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Heat-induced conformation transition of the comb-branched ˇ-glucan in dimethyl sulfoxide/water mixture Shuqin Xu a,b , Xiaojuan Xu b , Min Xu c , Timothy R. O’Leary d , Lina Zhang b,∗ a

School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, China College of Chemistry & Molecule Sciences, Wuhan University, Wuhan 430072, China School of Physics and Materials Science & Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062, China d Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA b c

a r t i c l e

i n f o

Article history: Received 9 September 2016 Received in revised form 4 November 2016 Accepted 6 November 2016 Available online xxx Keywords: ˇ-Glucan Conformation transition Stiff chain Light scattering Differential scanning calorimeter Hydrogen bonds

a b s t r a c t We studied the chain conformation transition of the comb-branched ˇ-glucan (AF1) isolated from Auricularia auricula-judae by heating associated with dimethyl sulfoxide (DMSO). The results from 1 H NMR and differential scanning calorimeter (DSC) indicated that the reversible hydrogen bonds between side chains of AF1 and water clusters formed at relatively low temperatures. With increasing vDMSO to 0.70, the transition temperature (Tm ) increased from 9 to 71 ◦ C, and then decreased to 57 ◦ C with continuously increasing vDMSO due to the competition between DMSO and water for forming hydrogen bonds. Additionally, the combined analysis of 13 C NMR, viscosity and light scattering revealed an obvious stiff-toflexible chain conformation transition of AF1, which occurred at 95–130 ◦ C, 120–145 ◦ C and 130–160 ◦ C with vDMSO of 0.90, 0.85 and 0.70, respectively. This work demonstrated that AF1 has complex structure under different conditions, and the results obtained herein would benefit us to understand its specific behaviors including hollow fibril and anti-hepatoma activity. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction In recent years, ˇ-glucans have attracted increasing attention of researchers because of their structural diversity as well as versatile physico-chemical and biological properties. Particularly, studies about their static and dynamic behavior in solutions can provide intrinsic information for exploring new functions in food and pharmaceutical fields (Moreno et al., 2016; Brunchi, Morariu, & Bercea, 2014; Marieta, Ibarburu, Duen˜as, & Irastorza, 2009). As an essential constituent of organisms, polysaccharides possess an abundance of hydroxyl groups, which result in multiple hydrogen bonds (Hbonds) affecting their water-solubility, stiffness and aggregation (Li, Cui, Wang, & Yada, 2010). Previously, a neutral water-soluble polysaccharide coded as AF1 was isolated from Auricularia auriculajudae, a traditional edible mushroom in Asia. Its main-chain was determined to be a ˇ-(1 → 3)-d-glucan, which was substituted at O6 by side chains of single ˇ-(1 → 6)-d-glucosyl residues with 39.5% non-reducing terminal units ((Xu, Xu, & Zhang, 2012). It is worth noting that the AF1 has displayed significant anti-hepatoma activity against mouse H22 tumour without cytotoxicity on normal tissues,

∗ Corresponding author. E-mail address: [email protected] (L. Zhang).

depending on its molecular weight and dosage or concentration (Ping et al., 2016). AF1 has the same backbone as some well-known ˇ-glucans including schizophyllan (SPG) (Malik, Fujita, Numata, Ogura, & Shinkai, 2010), lentinan (Zhang, Zhang, & Xu, 2004) and curdlan (Jin, Zhang, Yin, & Nishinari, 2006). These glucans have ˇ-dGlcp (1 → 3)-linked backbone with different substitution patterns and thus different water-solubility, but they all adopt stiff chain conformation under certain conditions. For example, curdlan with no side chain and SPG with one (1 → 6)–glycosidic bonds for every three (1 → 3)-linked main chain sugar units form a triple helix in aqueous solution, which is stabilized by a triangular network of interchain H-bonds among the hydroxyl groups linked to the C-2 atoms of the glucose units of the backbone (Atkins & Parker, 1968; Malik et al., 2010). The triple helix provides a hydrophobic center cavity and a hydrophilic outer shell, which can be used as a watersoluble carrier for hydrophobic drugs (Takedatsu et al., 2012). The helical structure can be dissociated into single strand under specific condition including alkaline (Zhang et al., 2004), dimethyl sulfoxide (DMSO) (Wang, Zhang, Zhang, & Ding, 2009) or heating (Ikeda & Shishido, 2005). Also, differences in branching degree (BD) observed in ˇ-d-glucans imply the diversity of their bioactivity. Regarding to their immunomodulating capacity, ˇ-d-glucans with a (1 → 3)-linked backbone with ˇ-d-Glcp units (1 → 6)–linked as branches are able to produce inhibition of tumour growth (Ping

http://dx.doi.org/10.1016/j.carbpol.2016.11.020 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Xu, S., et al. Heat-induced conformation transition of the comb-branched ˇ-glucan in dimethyl sulfoxide/water mixture. Carbohydrate Polymers (2016), http://dx.doi.org/10.1016/j.carbpol.2016.11.020

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et al., 2016; Xu, Zou, Xu, & Zhang, 2016). Furthermore, those with moderate molecular weight (1 × 105 –2 × 105 ) seem to be most active (Hu, Jiang, Huang, & Sun, 2016; Zang, Li, Wang, Zhang, & Cheung, 2011). It is worth noting that the triple helical glucans can act as a host to interact with certain polynucleotide, in which the supramolecular helices consist of two glucan strands and one polynucleotide strand by H-bonding and hydrophobic interactions (Sanada et al., 2012). For example, a complex consisted of antisense macrophage-migration inhibitory factor (MIF) and two single SPG chains can be used as a novel polysaccharide-based delivery system to treat the inflammatory bowel disease (Takedatsu et al., 2012). Interestingly, the self-assembly of the supramolecular helices is based on the helix-coil transition. AF1 experienced the stiff-flexible transition, which is a common phenomenon among the helical glucans (Ikeda & Shishido, 2005; Wang et al., 2009). More interestingly, AF1 with even higher BD exhibited the unique self-assembly behavior including well defined nanofibers and hollow fibers. It was driven by multiple H-bonds, which has been investigated by the integrated methods in our previous work (Xu, Xu, & Zhang, 2013; Xu, Lin, et al., 2013). And the mechanism of natural mimic self-assembly has been proposed that the short branches of ˇ-glucan exhibit relative hydrophilicity, whereas the backbone shows relative hydrophobicity (Xu, Xu et al., 2013; Xu, Lin, et al., 2013). The stiff-flexible transition of AF1 occurred by heating up to 140 ◦ C or adding 90% DMSO, clarified that the intramolecular H-bonds sustained the extremely extending chains even with molecular mass of ca. 2 × 106 . Meanwhile, by adding small amount of DMSO, the gentle decrease in chain size was resulted from the dissociation of fibril aggregates formed by the network of inter-H bonds (Xu, Ping et al., 2016). On the other hand, most polysaccharides have irregular homopolymeric structures, where the regularity is usually interrupted by the random intrusion of varied types of linkage and/or sugar residues (Ogawa, Takahashi, Yajima, & Nishinari, 2005). However, cellulose and amylose are unbranched linear glucans with a ˇ-(1 → 4)-linked backbone and ␣-(1 → 4)-linked backbone, respectively. They can form crystal structures which are composed of highly ordered helical chains (Hsien-Chih & Sarko, 1978; Viswanathan, & Shenouda, 1971). Like the above mentioned ˇ-(1 → 3)-d-glucans including SPG, lentinan and curdlan, polysaccharides with regular chemical structures are able to assume an ordered or quasi-ordered structure such as a helix. The 6-OH group of side glucoses along the glucan backbone can interact with water molecules to form an associating water layer via inter-hydrogen bonding, which results in the water-solubility (Atkins & Parker, 1968; Okobira, Miyoshi, Uezu, Sakurai, & Shinkai, 2008). This associating structure can be disrupted with relatively lower energy by slightly heating, leading to the improved rotation of side chains in glucan (Kitamura & Kuge, 1989; Wang et al., 2009). The researchers have reported that a series of biosynthesized amylose with various branching length were used to study the side-chain-dependent helical conformation of polymers, which was stiffened by intramolecular hydrogen bonds (Terao et al., 2012). The stiff triple helices can be dissociated into single random coils by heating to 135 ◦ C, and subsequently renature as the linear and circular helical components in the samples (McIntire & Brant, 1998). A “phase diagram” for SPG in DMSO/water solution at different temperature was mapped by using scanning calorimetry, indicating a reversible transition at low temperature (Kitamura & Ozasa, 1990). A lot of studies were concentrated on the triple helical glucans (Antonov & Wolf, 2005; Kim, Takemasa, & Nishinari, 2006; Miyoshi, Takaya, & Nishinari, 1994), while the single comblike glucan was scarcely studied. Here, we attempted to study the conformational transition of AF1 by modulating the temperature or DMSO contents. The combined methods including NMR, DSC, static and dynamic light scattering (SLS/DLS) and viscometry were

used to clarify the multiple conformational transitions including a reversible transition at low temperature and an irreversible transition at high temperature. 2. Methods 2.1. Sample preparation Dried fruit bodies of Auricularia auricula-judae from Fangxian county (Hubei, China) were purchased from a market in China. The polysaccharide AF1 was extracted according to our previous work (Xu et al., 2012). Briefly, the defatted crushed fungi were immersed in 70% ethanol at room temperature for 24 h. The residues were dipped into 0.15 M NaCl solution at 80–100 ◦ C for 2 h and stirred overnight at room temperature, which was repeated three times. After centrifugation at 8000 rpm for 30 min, the collected supernatant was precipitated with 70% ethanol overnight. The precipitates were dissolved in water and deproteinized with Sevag reagent (chloroform/butanol, v/v = 5:1) for several times. The resulting solution was followed by dialyzing and lyophilizing to give ∼4 g crude polysaccharides. Further purification by ethanol precipitation provided a neutral fraction (∼300 mg) with high molecular weight, which was a white cotton-like polysaccharide and coded as AF1. The samples in this work were prepared by dissolving AF1 in DMSO/water mixtures with desired DMSO volume ratio (vDMSO ) for 24 h to give fresh neutral solutions. For light scattering and viscosity tests, the solutions were diluted by using solvents with corresponding vDMSO, which were purified with 0.45 m filters (NYL, 13 mm syringe filter, Whatman, Inc., USA). 2.2. Nuclear magnetic resonance spectroscopy (NMR) AF1 was dissolved in DMSO-d6 /D2 O mixtures with vDMSO values of 0.70, 0.85, 0.90, 0.95 and 1.00, respectively, at 60 ◦ C for over two days to obtain 0.04 g/mL concentrated solutions. 13 C NMR spectra of these samples were recorded with Bruker DRX (500 MHz) spectrometer (Bruker Co., Germany–Switzerland) at 25 ◦ C with tetramethyl silicon (TMS) as the internal standard. In order to improve the signal, the consistent collected times for each sample were over 10 h. 2.3. Three-dimensional (3D) modeling The 3D structure of polysaccharide AF1 with three repeated units was generated by GLYCAM-Web server (GLYCAM WebServer, 2015; Complex Carbohydrate Research Center, 2015), which is a widely and extensively used resource. The simulating parameters like the optimal bond lengths and bond angles were designed based on the GLYCAM force field (Yuriev & Ramsland, 2015). The result is the lowest energy structure after starting the minimization with the branch glucose monosaccharides with various combinations of the omega torsion angle. Then we used PyMOL software to display it. 2.4. Ultrasensitive differential scanning calorimeter (US-DSC) US-DSC measurements were performed on a VP-DSC microcalorimeter (VP-DSC, Microcal Inc., U.S.A.) with desired solvents as the references. The concentrations of AF1 in DMSO/water mixtures (vDMSO = 0.00–0.90) were ∼0.004 g/mL. The samples were degassed for 30 min at room temperature before use, which were heated from 5 to 110 ◦ C at a heating rate of 0.5 ◦ C/min. Data analysis was done by using Microcal software. The temperature located at the maximum excess specific heat capacity during the transition was determined to be transition temperature (Tm ). The “real” calorimetric enthalpy change (Hcal ) was the integral of

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the peak area (Kim et al., 2006; Miyoshi et al., 1994; Tiktopulo, Bychkova, Ricka, & Ptitsyn, 1994). The transition can be expressed as two states (such as the stiff or flexible states) in character, which obeys the van’t Hoff equation,

∂ ln K HvH ≡ RT 2 ∂T

(1)

where K = /(1 − ) is the equilibrium constant for the transition,  is the molar number of cooperative units in one of the two states, and HvH is the effective enthalpy that represents the heat absorbed due to conversion of 1 mol of cooperative units. If T = Tm and  = 1/2, the above equation transforms into: HVH = 4RT 2

Cp (Tm ) Qtr

(2)

Here, Cp (Tm ) is the maximal Cp of macromolecules, which is corresponded to the height of the endothermic peak in the DSC curve. Qtr is the total absorbed heat in the transition reaction for 1 g of polymer, which can be calculated by planimeter integration of the DSC trace. On the other hand, the “real” calorimetric enthalpy (Hcal ) associated with the conversion of 1 mol of the whole polymer molecule can be calculated by Hcal = M • Qtr

(3)

Where M is the molecular weight. 2.5. Viscometry The viscosities of the AF1 solutions with varied wDMSO volume ratios were measured at 25 ◦ C by using Ubbelohde capillary viscometers. All solutions for the viscosity tests under heating had the same concentration of ∼5 × 10−4 g/mL. At temperatures higher than 80 ◦ C, the AF1 solutions were heated at the desired temperature for 30 min. Later, the samples were quenched in the ice bath immediately, and kept at room temperature for 2 h, which were then measured at 25 ◦ C to give an inherent viscosity value of lnhr /c (Ohshima, Yamagata, Sato, & Teramoto, 1999). 2.6. Laser Light Scattering (SEC-LLS/DLS) The laser light scattering study was performed as our previous work (Xu, Zou, Xu, Zhang, 2016; Xu, Ping, Xu, Zhang, 2016; Xu, Moon et al., 2016). In briefly, the Mw values of AF1 were determined by using size exclusion chromatography combined with static laser light scattering (SEC-LLS) equipped with a He-Ne laser at  = 633 nm (DAWN® DSP, Wyatt Technology Co., USA) and a differential refractive index detector (DAWN DSP, Wyatt Technology). A Shodex-OHpak SB–806 M HQ (8.0 mm × 300 mm) column was used with 0.9% NaCl elution solution. The specific refractive index increment (dn/dc) of AF1 was 0.136 mL/g in water at 633 nm and 25 ◦ C (Xu et al., 2012). The LLS signal at scattering angle  = 90◦ and concentration signal of sample were collected and analyzed by using the Astra software. Dynamic Light Scattering (DLS) tests were carried out on a modified commercial light scattering spectrometer (ALV/SP-125, ALV, Germany) equipped with an ALV-5000/E multi- digital time correlator and a He-Ne laser (at  = 632.8 nm). The hydrodynamic radius distribution f(Rh ) of AF1 in solutions at 25 ◦ C was calculated directly from the CONTIN Laplace inversion algorithm (Berne & Pecora, 1976; Chu, 1991). The AF1 samples for measuring Mw and Rh were prepared by heating AF1 solutions with different vDMSO at the corresponding highest temperature, which were then dialyzed against pure water for enough long time at room temperature. All samples were purified by 0.22 m membranes before use.

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3. Results and discussion 3.1. Conformational transition at low temperature 3.1.1. NMR analysis As previously reported, AF1 dissolved directly in DMSO/water mixed solution displayed a stiff-to-flexible chain conformational transition by increasing DMSO volume ratio (vDMSO ) from 0.80 to 1.00 at room temperature (Xu, Zou et al., 2016; Xu, Pinget al., 2016; Xu, Moon et al., 2016). And this remarkable transition was mainly ascribed to the complete destruction of inter- and intra-molecular association among AF1 chains. Then how the molecular interactions contribute to the conformational change? We attempted to monitor the changes in the binding nature of AF1 in mixed solutions during transition by using NMR. It was difficult to collect carbon atom signals for AF1 with relatively low DMSO content (vDMSO ), which was caused by gel-like consistency with such high molar mass (Mw > 1.0 × 106 ) at room temperature (Xu et al., 2012). As shown in Fig. 1, 1 H NMR spectra for AF1 in mixed solutions were collected at different temperatures to determine the optimal scanning condition. For vDMSO = 1.00–0.90, well-resolved peaks were obtained at room temperature as shown in Fig. 1a. Taken AF1 in DMSO for an example, the peak at 4.2 ppm was ascribed to the anomeric proton H1, while the other well separated peaks located in the region of 3.0–3.7 ppm were attributed to H2 to H6 of the saccharide unit (Xu et al., 2012). The whole 1 H signals shifted slightly to higher field due to no correction with TMS, which hardly affected our analysis (Storseth, Hansen, & Skjerrrno, 2004). It suggested that AF1 exhibited relatively flexible chains in this vDMSO region resulting in more freedom in rotational motion. However, for vDMSO = 0.85 and 0.70, few hydrogen atom signals for the glucan were collected at 25 ◦ C, as well as the anomeric region overlapping with the broad HDO peak. It indicated that the highly motional restrictions occurred in all the saccharide components of AF1 not only for the backbone but also for the side chains of glucans (Kim et al., 2006) due to its gelation. By stepping up the temperature, the proton signals for AF1 in Fig. 1b and c appeared gradually, and most of them agreed well with the chemical shifts in those of AF1 in pure DMSO. The peaks at 4.1 ppm and 3.0–3.5 ppm for vDMSO = 0.85 at 65 ◦ C were attributed to H1 and H2–H5, respectively. Clearly, it had a good resolution, suggesting the optimal temperature for 13 C NMR scanning. However, the 1 H signals for vDMSO = 0.70 did not appear until 85 ◦ C, and enhanced slightly at 100 ◦ C, which was the maximum limitation for collecting the carbon atom signal. Based on the above analysis, the improved proton signals for AF1 was resulted from the stiff-toflexible conformation transition induced by increasing vDMSO from 0.70 to 1.00, which led to the enhanced mobility of sugar units (Storseth et al., 2004). And heating can dissociate the strong synergistic interaction of AF1 mixtures with micro-gel and high viscosity (Takemasa & Nishinari, 2016). Interestingly, the strong HDO peak at 3.3–3.5 ppm in DMSO sample showed obvious dependence on vDMSO as well as temperature. It shifted to higher field by increasing DMSO content or heating, indicative of the dissociation of hydrogen bonding between HDO and glucans (Benesi, Grutzeck, O’Hare, & Phair, 2005). This result clarified the equivalent effect between DMSO and temperature on synergistic association in AF1 solutions (Ouyang et al., 2008). As reported, gel contains different states of water such as a large amount of “bulk” water and a small amount of “associated” water (Katayama & Fujiwara, 1979). The former is a free state, and the latter is partially restricted in motion compared to the former (Kim et al., 2006). In AF1 solutions, there were multiple hydrogen bonds including inter- and intra-molecular hydrogen bonds among glucan chains, as well as the possible “associated water layer” formed between water and sugar units in side chains.

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Fig. 1. 1 H NMR spectra for AF1 in DMSO/water mixtures at different temperatures. The samples in DMSO/water mixtures with vDMSO of 0.90, 0.95 and 1.00 were measured at 25 ◦ C from top to bottom (a). The temperatures were 25, 45, 55 and 65 ◦ C for vDMSO = 0.85 (b) and 25, 65, 85 and 100 ◦ C for vDMSO = 0.70 (c) from top to bottom. The peak at 2.6 ppm was attributed to the DMSO-d6 . The temperature-dependent broad peak at 3.5 4.2 ppm was attributed to HDO.

More evidence is required to clarify whether the H-bonds disrupted by moderate heating was related to the “associated” water. Fig. 2 shows the 13 C NMR spectra for AF1 in water/DMSO mixtures with vDMSO ranging from 0.70 to 1.00. The concentrations of all samples were ∼0.04 g/mL, giving a highly viscous solution. All carbon atom signals for AF1 in mixtures with vDMSO = 0.90, 0.95 and 1.00 showed good resolution at 25 ◦ C, suggesting the individual molecules were present in the solution. However, the resolution ratio of carbon atom decreased for sample in vDMSO = 0.85 at 55 ◦ C, and the signals were hardly observed for vDMSO of 0.70 even heating to 100 ◦ C. These results agreed well with the stiff-to-flexible conformation transition range determined by viscosity and light scattering (Xu, Zou et al., 2016; Xu, Pinget al., 2016; Xu, Moon et al., 2016). Again it suggested that the mobility of glucan chains decreased with increasing water content, which was caused by the stiff backbone and aggregation formation. A typical 13 C NMR spectrum for branched glucan is shown in Fig. 2a. The single peak for anomeric carbon C1 at 103 ppm indicated a pure fraction, and all peaks were definitely ascribed to the six carbon atoms as indicated in Fig. 2a. And the signal of uronic acid at ∼176 ppm was not observed and thus not shown here, suggesting AF1 was a neutral polysaccharide. The NMR results proved that AF1 consisted of

ˇ-(1 → 3)-d-glucan backbone with two single ˇ-(1 → 6)-d-glucosyl residues every three residues (Xu et al., 2012). Despite increasing reports on co-crystal structures of proteincarbohydrate complexes, it’s still hard to obtain the crystals of carbohydrates for its highly heterogeneous structure and polydispersity (Xu, Zou et al., 2016; Xu, Pinget al., 2016; Xu, Moon et al., 2016). In recent years, conformational analysis by modeling methods has become an important approach in determining reasonable 3D structures of complicated carbohydrates. Fig. 3 displays the molecular modeling of AF1 with three repeating units by using GLYCAM builder (GLYCAM Web-Server, 2015). The simulation started the minimization with them rotated at 60◦ , −60◦ , and 180◦ . This is called the ’ω’ torsion because it involves the glycosidic bond to the 6 position of another sugar: O1 -C 6 -C 5 -H 5 . However, it was only the rotation of the glucose branches that was varied. The rotation of the backbone was based on the default ϕ/ values of a ˇ(1 → 3)-d-glycosidic bond between two glucose residues (Complex Carbohydrate Research Center, 2015; Fu, Chipot, Shao, & Cai, 2015). Then, the simulation result with lowest energy was chosen for the most stable conformation, giving an extended chain with helical tendency. It was coherent with that AF1 exhibited similar chemical structure and conformational transition with those helical glucans.

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Fig. 4. Temperature dependence of specific heat capacity (Cp ) for AF1 in DMSO/water mixtures detected by US-DSC. All samples have the concentrations of ∼0.004 g/mL, in which the solvent composition is indicated in the figure.

Fig. 2. 13 C NMR spectra of glucan AF1 in DMSO/water mixtures with different vDMSO values of 1.00 at 25 ◦ C (a), 0.95 at 25 ◦ C (b), 0.90 at 25 ◦ C (c), 0.85 at 55 ◦ C (d) and 0.70 at 100 ◦ C (e). All samples have the concentrations of 0.04 g/mL. The carbon signals are marked in a, and m, d and t represent the 1,3-linked glucose, 1,3,6-linked glucose, and terminal glucose, respectively.

Fig. 3. Molecular model of polysaccharide AF1 with three repeating units which consisted of every three ˇ-(1 → 3)-d-glucosyl residues in the backbone carrying two single ˇ-(1 → 6)-d-glucosyl residues. The color labeling is the following: O (red), C (green), H (grey). Stereo diagram was generated by GLYCAM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

We analyzed the NMR spectra by taking the strength of C1 peak as standard. Clearly, almost all other peaks decreased with decreasing vDMSO , and finally disappeared when vDMSO was 0.70. This suggested that the AF1 chains became stiffer and tended to form a micro-gel in solutions via inter- and intra-molecular hydrogen bonds, leading to less motion of glucan chains (Katayama & Fujiwara, 1979; Storseth et al., 2004). This change was different from NMR analysis of the triple helical lentinan, in which the C3 signal at 86 ppm decreased significantly and disappeared in 0.30 DMSO solution, while major signals distributed in 70–80 ppm became stronger (Zhang, Li, Zhou, Zhang, & Chen, 2002). It was noted that the relative intensities of C2 t (74.4 ppm) and C4 t (70.7 ppm) in the side glucose residue decreased with an increase of water content, suggesting restriction of the side chains. Moreover, the intensity of C4 t decreased faster than that of C2t, which might be ascribed to much more H-bonds coming from the C4 site of side chains (Saito, Yoshioka, Yokoi, & Yamada, 1990). From the

conformational modeling of Fig. 3, the hydroxyl groups at C2t and C4t protruded outside of the side chains which could provide more chance for forming H-bonds with other sugar units or water clusters. The side-side hydrogen bonds changed the side chains from disorder to order and formed aggregates. The side-water hydrogen bonds might result in an associating water layer, which was discussed in the following work. 3.1.2. DSC analysis Fig. 4 shows the temperature dependence of the specific heat capacity (Cp ) for AF1 as detected by DSC in DMSO/water mixtures at a range of vDMSO (0.00–0.90). Obviously, there was an endothermic peak that appeared in all of the DSC curves from water up to mixture with vDMSO of 0.90. However, the temperature ranges of peaks were much lower than 140–160 ◦ C corresponding to the stiffto-flexible transition for AF1 in water (Xu, Xu et al., 2013; Xu, Lin, et al., 2013). It suggested a distinct conformational transition in AF1 chains at temperature lower than 110 ◦ C. It has been reported that triple helical glucans such as SPG and lentinan experienced a similar phenomenon in DSC in this temperature range (Wang et al., 2009). The transition was ascribed to dissociation of the hydrogen bonding between the side glucose of polysaccharide and water cluster, resulting in a highly immobile backbone and stiffer chains. The weak associated interaction with water could be broken by heating, leading to the free rotation of side chains (Jin et al., 2006). For vDMSO < 0.70, the transition temperature in the low temperature range named as Tm for AF1 was increased from 9.3 to 70.6 ◦ C, and the peak intensity was increased. However, for vDMSO > 0.70, Tm decreased gradually to 56.6 ◦ C at vDMSO = 0.90, and the peak intensity was decreased. The viscosity of AF1 solutions hardly changed with temperature in the low temperature range, suggesting that the gross morphology and size of the polysaccharide chains remained stable, and the indistinctive change could not be detected by viscometry (Xu et al., 2012). The results of 1 H NMR spectra, for vDMSO at 0.85 and 0.70 were correlated with the signals appearing at 55 and 85 ◦ C in the transition temperature measured by DSC. This result suggested that the transition was caused by the same interaction broken, which was a water association layer formed between backbone side glucoses and water clusters (Moreno et al., 2016; Xu, Zou et al., 2016; Xu, Pinget al., 2016; Xu, Moon et al., 2016; Zhang et al., 2002). However, the second peak representing the heat-induced stiff-to-flexible conformation transition at high tem-

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Fig. 5. US-DSC curves for transitions of AF1 in a DMSO/water mixture at vDMSO = 0.85. The sample was heated up to 77 ◦ C (. . .. . .), and then reheated up to 110 ◦ C after being cooled at 5 ◦ C for 1 h. The second heating (----), the third heating (—) (a). Dependence of Tm for AF1 in DMSO/water mixtures on vDMSO based on the DSC curves in Fig. 4.

perature named as T2 did not appear in the curves. It was noted that there was a trend of a peak appearing at ∼100 ◦ C for vDMSO = 0.90, which was closely coincided with the transition temperature (T2 ) determined by the following viscosity tests. We can speculate that the peak of T2 at high temperature would occur upon continuous heating. This could be a limitation due to the maximum detection temperature of the instrument, where the stiff-to-flexible conformation transition temperature of AF1 might be higher than 110 ◦ C in the range of vDMSO < 0.90. Consequently, the endothermic peak was related with the side chains along the glucan backbone. In order to investigate the reversibility of the associating water layer, the repeated heating-cooling scanning was conducted on the AF1 solution at vDMSO = 0.85. As shown in Fig. 5a, the sample was first scanned by heating from 5 to 77 ◦ C to force the complete transition at low temperature. Then it was rescanned for twice to 110 ◦ C after naturally cooling to 5 ◦ C. Obviously, the three endothermic curves were almost identical with each other, indicating that the transition for AF1 at lower temperature was reversible (Xu, Zou et al., 2016; Xu, Pinget al., 2016; Xu, Moon et al., 2016). The result further confirmed that the associating water layer self-assembled by water clusters and side chains of AF1 needed relatively low energy, which was a dynamic process (Antonov & Wolf, 2005). It was noted that a very small peak occurred at higher temperature (∼87 ◦ C), which was also reversible during the cycling scanning. It might be contributed to water crystals in the system (Ito, Yoshida, Ujimoto, & Yamaguchi, 2013). Fig. 5b shows the dependence of Tm on vDMSO for AF1 solutions obtained from DSC curves. The temperature at which the maximal excess specific heat capacity appeared in the transition process is Tm . As vDMSO varied, the Tm increased for vDMSO < 0.70, whereas decreased for vDMSO >0.70, and gave a maximum value of Tm at vDMSO = 0.70. When vDMSO > 0.70, DMSO was likely to compete with water to bind the hydroxyl group of glucan (Kitamura & Kuge, 1989; Li et al., 2010; Wang et al., 2009). This polar solvent largely destroyed the hydrogen bonding between the side glucose residues and water molecules, leading to decreasing Tm with a decrease of water content. However, the associated network remained intact when the DMSO content was relatively low. Due to solvation, DMSO was unable to penetrate the associated structure of stiff chainwater interactions, resulting in a shift of Tm to higher temperature (Xu, Zou et al., 2016; Xu, Pinget al., 2016; Xu, Moon et al., 2016; Kitamura & Kuge, 1989) The thermodynamic parameters for AF1 in DMSO/water mixtures at low temperature are listed in Table 1. The results indicated that the energy required for the transition by interrupting the

−H bonds with the associating water layer was relatively low. Moreover, when vDMSO < 0.70, DMSO as a protector surrounded the associating water layer, leading to higher Tm . Thus, the energy to destroy the hydrogen bonds was also increased. When vDMSO > 0.70, DMSO largely broke the associated interaction, resulting in the decrease of Tm , corresponding to the decrease of energy required for destruction of the hydrogen bonds (Xu, Zou et al., 2016; Xu, Pinget al., 2016; Xu, Moon et al., 2016). The ratio of Hcal to HvH (Hcal /HvH ) was employed to discuss the cooperativity of AF1 during the transition. Here, an all-or-none transition is always considered in a strictly two-state process (Kitamura & Ozasa, 1990; Tiktopulo et al., 1994). The ratio conventionally represents the cooperative unit size, namely, when Hcal /HvH = 1, the cooperative unit encompasses the whole molecule. But in fact, the all-or-none transition do not apply to those macromolecules with high molecular weight as a result of energy transfer processes (Kitamura & Kuge, 1989; Miyoshi et al., 1994). As shown in Table 1, the ratio value represents the effective number of cooperative units, which gave its molecular weight of about (1.4–14) × 105 based on equation 2 and 3, corresponding to about 170–1975 repeating units (Wang et al., 2009). This indicated that the cooperativity of AF1 in the low temperature region was high, suggesting the completed destruction of the associated water layer. Moreover, the ratio values increased slowly with increasing DMSO content, which suggested a decrease in cooperative unit size. It could be contributed to the enhanced solvation of DMSO in the mixed solvent, which surrounded outside of the associated structure to produce smaller cooperative units. 3.2. Stiff-to-flexible chain conformation transition at high temperature Viscosity We have confirmed that AF1 experienced the stiff-to-flexible chain transition in DMSO/water solutions. Meanwhile, we have investigated that heating could break the hydrogen bonding which sustains the chain stiffness. In the above work, this remarkable transition was not observed in the whole vDMSO range under moderate heating. Then, how does DMSO coupled with heating affect the chain conformation, and also the hydrogen bonds? In order to give the answer, a series of AF1 mixtures with vDMSO in the range of 1.00–0.70 were prepared with same concentration (c = ∼ 5.0 × 10−4 g/mL), which made the (ln r )/c values comparable (Wang et al., 2009; Ohshima et al., 1999). Fig. 6a shows the dependence of (ln r )/c for AF1 in the DMSO mixtures on tem-

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Table 1 Calorimetric Parameters of the transition at low temperature for AF1 in DMSO/water mixtures.

vDMSO

c (mg/mL)

Tm1 (◦ C)

Qtr (J/g)

Hcal (kJ/mol)

HvH (kJ/mol)

DHcal /DHvH

0.00 0.10 0.30 0.50 0.70 0.80 0.85 0.90

4.74 3.71 4.29 3.85 4.03 3.87 3.88 4.19

9.28 20.21 41.91 60.92 70.59 68.90 65.79 56.62

0.51 0.71 2.37 4.62 4.34 3.99 4.33 2.44

1028.6 1424.2 4744.5 9242.3 8683.7 7984.5 8665.5 4874.8

819.7 636.7 2349.8 2507.1 1218.9 877.2 596.2 448.6

1.25 2.24 2.02 3.69 7.12 9.10 14.53 10.87

Fig. 6. Temperature dependence of lnhr /c for AF1 in DMSO/water mixtures with vDMSO = 0.70 (䊉), 0.85 (), 0.90 (), 0.95 ( ), 1.00 (䊏) at given concentrations of ca. 5 × 10−4 g/mL (a). The Mw values obtained from SEC-LLS at 25 ◦ C for the AF1 in different vDMSO mixtures (0.95, 0.90, 0.85 and 0.70) (䊏) used in the viscosity measurements by heating at the corresponding highest temperature, which were then dialyzed against pure water for enough long time. The Mw values of AF1 in pure water shown in solid circle (䊉) sourced from reference (Xu et al., 2012) (b).

perature. The viscosity of AF1 samples under temperature lower than 90 ◦ C hardly changed. They increased with decreasing vDMSO , and the viscosity of AF1 in 0.70 DMSO mixtures was closed to that in water, which was coincided with the results in Fig. 3. The (ln r )/c values for AF1 in DMSO and 0.95 DMSO mixtures were very small and decreased slightly at elevating temperature, which was caused by heating leading to more flexible chains with smaller size (Xu, Xu et al., 2013; Xu, Lin, et al., 2013; Zhang et al., 2002). For vDMSO ≤ 0.95, all of the (ln r )/c underwent a sharp decrease at different high temperatures, and the transition temperature in high temperature range (T2 ) increased with the decrease of vDMSO . The ultimate viscosity values were closed to that in DMSO. The transition in viscosities revealed that the substantial hydrogen bonds sustained the chain stiffness were broken in the case of high temperature associated with larger vDMSO , leading to a flexible chain conformation (Brunchi et al., 2014). The T2 for AF1 in DMSO mixtures with vDMSO of 0.90, 0.85 and 0.70 were drop in 95–130 ◦ C, 120–145 ◦ C and 130–160 ◦ C, respectively, while the T2 for AF1 in water was 140–160 ◦ C (Xu, Xu et al., 2013; Xu, Lin, et al., 2013; Ikeda & Shishido, 2005). It indicated that T2 shifted to higher temperature range, and was finally up to that of in water. The wider range of T2 for vDMSO = 0.90 could be ascribed to the multiple hydrogen bonds among the aggregates (Xu, Zou et al., 2016; Xu, Pinget al., 2016; Xu, Moon et al., 2016). The conformational transition occurred under high temperature was that the stiff chains transformed into flexible chains resulted from the complete interruption of inter- and intra-molecular hydrogen bonds. 3.2.1. Light scattering (SEC-LLS/DLS) Since the viscosity usually reflects the change in hydrodynamic volume of polymer. It is needed to clarify if obvious degradation in chain companied with the conformational transition in such

rigorous environment. Then, the final AF1 mixtures experienced the conformational transition in the viscosity tests were dialyzed against water to get aqueous solutions. As shown in Fig. 6b, the Mw values in solid square for samples (vDMSO = 0.95, 0.90, 0.85 and 0.70) determined by SEC-LLS were fluctuated in the range of (2.2–1.7) × 106 , which were coherent with the value in solid circle from AF1 in pure water as reported previously (Xu et al., 2012). The results indicated that no marked degradation happened in the heating process. DLS measurements also suggested a change in coil dimensions of AF1 by heating coupled with high DMSO contents. As shown in Fig. 7, the corresponding hydrodynamic radius distributions (Rh ) of AF1 samples showed single symmetric peaks at different scattering angles (), which suggested a good molecular dispersion (Li et al., 2010). Usually, for stiff linear chains, Rh increases with the decrease of . But for the random coils, Rh is independence of . Here, for AF1 in vDMSO = 0.70 preheated to 165 ◦ C, the Rh values increased slightly from 20 nm to 27 nm with the decrease of  from 90◦ to 50◦ , which was much smaller than the stiff AF1 chain size of ∼90 nm. This result was coherent with the sharp decrease in viscosity at around 140–160 ◦ C, suggesting that the stiff AF1 chains transformed into flexible chains since the totally broken of intermolecular hydrogen bonds. 4. Conclusion This work demonstrated that AF1 in aqueous solutions experienced multiple conformation transitions in DMSO/water mixture by heating. The peaks of DSC curves at low temperatures indicated a reversible transition, which was caused by the disruption of water associated layer composed of side chains along backbone and the water clusters. The existence of the associated interaction was ver-

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Fig. 7. The corresponding hydrodynamic radius distributions calculated from CONTIN analysis at different scattering angles ( = 90◦ , 70◦ and 50◦ ) for AF1 in DMSO/water solution at 25 ◦ C: vDMSO = 0.70, preheated to 165 ◦ C, c = 5 × 10−4 g/mL.

ified based on the increase of (Ikeda & Shishido, 2005)C NMR signal intensity for C2t and C4t as well as the improved proton signals by increasing DMSO content. For vDMSO < 0.7, transition temperature (Tm ) shifted to higher temperatures with increasing vDMSO . It was caused by the solvation effect of DMSO which protected the associated structure of AF1. While for vDMSO > 0.7, DMSO destroyed the hydrogen bonds, leading to the decreased Tm . On the other hand, the drop in viscosity and chain size at high temperatures proved that the stiff-to-flexible chain conformation transition would happen only under rigidly heating with a large amount of DMSO. The transition temperature (T2 ) decreased with the increase of vDMSO due to strong solation of DMSO. These results in this work were similar to those of helical ˇ-glucans, implying AF1 might adopt helical structure in water, which will be further studied in our near future work. Acknowlegement This work was supported by the major grant of the National Natural Science Foundation of China (21334005 and 21604033). References Antonov, Y. A., & Wolf, B. A. (2005). Calorimetric and structural investigation of the interaction between bovine serum albumin and high molecular weight dextran in water. Biomacromolecules, 6, 2980–2989. Atkins, E. D. T., & Parker, K. D. (1968). Cyclic triad of hydrogen bonds in a helical polymer. Nature, 220, 784–785. Benesi, A. J., Grutzeck, M. W., O’Hare, B., & Phair, J. W. (2005). Room-temperature icelike water in kanemite detected by 2 H NMR T1 relaxation. Langmuir, 21, 527–529. Berne, B., & Pecora, R. (1976). Dynamic light scattering. New York: Plenum Press. Brunchi, C.-E., Morariu, S., & Bercea, M. (2014). Intrinsic viscosity and conformational parameters of xanthan in aqueous solutions: Salt addition effect. Colloids and Surfaces B: Biointerfaces, 122, 512–519. Chu, B. (1991). Laser light scattering (2nd ed.). New York: Academic Press. Complex Carbohydrate Research Center. (2015). Available from. http://www.ccrc. uga.edu/research/index.php/ Fu, H., Chipot, C., Shao, X., & Cai, W. (2015). Why do the structural properties of complexes formed by glucans and carbon nanotubes differ so much? RSC Advances, 5, 95682–95689. GLYCAM Web-Server (2015). Available from: http://glycam.org/ Hsien-Chih, H. W., & Sarko, A. (1978). The double-helical molecular structure of crystalline a-amylose. Carbohydrate Research, 61, 27–40. Hu, T., Jiang, C., Huang, Q., & Sun, F. (2016). A comb-like branched ␤-d-glucan produced by a Cordyceps sinensis fungus and its protective effect against cyclophosphamide-induced immunosuppression in mice. Carbohydrate Polymers, 142, 259–267. Ikeda, S., & Shishido, Y. (2005). Atomic force microscopy studies on heat-induced gelation of curdlan. Journal of Agricultural and Food Chemistry, 53, 786–791.

Ito, K., Yoshida, K., Ujimoto, K., & Yamaguchi, T. (2013). Thermal behavior and structure of low-temperature water confined in sephadex G15 gel by differential scanning calorimetry and X-ray diffraction method. Analytical Sciences, 29, 353–359. Jin, Y., Zhang, H., Yin, Y., & Nishinari, K. (2006). Comparison of curdlan and its carboxymethylated derivative by means of rheology, DSC and AFM. Carbohydrate Research, 341, 90–99. Katayama, S., & Fujiwara, S. (1979). NMR study of the spatial effect of polyacrylamide gel upon the water molecules confined in it. Journal of the American Chemical Society, 101, 4485–4488. Kim, B.-S., Takemasa, M., & Nishinari, K. (2006). Synergistic interaction of xyloglucan and xanthan investigated by rheology, differential scanning calorimetry and NMR. Biomacromolecules, 7, 1223–1230. Kitamura, S., & Kuge, T. (1989). A differential scanning calorimetric study of the conformational transitions of schizophyllan in mixtures of water and dimethylsulfoxide. Biopolymers, 28, 639–654. Kitamura, S., & Ozasa, M. (1990). A differential scanning calorimetric study of the conformational transitions of several kinds of (1 → 6) branched (1 → 3)-␤-d-glucans in a mixture of water and dimethylsulfoxide. Thermochimica Acta, 163, 89–93. Li, W., Cui, S. W., Wang, Q., & Yada, R. Y. (2010). Studies of aggregation behaviors of cereal ␤-glucans in dilute aqueous solutions by light scattering: Part I. structure effects. Food Hydrocolloids, 25, 189–195. Malik, S., Fujita, N., Numata, M., Ogura, K., & Shinkai, S. (2010). Creation of supramolecular assemblies from a dipolar dye molecule by the template effect of ␤-1, 3-glucan polysaccharide. Journal of Material Chemistry, 20, 9022–9024. Marieta, C., Ibarburu, I., Duen˜as, M., & Irastorza, A. (2009). Supramolecular structure and conformation of a (1 → 3) (1 → 2)-␤-d-glucan from Lactobacillus suebicus CUPV221 as observed by tapping mode atomic force microscopy. Journal of the American Chemical Society, 57, 6183–6188. McIntire, T. M., & Brant, D. A. (1998). Observations of the (1 → 3)-␤-d-glucan linear triple helix to macrocycle interconversion using noncontact atomic force microscopy. Journal of the American Chemical Society, 120, 6909–6919. Miyoshi, E., Takaya, T., & Nishinari, K. (1994). Gel—sol transition in gellan gum solutions. II. DSC studies on the effects of salts. Food Hydrocolloids, 8, 529–542. Moreno, R. B., Ruthes, A. C., Baggio, C. H., Vilaplana, F., Komura, D. L., & Iacomini, M. (2016). Structure and antinociceptive effects of ␤-D-glucans from Cookeinatricholoma. Carbohydrate Polymers, 141, 220–228. Ogawa, E., Takahashi, R., Yajima, H., & Nishinari, K. (2005). Thermally induced coil-to-helix transition of sodium gellan gum with different molar masses in aqueous salt solutions. Biopolymers, 79, 207–217. Ohshima, A., Yamagata, A., Sato, T., & Teramoto, A. (1999). Entanglement effects in semiflexible polymer solutions: 3 Zero-shear viscosity and mutual diffusion coefficient of poly(n-hexylisocyanate) solution. Macromolecules, 32, 8645–8654. Okobira, T., Miyoshi, K., Uezu, K., Sakurai, K., & Shinkai, S. (2008). Molecular dynamics studies of side chain effect on the ␤-1, 3-d-glucan triple helix in aqueous solution. Biomacromolecules, 3, 783–788. Ouyang, J., Qiao, K., Zhang, W., Deng, Z., Li, L., & Lin, W. (2008). NMR structural elucidation of 3,4-seco-olean-11,13-dien-4,15␣,22␤,24-tetraol-3-oic acid with modulation of water peak. Acta Chemical Sinica, 66, 1327–1332. Ping, Z., Xu, H., Liu, T., Huang, J., Meng, Y., Xu, X., et al. (2016). Anti-heparoma activity of the stiff branched ␤-d-glucan and effects of molecular weight. Journal of Material Chemistry B, 4, 4565–4573. Saito, H., Yoshioka, Y., Yokoi, M., & Yamada, J. (1990). Distinct gelation mechanism between linear and branched (1 → 3)-␤-d-glucans as revealed by high-resolution solid-state 13 C NMR. Biopolymers, 29, 1689–1698. Sanada, Y., Matsuzaki, T., Mochizuki, S., Okobira, T., Uezu, K., & Sakurai, K. (2012). ␤-1, 3-d-Glucan schizophyllan/poly(dA) triple-helical complex in dilute solution. The Journal of Physical Chemistry B, 116, 87–94. Storseth, T. R., Hansen, K., & Skjerrrno, J. (2004). Characterization of a ␤-d-(1 → 3)-glucan from the marine diatom Chaetocerosmulleri by high-resolution magic angle spinning NMR spectroscopy on whole algal cells. Carbohydrate Research, 339, 421–424. Takedatsu, H., Mitsuyama, K., Mochizuki, S., Kobayashi, T., Sakurai, K., Takeda, H., et al. (2012). A new therapeutic approach using a schizophyllan-based drug delivery system for inflammatory bowel disease. Molecular Therapy, 20, 1234–1241. Takemasa, M., & Nishinari, K. (2016). Solution structure of molecular associations investigated using NMR for polysaccharides: Xanthan/galactomannan mixtures. The Journal of Physical Chemistry B, 120, 3027–3037. Terao, K., Maeda, F., Oyamada, K., Ochiai, T., Kitamura, S., & Sato, T. (2012). Side-chain-dependent helical conformation of amylose alkylcarbamates: Amylose tris (ethylcarbamate) and amylose tris (n-hexylcarbamate). The Journal of Physical Chemistry B, 116, 12714–12720. Tiktopulo, E. I., Bychkova, V. E., Ricka, J., & Ptitsyn, O. B. (1994). Cooperativity of the coil-globule transition in a homopolymer: Microcalorimetric study of poly(N-isopropylacrylamide). Macromolecules, 27, 2879–2882. Viswanathan, A., & Shenouda, S. G. (1971). The helical structure of cellulose I. Journal of Applied Polymer Science, 15, 519–535. Wang, X., Zhang, Y., Zhang, L., & Ding, Y. (2009). Multiple conformation transitions of triple helical lentinan in DMSO/water by microcalorimetry. The Journal of Physical Chemistry B, 113, 9915–9923. Xu, S., Xu, X., & Zhang, L. (2012). Branching structure and chain conformation of water-soluble glucan extracted from Auricularia auricula-judae. Journal of Agricultural and Food Chemistry, 60, 3498–3506.

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G Model CARP-11731; No. of Pages 9

ARTICLE IN PRESS S. Xu et al. / Carbohydrate Polymers xxx (2016) xxx–xxx

Xu, S., Lin, Y., Huang, J., Li, Z., Xu, X., & Zhang, L. (2013). Construction of high strength hollow fibers by self-assembly of a stiff polysaccharide with short branches in water. Journal of Matererial Chemistry A, 1, 4198–4206. Xu, S., Xu, X., & Zhang, L. (2013). Effect of heating on chain conformation of branched ␤-glucan in water. The Journal of Physical Chemistry B, 117, 8370–8377. Xu, H., Zou, S., Xu, X., & Zhang, L. (2016). Anti-tumor effect of ␤-glucan from Lentinus edodes and the underlying mechanism. Scientific Reports, 6(28802), 1–13. Xu, S., Ping, Z., Xu, X., & Zhang, L. (2016). Changes in shape and size of the stiff branched beta-glucan in dimethlysulfoxide/water solutions. Carbohydrate Polymers, 138, 86–93. Xu, Y., Moon, A. F., Xu, S., Krahn, J. M., Liu, J., & Pedersen, L. C. (2016). Structural based substrate specificity analysis of heparan sulfate 6-O-sulfotransferase. ACS Chemical Biology,. accepted.

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Yuriev, E., & Ramsland, P. A. (2015). Carbohydrates in cyberspace. Frontiers in immunology, 6(300). Zhang, L., Li, X., Zhou, Q., Zhang, X., & Chen, R. (2002). Transition from triple helix to coil of lentinan in solution measured by SEC, viscometry and 13 C NMR. Polymer Journal, 34, 443–449. Zhang, X., Zhang, L., & Xu, X. (2004). Morphologies and conformation transition of lentinan in aqueous NaOH solution. Biopolymers, 75, 187–195. Zhang, Y., Li, S., Wang, X., Zhang, L., & Cheung, P. C. K. (2011). Advances in lentinan: Isolation, structure: Chain conformation and bioactivities. Food Hydrocolloids, 25, 196–206.

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