Letter pubs.acs.org/macroletters
Cohelical Crossover Network by Supramolecular Polymerization of a 4,6-Acetalized β‑1,3-Glucan Macromer Junji Sakamoto,*,† Rio Kita,‡ Isala Duelamae,‡ Masashi Kunitake,§ Megumi Hirano,§ Daisuke Yoshihara,† Tatsuhiro Yamamoto,† Takao Noguchi,∥ Bappaditya Roy,∥ and Seiji Shinkai†,∥,⊥ †
Laboratory of Nanotechnology, Institute of Systems, Information Technologies and Nanotechnologies, 4-1 Kyudai-Shinmachi, Nishi, Fukuoka 819-0388, Japan ‡ Department of Physics, School of Science, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan § Department of Applied Chemistry and Biochemistry, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo, Kumamoto 860-8555, Japan ∥ Institute for Advanced Study, Kyushu University, 4-1 Kyudai-Shinmachi, Nishi, Fukuoka 819-0388, Japan ⊥ Department of Nanoscience, Faculty of Engineering, Sojo University, 4-22-1 Ikeda, Nishi, Kumamoto 860-0082, Japan S Supporting Information *
ABSTRACT: Natural polysaccharides represent a renewable resource whose effective utilization is of increasing importance. Chemical modification is a powerful tool to transform them into processable materials but usually sacrifices the original structures and properties of value. Here we introduce a chemical modification of Curdlan, a β-1,3-glucan, via 4,6-acetalization. This modification has successfully combined a helix-forming ability of Curdlan with new solubility in organic media. Furthermore, it has operationalized efficient cohelical crossovers (CCs) among the helices to demonstrate the formation of an extensive supramolecular network that goes well beyond the nanoscopic regime, allowing for preparation of flexible self-supporting films with macroscopic dimensions. This protocol, which is now viewed as supramolecular polymerization of a helical polysaccharide macromer, can add a new dimension to “polysaccharide nanotechnology”, opening a door for the creation of unconventional polymer materials based on the cohelical crossover network (CCN).
A
considered a polysaccharide for use as the macromer with a view to exploring bottom-up “polysaccharide nanotechnology”. The homogeneous sequence of the naturally occurring polysaccharide indeed cannot match the sequence of the artificial DNA specifically designed for the sophisticated nanoconstruction. However, the ready availability and modifiability of the polysaccharide can open attractive opportunities for practical applications. This paper describes the initial important steps in this direction. CCs can operate not only with DNA but in principle also with any other natural or synthetic polymers that are able to form cohelices. Hence, all such polymers can be considered as potential candidates for the macromer. Polymers usually exist as a mixture of molecular chains with unequal lengths. Therefore, the cohelices by necessity have overhangs at the ends that are available for spontaneous growth by CCs. This perspective is indeed supported by a gelation mechanism of some doublehelix-forming polysaccharides proposed by Morris et al.3−5
part from the vital roles biopolymers play in Nature, there are an increasing number of attempts to take advantage of their structure-forming characters for abiotic applications. A famous example is represented by the structural DNA nanotechnology coined by Seeman et al.1 This makes use of DNA molecules as building blocks for the bottom-up nanoconstruction. The DNA sequences are artificially designed such that double-stranded helices carry single-stranded overhangs at the ends. Thereby, the double helices can be mutually connected by cohelical crossovers (CCs) of the overhangs if their sequences are complementary to each other. Because of the specific base pairing combined with the diverse sequences available by DNA synthesis, the usage of DNA offers a substantial advantage over the other biopolymers in assembling the building blocks in a predictable manner. However, the synthesis can hardly be scaled up, which limits the applications only to the small scale. From a viewpoint of polymer chemistry, we consider the helical building blocks and the successive CCs as a special set of (macro)monomers and supramolecular polymerization, respectively.2 We have thus set out to make this protocol more general and versatile for practical applications to materials. In this study, we have © XXXX American Chemical Society
Received: September 16, 2016 Accepted: December 6, 2016
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DOI: 10.1021/acsmacrolett.6b00706 ACS Macro Lett. 2017, 6, 21−26
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ACS Macro Letters Note that CCs usually connect double helices for their linear extension,1 and bifurcation occurs only occasionally.3 In contrast, the CCs of triple helices should bring about an efficient network formation because each end carries two overhangs available for bifurcation (Scheme 1). Scheme 1. Idealized Cartoon Representation of (a) Linear Extension of Double Helices and (b) Bifurcation Cascade of Triple Helices by CCs of the Overhangs at the Ends
Figure 1. Chemical modification of CUR: (a) synthetic scheme of 4,6-acetalization of 1 with the aldehyde 2 to afford the derivative 3 and representation of the chemical structures of two reference compounds 4 and 5; (b) simplified cartoon illustration of the right-handed 61 triple helix of 1; (c) cross-sectional structures of the triple helix (left) and its hypothetical homologue formed by 3 (right). Three strands of the triple helix are distinguished by color.
Here we employed Curdlan (CUR, 1), i.e., a β-1,3-glucan, as a precursor of the macromer (Figure 1a). CUR is a bacterial polysaccharide discovered by Harada et al. in 1966 and nowadays is produced on an industrial scale for use as food additives.6 CUR is known to form a right-handed 61 triple helix by itself (Figure 1b).7,8 It is also able to form unnatural hybrid-type cohelices with other polymers such as DNA and polythiophene.9,10 The strong helix-forming propensity even allows for inclusion complexation with a range of guests.10 Such a great functional versatility as well as the ready availability from commercial sources meet the requirements for practical applications. However, CUR can hardly be used as-is because it is insoluble in common solvents except alkaline water and dimethyl sulfoxide (DMSO) that unwind the helix upon dissolution.11 Aqueous suspensions of CUR turn into a gel by heating, while the gelation mechanism remains largely unclear.12 It is well-known that rigid rod-like polymers have a general tendency to aggregate by lateral cohesion.13 In order to circumvent this problem, we have introduced oligo(ethyleneoxy) side chains to CUR by using the 4,6-acetalization chemistry.14−16 Note that 4,6-acetalization cannot be applied to major polysaccharides such as cellulose, amylose, and dextran (i.e., 1,4- and 1,6-glucans) because they lack either a 4- or 6-OH group in the glucose repeat unit.17 Therefore, the present chemical modification of polysaccharides is quite unique to 1,3-glucans like 1. This modification can provide a special advantage to maintain both the 4C1 conformation of the glucose repeat unit and the 2-OH groups that are considered essential
for the triple helix formation of 1.7,8,18 We thus hypothesize that the derivative 3 retains the helix-forming character similar to 1 (Figure 1c). If this is indeed the case, the flexible side chains emanate from the outer surface of the helices like “hairy rods” to hamper their lateral cohesion and mediate the solubility.13 The average molecular weights of 1 were determined as Mw = 635 kDa (n = 3920) and Mn = 292 kDa (n = 1800) by gel permeation chromatography (GPC) using pullulan standards and DMSO as eluent. This material was subjected to the acetalization with 4-(1,4,7,10-tetraoxaundecanyl)benzaldehyde (2) using 10-camphorsulfonic acid (CSA, racemic form) as catalyst, trimethyl orthoformate (TMOF) as dehydration agent,19,20 and anhydrous DMSO as solvent (Figure 1a). The reaction was carried out with stirring at 25 °C for 1 day. A control experiment suggested ca. 10% decrease of the average molecular weights during the reaction (Figure S1). The resultant solution was poured into diethyl ether to afford 3 as a white precipitate. A 2-O-acetylated analogue 4 and a lowmolecular-weight model compound 5 were prepared from 3 and methyl β-D-glucopyranoside, respectively, for use as references. The product was analyzed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy (Figure 2). DMSO-d6 was 22
DOI: 10.1021/acsmacrolett.6b00706 ACS Macro Lett. 2017, 6, 21−26
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ACS Macro Letters
Figure 2. 1H (a) and 13C NMR spectra (b) of 1, 3, 4, and 5 in DMSO-d6 at 80 °C. Residual solvents are marked with * and #. The signals from the glucose moieties of 1 and 5 and the acetyl group of 4 are specified.
used as solvent that dissolves 1 as random coils.21 In sharp contrast to 1, 4, and 5, both 1H and 13C signals of 3 suffered from significant broadening. The signals of the side chain were broader as they were closer to the main chain whose signals remained elusive even at elevated temperature up to 120 °C (Figure S2) but emerged only after the 2-O-acetylation (Figure 2).22 Despite excess use of 2 and TMOF for the acetalization (see SI), the degree of substitution (DS) was revealed as ca. 0.90 by direct hydrolysis of 3 in an NMR tube (Figure S3).23 The 13C NMR spectrum of 4 displays 12 side-chain and six main-chain signals of a single isomer in addition to two distinct signals of the acetyl group (Figures S4 and S5). Moreover, chemical shifts of C5, C6, and the acetal substituent of 4 agree well with those of 5 whose chemical structure was identified (Figure S6). These results indicate the selective 4,6acetalization as well as a stable helix formation of 3 in DMSOd6 where the 2-OH groups take a key role (see below).24 Helix formation was ascertained by circular dichroism (CD) spectroscopy and light scattering (LS) experiments as follows. Unlike 4 and 5, the spectrum of 3 displays a characteristic negative peak in the absorption region of the phenylene groups (Figure 3).25 The CD intensity slightly decreased by the temperature increase from 20 to 80 °C, while this change was reversible (Figure S7). In contrast, the negative peak vanished after the solution was heated with tetrabutylammounium hydroxide at 80 °C (Figure S8). This is somewhat akin to the known helix-to-coil transformation of 1 induced by the pH increase in aqueous media.26 The fact that 3 forms a stable helix in DMSO unlike 1 and 4 provides evidence that the 4,6-acetalization has even enhanced the helix-forming propensity of the polymer chain, and the 2-OH groups still play an integral role in the helix formation. This can be attributed to enlargement of the apolar faces of the repeat units that promotes their stacking in a polar environment to fold the polymer chain.27 The resultant compact helix could possibly shield the inner 2-OH groups from DMSO,
Figure 3. UV−vis absorption (a) and CD spectra of 3 (4.7 mM in repeat unit), 4 (7.6 mM in repeat unit), and 5 (6.4 mM) in DMSO at 25 °C (b). Black, dark gray, and light gray curves represent the spectra of 3, 4, and 5, respectively.
allowing for the formation of a hydrogen bonding network at the core.7,8,18 The hydrogen bonding stabilization is also suggested by the fact that 3 can form the helix even in an apolar solvent like chloroform (Figure S9). The results of LS experiments for a solution of 3 in DMSO (7.35 g/L, 25.0 °C) were plotted in the form of q2P(q) vs q (Kratky plot, Figure S10). Here, P(q) is the form factor and assumed to be proportional to the excess scattering intensity under the present conditions. The value of q2P(q) increased 23
DOI: 10.1021/acsmacrolett.6b00706 ACS Macro Lett. 2017, 6, 21−26
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ACS Macro Letters linearly with q (i.e., the magnitude of the scattering wave vector q) to reach a plateau at q = 2.6 × 107 m−1, which indicates a rod-like shape of 3 in solution. The persistence length of 3 was evaluated as 70 nm with the error range of 10% based on the Benoit−Doty equation for the Kratky−Porod model assuming 3 as a rigid rod (see SI).28−32 The persistence length of 70 nm is significantly higher than the reported value of a CUR random coil in DMSO (5.8 nm)21 and even surpasses a DNA double helix in water (50 nm).33 However, it does not reach the value of a schizophyllan triple helix in water (150 nm) that is homologous to the triple helix of 1.31,32 This result points to a possibility that the solution contains not only a triple helix but also less rigid helical species that pull the mean value down. Indeed, a careful inspection of the 1H NMR spectrum showed the presence of at least two different helical species in solution (Figure 2a, inset). The aromatic peaks at 6.5 and 6.9 ppm are assigned to the major species and those at 6.7 and 7.2 ppm to the minor. As the corresponding signals of references appear at 6.9 and 7.3 ppm, the aromatic peaks of the major species are significantly shifted upfield. This can be rationalized by the anisotropic effect caused by stacking of the phenylene groups. We conclude that the major species most likely represents the triple helix. The less shielded 1H signals are ascribed to less compact helices such as single- or doublestranded helices (see below).34,35 In contrast with 1, low-boiling-point solvents such as chloroform (containing 0.5 v/v % of triethylamine) could also dissolve 3. However, the solubility of 3 diminished after drying in a vacuum, while the solubility of 4 and 5 remained little changed. This is an indication of a cross-linking event that occurred specifically to 3 during the drying process.36 The dried material was nevertheless redissolved in DMSO by heating. This points to a noncovalent reversible nature of the cross-links. To elucidate the cross-linking mechanism, chloroform solutions with varied concentrations of 3 were spread on mica and analyzed by atomic force microscopy (AFM). Figure 4 represents a height image of the sample prepared from the solution of 7 mg/L. It shows the formation of an extensive 2D network composed of straight stems with junctions at the ends. It is noteworthy that the stems have a nearly uniform height of about 2.7 nm that agrees well with the crosssectional diameter of the triple helix.37 For reference, the AFM height of a CUR random coil is 0.65 nm.38 This indicates that the 2D network largely consists of the triple helices lying on the surface. The 2D confined geometry suggests that the network formed not in the solution but during adsorption and drying processes on mica. Note that the uniform height also applies to the junction points, which verifies the cross-links created by CCs and rules out other possibilities such as overlaying, supercoiling, or entanglements between the helices.39 The isotropic growth of the network can be accounted for by the bifurcation cascade during the polymerization as depicted in Scheme 1b.3 Another sample was prepared using a similar procedure, except that the substrate was spun at 3000 rpm while dropping the solution onto it. Mechanical stress was imposed purposely to disrupt network formation. Indeed, the AFM images show signatures of incomplete CCs between the bifurcated ends of the helices (Figure 5). Nevertheless, the overall connectivity of the network is more or less established, pointing to the high activity of 3 in CCs. AFM images from a higher dilution solution (0.7 mg/L) show discrete helices, indicating that the dilution suppressed the polymerization (Figure S11). However, the observation of
Figure 4. AFM analysis of a CCN of 3 on mica: (a) a height image and (b) height profiles along the blue lines indicated in the image.
toroidal features presents evidence that the ends of the helices still remain active in CCs and available for cyclization under the dilution condition.39,40 The helices have varied lengths as a result of the polydispersity of 3. The long helices appear to be persistent well over 100 nm, while the short ones look granular, which is likely due to the probe-broadening effect.41 The majority of the helices has a height of about 2.7 nm, but lower helices, which most likely correspond to the single- or doublestranded helices, also exist either as independent entities or as moieties of other rods or toroids. Note that neither such distinct rods nor network was observed in control experiments with 4 under comparable conditions; AFM shows only fuzzy features with the random coils (Figure S12). Furthermore, preliminary studies on film preparation have demonstrated that 3 can form flexible selfsupporting films with macroscopic dimensions, while 4 resulted in brittle solids under similar conditions (Figure S13). All these results are consistent with the strong cohelix-forming propensity of 3 that is responsible for the CCN formation. 24
DOI: 10.1021/acsmacrolett.6b00706 ACS Macro Lett. 2017, 6, 21−26
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Figure 5. AFM analysis of an unmatured CCN formed under the mechanical stress on mica: (a) a height image, (b) its magnification, and (c) height profiles along the blue lines indicated in (b).
Author Contributions
To conclude, helical polymers can be used as a macromolecular platform for creating a variety of functional nanomaterials. However, in respect to the creation of materials more generally, cohelix-forming polymers can offer even greater opportunity to make such discrete nanostructures grow into “real materials” that go well beyond the nanoscopic regime. Curdlan, a β-1,3-glucan polysaccharide, represents a biopolymer that has a strong cohelix-forming character. The present work has taken advantage of this character and demonstrated a new type of supramolecular polymerization resting on cohelical crossovers (CCs). This protocol has thus opened a continuous avenue that bridges the gap between nano- and macroscopic material worlds. Further studies are now in progress with a view to creating novel functional materials based on the cohelical crossover network (CCN).
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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.
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ACKNOWLEDGMENTS The authors thank Prof. Y. Miura and Mr. M. Nagao (Kyushu Univ., Japan) for access to the GPC apparatus in their laboratory and kind instruction. This work was supported partly by JSPS Grant-in-Aid for Scientific Research(B) JP26288102 and MEXT-Supported Program for the Strategic Research Foundation at Private Universities.
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ASSOCIATED CONTENT
S Supporting Information *
(1) Seeman, N. C. Nature 2003, 421, 427. (2) de Greef, T. F. A.; Meijer, E. W. Nature 2008, 453, 171. (3) Gunning, A. P.; Morris, V. Int. J. Biol. Macromol. 1990, 12, 338. (4) Gunning, A. P.; Kirby, A. R.; Ridout, M. J.; Brownsey, G. J.; Morris, V. J. Macromolecules 1996, 29, 6791. (5) Ikeda, S.; Morris, V. J.; Nishinari, K. Biomacromolecules 2001, 2, 1331. (6) Harada, T.; Masada, M.; Hidaka, H.; Takada, M. Hakko Kogaku Zasshi 1966, 44, 20. (7) Deslandes, Y.; Marchessault, R. H.; Sarko, A. Macromolecules 1980, 13, 1466. (8) Chuah, C. T.; Sarko, A.; Deslandes, Y.; Marchessault, R. H. Macromolecules 1983, 16, 1375.
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00706.
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REFERENCES
Experimental and Figures S1−S13 (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Junji Sakamoto: 0000-0003-2702-7425 25
DOI: 10.1021/acsmacrolett.6b00706 ACS Macro Lett. 2017, 6, 21−26
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(41) Morris, V. J.; Kirby, A. R.; Gunning, A. P. Atomic Force Microscopy for Biologists; Imperial College Press: London, U. K. 1999.
(9) Sakurai, K.; Uezu, K.; Numata, M.; Hasegawa, T.; Li, C.; Kaneko, K.; Shinkai, S. Chem. Commun. 2005, 41, 4383. (10) Numata, M.; Shinkai, S. Chem. Commun. 2011, 47, 1961. (11) Zhang, R.; Edgar, K. J. Biomacromolecules 2014, 15, 1079. (12) Ikeda, S.; Shishido, Y. J. Agric. Food Chem. 2005, 53, 786. (13) Wegner, G. Macromol. Chem. Phys. 2003, 204, 347. (14) Clode, D. M. Chem. Rev. 1979, 79, 491. (15) Amanokura, N.; Yoza, K.; Shinmori, H.; Shinkai, S.; Reinhoudt, D. N. J. Chem. Soc., Perkin Trans. 2 1998, 2, 2585. (16) Yoza, K.; Ono, Y.; Yoshihara, K.; Akao, T.; Shinmori, H.; Takeuchi, M.; Shinkai, S.; Reinhoudt, D. N. Chem. Commun. 1998, 8, 907. (17) For example, isopropylidene acetalization of dextran by using 2methoxypropene and pyridinium p-toluenesulfonate was previously reported: Bachelder, E. M.; Beaudette, T. T.; Broaders, K. E.; Dashe, J.; Fréchet, J. M. J. J. Am. Chem. Soc. 2008, 130, 10494. (18) Miyoshi, K.; Uezu, K.; Sakurai, K.; Shinkai, S. Chem. Biodiversity 2004, 1, 916. (19) Geng, Y.; Faidallah, H. M.; Albar, H. A.; Mhkalid, I. A.; Schmidt, R. R. Eur. J. Org. Chem. 2013, 2013, 7035. (20) Ono, F.; Hirata, O.; Ichimaru, K.; Saruhashi, K.; Watanabe, H.; Shinkai, S. Eur. J. Org. Chem. 2015, 2015, 6439. (21) Zhang, H.; Nishinari, K. Food Hydrocolloids 2009, 23, 1570. (22) The main-chain signals were still weak and broad even after the acetylation, and some of them were overlapped by large side-chain signals. This made assignment of the main-chain signals difficult for 1H NMR. For assignment of 13C NMR signals, see Figure S6. (23) The 4,6-acetal linkages were more susceptible to the hydrolysis than the glucosidic bonds. The small peak at 8.1 ppm in the spectrum of the hydrolysate mixture is likely due to methyl formate, a hydrolysate of an orthoester from a side reaction with TMOF (Figure S3a). For the side reaction, see ref 19. (24) Saito, H.; Ohki, T.; Sasaki, T. Biochemistry 1977, 16, 908. (25) Curdlan 1 having no chromophoric group in the structure is inactive in CD. For a CD study using a labeled Curdlan in DMSO, see: Fukuhara, G.; Inoue, Y. Chem. Commun. 2010, 46, 9128. (26) Ogawa, K.; Watanabe, T.; Tsurugi, J.; Ono, S. Carbohydr. Res. 1972, 23, 399. (27) Nelson, J. C.; Saven, J. G.; Moore, J. S.; Wolynes, P. G. Science 1997, 277, 1793. (28) Benoit, H.; Doty, P. J. Phys. Chem. 1953, 57, 958. (29) Kratky, O.; Porod, G. Recl. Trav. Chim. Pays-Bas. 1949, 68, 1106. (30) Yamakawa, H. Helical Wormlike Chains in Polymer Solutions; Springer: Berlin, 1997. (31) Kashiwagi, Y.; Norisuye, T.; Fujita, H. Macromolecules 1981, 14, 1220. (32) Sanada, Y.; Matsuzaki, T.; Mochizuki, S.; Okobira, T.; Uezu, K.; Sakurai, K. J. Phys. Chem. B 2012, 116, 87. (33) Baumann, C. G.; Smith, S. B.; Bloomfield, V. A.; Bustamante, C. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 6185. (34) Okuyama, K.; Otsubo, A.; Fukuzawa, Y.; Ozawa, M.; Harada, T.; Kasai, N. J. Carbohydr. Chem. 1991, 10, 645. (35) Zhang, L.; Wang, C.; Cui, S.; Wang, Z.; Zhang, X. Nano Lett. 2003, 3, 1119. (36) Restructuring of the acetal was unlikely during the drying process without acid catalysis. The dried material could redissolve in DMSO, which rules out the possibility of chemical cross-linking. (37) The hypothetical triple helix of 3 described in Figure 1c is supposed to have a cross-sectional diameter larger than that of the anhydrous form of the triple helix of 1 (2.3 nm) reported in ref 10. The increase of the diameter can be explained by the side groups introduced by the 4,6-acetalization. (38) Jin, Y.; Zhang, H.; Yin, Y.; Nishinari, K. Colloid Polym. Sci. 2006, 284, 1371. (39) Stokke, B. T.; Elgsaeter, A.; Brant, D. A.; Kitamura, S. Macromolecules 1991, 24, 6349. (40) Stokke, B. T.; Elgsaeter, A.; Brant, D. A.; Kuge, T.; Kitamura, S. Biopolymers 1993, 33, 193. 26
DOI: 10.1021/acsmacrolett.6b00706 ACS Macro Lett. 2017, 6, 21−26
