Molecular design and synthesis of cellobiose(oNB2)-pNBWe designed and synthesized cellobiose derivatives bearing two o-nitrobenzyl (oNB) groups at the C6 positions to confer photo-responsiveness on the self-assembled glyco-materials, as displayed in Fig. 1a. Previous studies on the aqueous self-assembly of amino acid or peptide derivatives17,18,19 have suggested that the p-nitrobenzyl (pNB) group introduced at the C1 atom (the reducing end of cellobiose) in this study could promote self-assembly under aqueous conditions. This is probably due to the stronger π–π interactions expected for electron-deficient aromatic groups capable of adopting offset (parallel displaced) stacking20,21. Cellobiose(oNB2)-pNB was synthesized according to the concise scheme displayed in Fig. 1b (see also, Supplementary Scheme S1). In brief, 6-O-oNB-modified d-glucose donor 1 and 6-O-oNB-modified d-glucose acceptor 2 were glycosylated in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) to afford the corresponding cellobiose (3) in 64% yield. Subsequently, cellobiose 3 was glycosylated with pNB alcohol under the NIS(N-iodosuccinimide)–TfOH(trifluoromethanesulfonic acid) promoter system, affording pNB-glycosylated cellobiose (4) in 83% yield. Finally, the deprotection of 4 over two steps afforded cellobiose(oNB2)-pNB. The synthesized cellobiose(oNB2)-pNB was unambiguously characterized by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry measurements (for NMR spectra see Supplementary Figs. S7-S22). Notably, the present synthetic scheme can be employed for the systematic introduction of a variety of functional groups, including pNB in this study, to the reducing end of the cellobiose(oNB2) scaffold (at glycosidation steps from 3 to 4, Fig. 1b), without the loss of potential photo-responsiveness.Fig. 1: Molecular design and synthesis of cellobiose(oNB2)-pNB.a Chemical structure of cellobiose(oNB2)-pNB [Electron diffraction structure (CCDC: 2369905) of cellobiose(oNB2)-pNB is also shown, vide infra (Figs. 4, 5)]. b Synthesis scheme of cellobiose(oNB2)-pNB from glucose derivatives (1 and 2) (Supplementary Scheme S1 for the complete synthesis scheme).Properties of aqueous self-assembled cellobiose(oNB2)-pNBTo control the self-assembly structural morphology of cellobiose(oNB2)-pNB under aqueous conditions and investigate the effect of the concentration and cooling rate on its morphology, cellobiose(oNB2)-pNB at different concentrations [0.05, 0.10, and 0.20 wt% in 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-NaOH (pH 7.4) containing dimethyl sulfoxide (DMSO), as typical aqueous solutions] was subjected to thermal treatment. The treatment procedure was as follows: heating at 95 °C for 5 min, followed by cooling at 5.0, 20, and 30 °C min–1 using a conventional thermal cycler, according to the protocol depicted in Fig. 2a. Subsequently, the morphology of the self-assembled structures of cellobiose(oNB2)-pNB in the suspensions obtained after thermal treatment was determined by optical microscopy (OM) and polarized optical microscopy (POM, under cross-polarizers). The morphology was dependent on not only the concentrations but also the cooling rate in the preparation of the aqueous suspension, as summarized in Fig. 2b (Supplementary Fig. S1 for the images). In brief, at low concentrations and high cooling rates, such as 0.05 wt% (0.67 mM) and 30 °C min–1, microspheres were found (Fig. 2c), the average diameter of which was 2.04 ± 0.97 µm (n = 70). In contrast, at high concentrations and low cooling rates, such as 0.10 wt% (1.3 mM) and 5.0 °C min–1, long (over several hundred µm) microfibers were observed, as shown in Fig. 2c–i. In addition, from the POM images shown in Fig. 2c–ii, the microfibers exhibit stronger birefringence than the microspheres, suggesting that the cellobiose(oNB2)-pNB molecules in the microfibers have crystalline and/or ordered structures. These results motivated us to investigate the time-dependent morphological transformation of the microspheres, which would be considered as molecule-rich condensates22,23. As anticipated, the emergence and growth of long microfibers, i.e., the structural transformation from microspheres to microfibers, was observed (Fig. 2c, from “iii & iv” to “v & vi”). As shown in Fig. 2c–v, the length of the microfibers increased and reached more than 100 µm after 6 h. To obtain further insight into the structural transformation, the influence of the Hofmeister effect24,25 was examined. Without using the buffering component (i.e., HEPES), microfibers were formed directly after cooling (Supplementary Fig. S2). Conversely, the presence of SCN−, as a chaotropic anion, resulted in the transformation of the initially obtained microspheres into microfibers, which is comparable to the result for the aqueous HEPES buffer described above. In contrast, microfibers were directly formed in the presence of Cl−, as a representative kosmotropic anion (Supplementary Fig. S2). These results suggest that the dehydration of the self-assembled structures plays an important role in their transformation from microspheres into microfibers. Collectively, the proposed mechanism of the thermally induced aqueous self-assembly of cellobiose(oNB2)-pNB to form microfibers is depicted in Fig. 2d. A similar structural transformation from spherical to fibrous architecture has been reported for peptide- and protein-based self-assemblies22,23. According to the reports, Ostwald ripening and nucleation-induced crystallization in liquid–liquid phase separated state could play important roles in the formation of supramolecular fibrous architectures26.Fig. 2: Thermally induced aqueous self-assembly of cellobiose(oNB2)-pNB.a Representative procedure for the preparation of the self-assembled structures of cellobiose(oNB2)-pNB through a thermal process under aqueous conditions. b Morphology of cellobiose(oNB2)-pNB observed within 5 min after cooling to 25 °C at different rates, depending on its concentration. c Representative optical microscopic (OM, panels i, iii, v) and polarized optical microscopic (POM, under cross-polarizers, panels ii, iv, vi) images of the self-assembled structures of cellobiose(oNB2)-pNB. Conditions: [cellobiose(oNB2)-pNB] = 0.05, 0.10, and 0.20 wt%; 100 mM HEPES-NaOH (pH 7.4) containing 1, 2, and 4 vol% DMSO, rt. See Supplementary Fig. S1 for more details. d Schematic representation of cooling rate dependent formation of microspheres and microfibers through aqueous self-assembly and the isothermal transformation from microspheres into microfibers.Structural elucidation of the glyco-microfibers consisting of cellobiose(oNB2)-pNBTo obtain further insight into the structure of the cellobiose(oNB2)-pNB microfibers at the nanoscale, a series of microscopic analyses [transmission and scanning electron microscopy (TEM, SEM) and atomic force microscopy (AFM), Fig. 3] were performed. As shown in Fig. 3a, ribbon-like nanostructures were observed in the TEM images. Moreover, the magnified TEM image (Fig. 3a–ii) revealed the presence of stripes along the long axis of the ribbon-like nanostructures, and relatively thin fibers were concurrently observed with the ribbon-like nanostructures in the low-magnification TEM image (Fig. 3a–i). These indicate that the microfibers observed in the OM images (Fig. 2c) can be attributed to the flat ribbon-like nanostructures assembled from thin nanofibrils. A similar morphology was observed in the SEM images (Fig. 3b) and AFM height images (Fig. 3c–i), which further supports the view that the nanofibril structures can be assembled into ribbon-like nanostructures. In addition, the height of the nanofibrils was evaluated to be approximately 2 nm from the AFM cross-sectional profile (Fig. 3c–ii), which is almost consistent with the atomic-scale structural analysis result based on the X-ray structure (vide infra).Fig. 3: Microscopic observations of the glyco-microfibers consisting of cellobiose(oNB2)-pNB.a Representative (i, ii) TEM images of the microfibers on an elastic-carbon-coated grid. b Representative (i, ii) SEM images of the microfibers on a silicon wafer and the corresponding energy-dispersive X-ray spectroscopy (EDS) pattern (inset in i) recorded from the yellow mark in the image (i). c Representative (i) AFM height image (tapping mode) of the microfibers on freshly cleaved mica and (ii) the corresponding cross-sectional profile along the yellow line in the image (i).To obtain insights into the atomic-scale self-assembly mode, electron diffraction (ED) structural analyses27 for the cellobiose(oNB2)-pNB microfibers were conducted at room temperature (Supplementary Fig. S24). Furthermore, X-ray structural analyses were performed for thin-needle single crystals obtained from 2-propanol, although the crystals were too thin to obtain complete data (Supplementary Fig. S23). The analyzed crystal structures of cellobiose(oNB2)-pNB provided valuable insights into the individual molecular conformation as well as the self-assembled structure. First, the ED structure (Figs. 4, 5) was compared with those of cello-oligosaccharides (such as cellobiose28) and cellulose polymorphs (Iα29, Iβ30, II31, IIII32,33), whose typical structural features are summarized in Table 1. In the cellulose polymorphs, the cellulose main chain aligns with the long axis of the fibrils, whereas the packing direction (parallel or antiparallel) is dependent on the polymorphs (Fig. 5d). That is, parallel packing was observed for triclinic cellulose Iα and monoclinic celluloses Iβ and IIII, whereas antiparallel packing was observed for monoclinic cellulose II. In addition, celluloses II and IIII showed eclipsed chain packing, whereas the others showed quarter staggered chain packing (Fig. 5d). Accordingly, the parallel and eclipsed packing modes of the cellobiose(oNB2)-pNB chains along the a-axis disclosed in this study for both of the ED and X-ray structures (Fig. 4a, Supplementary Figs. S3,4,5) are comparable to those of cellulose IIII. The molecular conformations of the cellobiose scaffolds (in which pyranose rings appear in 4C1 conformation) constituting cellobiose(oNB2)-pNB are displayed in Fig. 5c–i (Supplementary Fig. S5c for both of the ED and X-ray structures). For instance, the glycosidic torsion angles [φ (O5–C1–O–C4′) and ψ (C1–O–C4′–C3′), see Table 1 and Fig. 5a for definitions] of cellobiose(oNB2)-pNB (X-ray) are highly similar to those of cellulose IIII and dissimilar to those of cellobiose. On the other hand, the ED structure of cellobiose(oNB2)-pNB is more similar to those of cellobiose. In general, φ is largely influenced by the exo-anomeric effect, whereas ψ is dependent on the noncovalent bonding interactions with nearby residues and solvents33. In addition, the orientation of the C6–O6 bonds (Fig. 5b), which could be dependent on the hydrogen bonding network patterns of the cellulose polymorphs29,30,31,32,33 and the gauche effect against the equatorial C4–O4 bond34, was gt (gauche-trans) for both cellobiose(oNB2)-pNB (X-ray) and cellulose IIII (Fig. 5c, Supplementary Fig. S5c). On the other hand, the combination of gg and gt orientations was identified for the ED structure of cellobiose(oNB2)-pNB. Hydrogen bonding interactions between NO2 group and O(3)H group (~3.0 Å) may stabilize the gg orientation while the disorder of the two oNB groups in cellobiose(oNB2)-pNB (ED) would be attributed to their intrinsic flexibility. The presence of co-crystallized water in the X-ray structure but not in the ED structure (Supplementary Fig. S5 for the comparison) also suggests that the two oNB groups are flexible with respect to the rigid cellobiose core. Such intrinsically disordered groups may be related to the transient formation of non-crystalline microspheres (Fig. 2d).Fig. 4: Structural analysis of the glyco-microfibers consisting of cellobiose(oNB2)-pNB.a Electron diffraction (ED) structures (gray: carbon, red: oxygen, blue: nitrogen, white: hydrogen. Top view: stick model; all hydrogens are omitted for clarity. Side view: space-filling model) of the microfibers of cellobiose(oNB2)-pNB (Supplementary Fig. S3 for long axis of the microfibers). b (i) Experimental PXRD pattern (three representative peaks are picked up as d-spacing) of cellobiose(oNB2)-pNB microfibers and (ii, iii) simulated patterns for (ii) the microfiber electron diffraction data and (iii) the needle-like single-crystal X-ray data of cellobiose(oNB2)-pNB obtained from 2-propanol (Supplementary Fig. S5 for the comparison of the ED and X-ray structures).Fig. 5: Structural parameters of the cellobiose-based compounds.a Glycosidic torsion angles defined by the International Union of Pure and Applied Chemistry (IUPAC) convention46 or the C–1 crystallographic style47: φ (O5–C1–O–C4′) and ψ (C1–O–C4′–C3′)] for the cellobiose unit (β1 → 4-linked). b Orientation of the C6–O6 bond (tg, gg, gt: t and g denote trans and gauche conformations, respectively) for the β-glucose unit. The first g or t refers to the position of O6 against O5 (O5–C5–C6–O6), whereas the second g or t refers to the position of O6 against C4 (C4–C5–C6–O6). c Glycosidic torsion angles and orientation of the C6–O6 bond of (i) cellobiose(oNB2)-pNB (ED), (ii) cellobiose, (iii) cellulose Iα, and (iv) cellulose IIII. d Schematic representations of the packing manners of the main chains and their orientations against or along the fiber main axis.Table 1 Glycosidic torsion angles (degrees) [C–1 crystallographic style: φ (O5–C1–O–C4′) and ψ (C1–O–C4′–C3′)] and the orientation of the C6–O6 bond (tg, gg, gt) for the cellobiose units of cellobiose(oNB2)-pNB, β-cellobiose, and cellulose polymorphs (Iα, Iβ, II, IIII) (see also Fig. 5a, b for the definitions) and packing arrangement of the cellobiose or cellulose main chainsSecond, to further compare the ED and X-ray structures of cellobiose(oNB2)-pNB, the cellobiose(oNB2)-pNB microfibers obtained by thermal annealing under aqueous conditions (0.10 wt%, 5 °C min–1, in Milli-Q water) were subjected to powder X-ray diffraction (PXRD) analyses. As shown in Fig. 4b, there are reasonably overlapped peaks between the obtained PXRD (Fig. 4b–i) and the simulated patterns (Fig. 4b–ii) for the ED structure of cellobiose(oNB2)-pNB, indicating that the ED structure could represent the atomic-scale self-assembly mode in the cellobiose(oNB2)-pNB microfibers. Interestingly, each long axis of the needle-like crystal (X-ray) and microfibers (ED) of the cellobiose(oNB2)-pNB is parallel to the a-axis (Supplementary Figs. S3,4). Such a molecular arrangement in the fibrous structure is reminiscent of amyloid peptide fibers/fibrils having a cross-β-amyloid structure4,35. Hereinafter, we thus refer to this self-assembled architecture of cellobiose(oNB2)-pNB microfibers as the cross-β-glucan structure. Notably, the self-assembled (fibrous) architectures of cellobiose28 and cello-oligosaccharides11,13 reported previously also have the cross-β-glucan structure. Nonetheless, antiparallel packing (cellulose II type) is frequently observed in the self-assembly structures as far as without additional controlling factors, such as the introduction of alkyl groups at the reducing end of cello-oligosaccharides36.Third, there are intriguing differences between the present self-assembled fibrous structure of cellobiose(oNB2)-pNB and that of a closely related disaccharide (gentiobiose: β−1,6-linked glucose, not β−1,4-linked) derivative bearing aromatic groups, which has recently been reported by another research group7,8. In particular, the mode of π interactions among the aromatic groups introduced into the disaccharide scaffolds is significantly different. That is, the crystal structures (ED and X-ray) of cellobiose(oNB2)-pNB shows offset parallel π–π stacking modes among aromatic rings (oNB and pNB), whereas the C–H⋯π type edge-to-face interaction mode was unveiled in the self-assembled structure of gentiobiose bearing aromatic groups7,8. As described in the molecular design section, the nitro group substituted at the phenyl ring could allow for enhanced π–π interactions, affording offset parallel π–π stacking modes17,18,19,20. In contrast, according to a Hunter–Sanders model37 (whose limitation has been actively discussed21), an electric quadrupole would play a crucial role in the phenyl ring without a nitro group, resulting in C–H⋯π type edge-to-face interactions. Although further investigation is required, it is clear that the presence or absence of a nitro group may, at least in part, account for the difference in the self-assembly mode between cellobiose(oNB2)-pNB bearing nitrophenyl groups and gentiobiose bearing pristine phenyl rings. The center-of-mass distances between the stacked NB rings were estimated to be 5.0 Å for ED and 4.5 Å for X-ray (distances to rings tool implemented in PyMOL was used) for the offset parallel π–π stacking, as displayed in Fig. 4a, b. These distances are almost comparable to previously reported distances for the center-to-center spacing of aromatic rings17,21. Interestingly, the offset parallel π–π stacking distance of the X-ray structure coincided with the face-to-face stacking distances of the glucose rings in the cellobiose scaffold (4.5 Å for X-ray)28 and that of cellulose IIII (4.4 Å)32. The π–π stacking distance of the ED structure was larger than that of the X-ray structure due to the oblique stacking of the cellobiose scaffold but matched with the face-to-face stacking distances of the glucose rings (5.0 Å for ED). These complementary distances may be crucial for the formation of the stable 1D self-assembled architectures of cellobiose(oNB2)-pNB microfibers having the cross-β-glucan structure.Photo-responsiveness of the cellobiose(oNB2)-pNB microfibersFinally, we evaluated the photo-responsiveness of the cellobiose(oNB2)-pNB microfibers [typical preparation conditions; 0.10 wt% (1.3 mM), 20 °C min–1, in 100 mM HEPES-NaOH (pH 7.4) containing 2 vol% DMSO]. The oNB groups introduced at the C6 positions of cellobiose(oNB2)-pNB can be removed by ultraviolet (UV)-light irradiation, as shown in the plausible scheme in Fig. 6a38,39. Such a photo-induced transformation of the chemical structure should increase the aqueous solubility and result in the disassembly of the cellobiose(oNB2)-pNB microfibers under aqueous conditions. For instance, pristine cellobiose exhibits low, but reasonable solubility in water at concentrations of 39.4 mM40 or 14 g/100 g41, well above the typical concentration of cellobiose(oNB2)-pNB (0.10 wt%, 1.3 mM) at which microfibers are formed. As expected, upon irradiation with UV light (365 nm, LED), the cellobiose(oNB2)-pNB microfibers were degraded within 3 min, as shown in Fig. 6b–i. Notably, a non-negligible amount of brown precipitates remained, probably owing to the formation of aqueous insoluble residues derived from the photo-removed 2-nitrobenzyl groups (e.g., 2-nitrosobenzaldehyde)39. Nonetheless, encouraged by this result, a film mask rendering a 20 µm width line was set in front of the UV light source. As displayed in Fig. 6b–ii, the spatio-controlled degradation of the cellobiose(oNB2)-pNB microfibers was demonstrated, which is one of the advantages of photo-responsiveness38. To evaluate the molecular transformation induced by the UV-light irradiation, 1H NMR spectroscopic analyses were performed after dissolving the lyophilized samples in a solution of DMSO-d6:D2O = 5:1 (v/v). Cellobiose(oNB2)-pNB (Fig. 6c–i) and cellobiose (Fig. 6c–iv) molecules can be dissolved in this mixed solution. After UV-light irradiation to complete the degradation of the cellobiose(oNB2)-pNB microfibers (Supplementary Fig. S6), the number of peaks assignable to the oNB groups (peaks: “b,c,d,f,h”) decreased significantly, whereas those for the pNB group (peaks: “a,e,g,i”) remained almost unchanged, as shown in Fig. 6c–ii. Furthermore, by membrane filtration (pore size: 0.45 µm) to remove precipitates before lyophilization, the obtained spectrum (Fig. 6c–iii) can almost be exclusively assigned to cellobiose-pNB, as displayed in Fig. 6a. Collectively, these results indicate that the two oNB groups introduced into cellobiose(oNB2)-pNB were selectively removed by UV-light irradiation, inducing the disassembly of the cellobiose(oNB2)-pNB microfibers.Fig. 6: Photo-responsiveness of the cellobiose(oNB2)-pNB microfibers.a Plausible photo-responsive transformation of cellobiose(oNB2)-pNB to afford a potentially water-soluble compound (i.e., cellobiose-pNB). b Representative OM images before and after UV-light irradiation (LED, 365 nm) (i) without or (ii) with a photomask. Scale bar: 50 µm. c 1H NMR spectra [400 MHz, DMSO-d6:D2O = 5:1 (v/v)] of the samples prepared from the cellobiose(oNB2)-pNB microfibers (i) before and (ii, iii) after UV-light irradiation (ii) without or (iii) with membrane filtration before lyophilization and (iv) cellobiose (see Supplementary Fig. S6 for the experimental protocols to prepare the samples).
