthesized cellulose derivatives were self-assembled into macroscopic network structures, regardless of the OEG chain length. The monomer conversions were estimated to be approximately 60%, 30%, 40%, and 40% for Glc-EGn primers with n = 2, 4, 6, and 8, respectively, based on the yields and average DP of the products (Table 1). The monomer conversion for GlcTable 1. Summary for the CDP-Catalyzed Synthesis of OEGBearing Cellulose Oligomers primer GlcEG2 GlcEG4 GlcEG6 GlcEG8
allomorphs
thickness of the nanoribbons (nm)
width of the nanoribbons (nm)
9
cellulose II
5.3 ± 0.3
30
10
cellulose II
5.8 ± 0.4
40
10
cellulose II
6.0 ± 0.3
several hundred several hundred 100−200
40
11
cellulose II
6.1 ± 0.4
< 100
conversion (%)
DP
60
EG2 primer was greater than that for D-glucose primer (∼35%),33 whereas those for other primers were comparable to that for D-glucose primer. The greater monomer conversion for Glc-EG2 primer suggested that CDP showed the greater recognition capability against Glc-EG2 primer, potentially due to the additional interactions of the EG2 moiety with the active site of CDP via hydrogen bonding and/or hydrophobic interactions. Meanwhile, the longer OEG chains of other primers (n = 4, 6, 8) did not promote the interactions with CDP, potentially due to steric hindrance of longer OEG moieties. Molecular-Level Characterization of OEG-Bearing Cellulose. The 1H NMR spectra of water-insoluble products dissolved in 4% NaOD/D2O showed signals corresponding to the proton signals of cellulose50 and OEG chains (δ ∼ 3.5), thus confirming the successful synthesis of OEG-bearing cellulose via the CDP-catalyzed reaction (Figure 3). The average DP of the cellulose chains were estimated to be 9, 10, 10, and 11 for Glc-EGn primers with n = 2, 4, 6, and 8, respectively, based on the integral ratios of internal (δ = 4.3) and terminal (δ = 4.2) anomeric protons (Table 1). The average DP of the cellulose chains slightly increased as the OEG chain length increased, potentially suggesting that the crystallization-driven self-assembly of the synthesized cellulose derivatives with longer OEG chains was suppressed due to their increased solubility and/or steric hindrance of OEG chains. MALDI-TOF-MS showed two series of peaks with a peak-topeak mass increase of 162 Da, which was equivalent to the mass of the glucosyl repeating unit (Figure S1 in the Supporting Information). The observed masses corresponded to the sodium or potassium ion adducts of the synthesized cellulose derivatives with a DP of 6−12, whereas those with smaller DP (
