Synthesis, Characterization, and Lectin Recognition of Hyperbranched

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Synthesis, Characterization, and Lectin Recognition of Hyperbranched Polysaccharide Obtained from 1,6-Anhydro-D-hexofuranose Nguyen To Hoai,† Akiyoshi Sasaki,§ Masahide Sasaki,§ Harumi Kaga,§ Toyoji Kakuchi,‡ and Toshifumi Satoh*,‡ †

Graduate School of Engineering and ‡Faculty of Engineering, Hokkaido University, Sapporo 060-8628, Japan § National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan

bS Supporting Information ABSTRACT: 1,6-Anhydro-D-hexofuranoses, such as 1,6-anhydroβ-D-glucofuranose (1), 1,6-anhydro-β-D-mannofuranose (2), and 1,6-anhydro-R-D-galactofuranose (3), were polymerized using a thermally induced cationic catalyst in dry propylene carbonate to afford hyperbranched polysaccharides (poly1
3) with degrees of branching from 0.40 to 0.46. The weight-average molecular weights of poly1
3 measured by multiangle laser light scattering varied in the range from (1.02 to 5.84)  104 g 3 mol
1, which were significantly higher than those measured by size exclusion chromatography. The intrinsic viscosities ([η]) of poly1
3 were very low in the range from 4.9 to 7.4 mL 3 g
1. The exponent (R) in the Mark
Houkwink
Sakurada equation ([η] = KMR) of the polymers was 0.20 to 0.33, which is 97.0%), sulfolane (99%), tetraethylene glycol dimethyl ether (TEGDME), Amberlyst 15(dry), and Dowex AG 50Wx2 were purchased from Sigma-Aldrich. Methyl-R,β-D-glucofuranose (MGF) and methyl-R-Dmannofuranose (MRMF) were prepared according to the reported procedures.31,32 The molecular sieve 4A was purchased from Merck (Art. 5708) and activated at 330 °C for 3 h in vacuo prior to its use.

Sulfolane was liquefied with 2% TEGDME for easy handling and used as ordinary sulfolane. Dry sulfolane was prepared by drying ordinary sulfolane over molecular sieve 4A for more than 7 days in a glovebox (mBRAUN, Ar atmosphere). Methanol, dimethylformamide (DMF), dimethyl sulfoxide, ethyl acetate (AcOEt), acetone, toluene, and chloroform were supplied from Kanto Chemical, and used as solvents. Propylene carbonate was purchased from Sigma-Aldrich and dried over CaO prior to fractional distillation under reduced pressure. Fluorescein isothiocyanate labeled Concanavalin A (FITC-ConA) and Concanavalin A (ConA) were purchased from Sigma-Aldrich and used without further purification. Lectin buffer (pH 7.5, 50 mM tris(hydroxymethyl)aminomethane (TRIS), 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2) was used to dissolve the HPs and FITC-ConA for lectin recognition. Poly4
6 were synthesized as reported in previous references.23,24 Measurements. The 1H NMR (400 MHz) and 13C NMR (100 MHz) were recorded using a JEOL JNM-A400II instrument. The inverse-gated decoupling 13C NMR spectra were obtained using a 15% (wt/vol) sample at 25 °C, 45° pulse angle, inverse-gated decoupling with a 7.0 s delay, and 4000 scans. The preparative size exclusion chromatography (SEC) was performed in H2O (10 mL 3 min
1) using a JAI LC-928 equipped with a JAI GEL-W253-40 column (linear, 40 mm 500 mm; exclusion limit, 5  104) and JAI RI-50 refractive index detector. The molecular weight values of poly1
3 were determined by SEC in an aqueous sodium nitrate solution (0.2 mol 3 L
1 NaNO3, 1.0 mL 3 min
1) at 25 °C using an Agilent 1100 series instrument equipped with two Tosoh TSKgel GMPWXL columns (linear, 7.5 mm 600 mm; exclusion limit, 5  107), a multiangle laser light scattering (MALLS) detector (Wyatt, DAWN 8), a viscosity detector (Wyatt, Viscostar), and a refractive index detector (Wyatt, Optilab rEX). The absolute molecular weights (Mw,MALLS), intrinsic viscosity ([η]), refractive index change (dn 3 dc
1), and Mark
Houwink
Sakurada 1892

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Biomacromolecules constants, R and K, were estimated by ASTRA 5.1.6.0 (Wyatt) software. The Mw,SEC values were determined using polyethylene oxide as a standard. The specific rotations were measured using a Jasco DIP-1000 digital polarimeter. The fluorescence (FL) spectra were measured in a 10 mm path length using a Jasco FP-6300 spectrofluorometer. Preparation of 1,6-Anhydro-β-D-glucofuranose (1). The preparation of 1,6-anhydro-β-D-glucofuranose (1) by microwave-assisted heating is described as follows. Ordinary sulfolane (5 mL) was added to 700 mg of MGF (R:β 46:54) in a 20 mL test tube. A K-type thermocouple through a capillary tube sealed with Bond SU adhesive (Konishi 04592, Konishi, Osaka, Japan) and a parallel side arm adapter (Claisen adapter) were placed in the center of the mixture. The test tube was placed in microwave equipment (Green Motif-I, IDX; single mode; output power 30
300 W) with an argon gas bubbler mounted on the side arm. The mixture was heated to a preset temperature of 240 °C with magnetic stirring for 3 min. The microwave-assisted heating of MGF was repeated 10 times at 240 °C for 3 min. The combined reaction mixtures were then directly purified by silica gel (SiO2) column chromatography, twice eluted with AcOEt/methanol (20/1) to give a faintly yellowish oil (2.25 g, 38.5%, Rf = 0.30), which was recrystallized from AcOEt/ chloroform to afford 1 (1.86 g, 31.8%). Mp 110
111 °C (ref 33, 111 to 112 °C). The 1H NMR and 13C NMR spectra of the obtained 1 were identical to those already reported.34 Preparation of 1,6-Anhydro-β-D-mannofuranose (2). The preparation of 1,6-anhydro-β-D-mannofuranose (2) using microwaveassisted heating was similar to that of 1. The microwave-assisted heating of methyl-R-D-mannofuranoside (MRMF, 100 mg) in dry sulfolane (5 mL) was repeated 10 times at 240 °C for 3 min. The combined reaction mixtures were then directly purified by SiO2 column chromatography eluted with AcOEt to give a colorless solid (450 mg, 53.9%, Rf = 0.21), which was crystallized from methanol/AcOEt to afford 2 (405 mg, 48.5%). Mp 185
188 °C (ref 35, 188
190 °C). The 1H NMR and 13 C NMR spectra of the obtained 2 were identical to those already reported.34 Preparation of 1,6-Anhydro-r-D-galactofuranose (3). The procedure for the preparation of 1,6-anhydro-R-D-galactofuranose (3) is described as follows. D-Galactose (15 g) in 300 mL of DMF was heated to 135 °C in the presence of amberlyst 15(dry) (15 g) for 1 h. The reaction mixture after removing DMF by evaporation was purified using a SiO2 column (eluted by AcOEt, then AcOEt/methanol: 20/1, and finally AcOEt/methanol: 10/1) to give a mixture of 3 and 6. The crude mixture was isolated by a reported procedure35 using the Dowex AG 50Wx2 (Caþþ) resin column to afford 3 (4.40 g, 32.6%). Mp 182
184 °C (ref 35, 185
188 °C). The 1H NMR and 13C NMR spectra of the obtained 3 were identical to those already reported.34

Cationic Ring-Opening Polymerization of 1,6-Anhydro-βD-glucofuranose (1). All procedures were performed under an argon

atmosphere. A typical procedure for the thermally induced cationic polymerization of 1 (entry 1 of Table 1 in the Suporting Information) is as follows: 7 in dry propylene carbonate (4.4 μL, 1.77  10
2 mmol) was added to a mixture of 1 (1.00 g, 6.2 mmol) in dry propylene carbonate (1.55 mL) at 150 °C using a microsyringe. After 20 min, the reaction mixture was quenched by large amount of methanol. After filtration, the residue was purified by reprecipitation in water and methanol to obtain a brownish white powder (poly1) in 60% yield. The brownish white color might be cause by trace amount of catalyst, which can be removed by preparative SEC, that remained in polymer. The Mw,MALLS and Mw,SEC values of poly1 were 3.47  104 (dn 3 dc
1 = 0.146) and 8.0  103 (Mw,SEC/Mn,SEC = 2.15), respectively. [R]D = þ79.3° (c 1.00, H2O, 25 °C). 1H NMR (400 MHz, D2O); δ 5.32
5.09 (H-1, m), 4.95 (R-H-1, br. s), 4.48 (β-H-1, br. s), 4.33
3.43 (H-2, H-3, H-4, H-5, H-6, m). 13C NMR (100 MHz, D2O); δ 109.03 (βC-1, glucofuranosyl unit), 105.55
97.99 (C-1, m) including the peaks at 103.47 (β-C-1, glucopyranosyl unit), 100.58 (R-C-1, glucopyranosyl

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unit), and 98.45 (R-C-1, (1f6)-linked glucopyranosyl unit), 80.39 (βC-2, glucofuranosyl unit), 78.49 (β-C-3, glucofuranosyl unit), 76.48 (βC-2, glucopyranosyl unit), 76.24 (β-C-3, glucopyranosyl unit), 73.71 (βC-4, glucopyranosyl unit), 73.50 (R-C-2, glucopyranosyl unit), 72.41 (R-C-3, glucopyranosyl unit), 72.09 (R-C-4, glucopyranosyl unit), 70.09 (R,β-C-5, glucopyranosyl unit), 66.07 (R-C-6, (1f6)-linked glucopyranosyl unit), 63.65 (β-C-6, glucofuranosyl terminal unit), 63.41 (R-C-6, glucofuranosyl terminal unit), 61.33 (β-C-6, glucopyranosyl terminal unit), and 61.08 (R-C-6, glucopyranosyl terminal unit).

Cationic Ring-Opening Polymerization of 1,6-Anhydro-β(2). All procedures were performed using the

D-mannofuranose

same steps for the cationic ring-opening polymerization of 1 (entry 3 of Table 1 in the Supporting Information). After reprecipitation, a brownish white powder (poly2) was obtained in 64% yield. The Mw,MALLS and Mw,SEC values of poly2 were 5.84  104 (dn 3 dc
1 = 0.153) and 3.12  104 (Mw,SEC/Mn,SEC = 8.47), respectively. [R]D = þ58.0° (c 1.00, H2O, 25 °C). 1H NMR (400 MHz, D2O); δ 5.69
5.06 (H-1, m), 5.01 (R-H1, br. s), 4.49 (β-H-1, br. s), 4.42
3.48 (H-2, H-3, H-4, H-5, H-6, m). 13 C NMR (100 MHz, D2O); δ 107.95 (R,β-C-1, mannofuranosyl unit), 105.55 (R,β-C-1, mannopyranosyl unit), 103.64
99.08 (C-1, m), 79.68 (R,β-C-4, mannofuranosyl unit), 76.84 (β-C-5, mannopyranosyl unit), 73.78 (R-C-3, mannopyranosyl unit), 73.32 (R-C-5, mannopyranosyl unit), 71.16 (β-C-3, mannopyranosyl unit), 70.87 (β-C-2, mannopyranosyl unit), 70.59 (R-C-2, mannopyranosyl unit), 67.35 (R,β-C-4, mannopyranosyl unit), 63.86 (R,β-C-6, mannofuranosyl terminal unit), and 61.55 (R,β-C-6, mannopyranosyl terminal unit).

Cationic Ring-Opening Polymerization of 1,6-Anhydro-r(3). All experiments were carried out using the

D-galactofuranose

same steps for the cationic ring-opening polymerization of 1 (entry 6 of Table 1 in the Supporting Information). The brownish white powder (poly3) was obtained in 55% yield after reprecipitation. The Mw,MALLS and Mw,SEC values of poly3 were 2.27  104 (dn 3 dc
1 = 0.136) and 6.6  103 (Mw,SEC/Mn,SEC = 2.06), respectively. [R]D = þ54.8° (c 1.00, H2O, 25 °C). 1H NMR (400 MHz, D2O); δ 5.48
4.89 (H-1, m), 4.43 (β-H-1, br. s), 4.37
3.46 (H-2, H-3, H-4, H-5, H-6, m). 13C NMR (100 MHz, D2O); δ 109.76 (β-C-1, galactofuranosyl unit), 108.44 (β-C-1, (1f6)-linked galactofuranosyl unit), 104.84 (β-C-1, galactopyranosyl unit), 103.92 (R-C-1, galactofuranosyl unit), 101.1 (R-C-1, galactopyranosyl unit), 99.18 (R-C-1, (1f6)-linked galactopyranosyl unit), 84.77
82.94 (R,β-C-4, galactofuranosyl unit), 82.37
80.97 (β-C-2, galactofuranosyl unit), 80.97
78.08 (R,β-C-3, galactofuranosyl unit), 78.08
76.25 (R-C-2, galactofuranosyl unit), 75.77 (β-C-5, galactopyranosyl unit), 73.25 (β-C-3, galactopyranosyl unit), 71.48 (β-C-2, galactopyranosyl unit and R-C-5, galactopyranosyl unit), 70.05 (R-C5, (1f6)-linked galactopyranosyl unit), 69.85 (R-C-3, (1f6)-linked galactopyranosyl unit and R-C-4, galactopyranosyl unit), 69.24 (β-C-4, galactopyranosyl unit and R-C-2, (1f6)-linked galactopyranosyl unit), 69.03 (R-C-3, galactopyranosyl unit and R-C-4, (1f6)-linked galactopyranosyl unit), 67.30 (R-C-6, (1f6)-linked galactopyranosyl unit), 63.87 (R-C-6, galactofuranosyl terminal unit), 63.35 (β-C-6, galactofuranosyl terminal unit), 61.77 (R-C-6, galactopyranosyl terminal unit), and 61.63 (β-C-6, galactopyranosyl terminal unit). Methylation Analysis. Methylation of the polysaccharide was carried out according to the method described by Tomoda et al.36 The methylated polysaccharides were converted to partially methylated D-glucitol acetates (PMGA), methylated D-mannitol acetates (PMMA), and methylated D-galactitol acetates (PMGaA), as described by Kenedy et al.37 The sample was analyzed by gas chromatography (GC) using a Shimadzu GC-17A chromatograph equipped with a BPX 70 capillary column (70% bis(cyanopropyl)poly(silylphenylenesiloxane), 30 m  0.25 mm, 0.25 μm film thickness, SGE) and a flame-ionization detector. The oven was heated to 190 °C as the initial temperature, then heated at the rate 1 °C 3 min
1 to the final temperature of 250 °C, and maintained for 10 min. The injection and detector temperatures were 260 °C. The 1893

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PMGA, PMMA, and PMGaA were identified by GC and mass spectrometry (GC-MS) analyses using a JEOL JMS-AX-500 equipped with a BPX capillary column and electron impact ionization at 70 eV (GC-MS & NMR Laboratory, Graduate School of Agriculture, Hokkaido University) and also by their retention times relative to myo-inositol hexaacetate, as described by Bacic et al.38 For the quantitative PMGA, PMMA, and PMGaA analyses using GC, the relative molar response factors were estimated from the theoretical calculations.39 The calculated response factors were 0.70 for di-O-acetyl-tetra-O-methyl-D-hexitol, 0.74 for the 1,2,5-, 1,3,5-, 1,4,5-, 1,2,4-, and 1,3,4-tri-O-acetyl-tri-Omethyl-D-hexitols, 0.75 for the 1,5,6- and 1,4,6-tri-O-acetyl-tri-O-methylD-hexitols, 0.79 for the 1,2,3,5-, 1,3,4,5-, and 1,2,4,5- tetra-O-acetyl-di-Omethyl-D-hexitols, 0.8 for the 1,2,5,6-, 1,3,5,6-, 1,4,5,6-, 1,2,4,6-, and 1,3,4,6-tetra-O-acetyl-di-O-methyl-D-hexitols, 0.84 for penta-O-acetylO-methyl-D-hexitol, and 0.89 for hexa-O-acetyl-D-hexitol. Degree of Branching. The DB of the polysaccharides prepared from the latent cyclic AB4-type monomers 1∼3 was calculated from the number of terminal units (T), linear units (L), semidendritic units (sD1 and sD2), and dendritic units (D) using Frey’s equation (eq 1).40 DB ¼

3D þ 2sD2 þ sD1 0:75ð4D þ 3sD2 þ 2sD1 þ LÞ

ð1Þ

The numbers of each unit were determined by the methylation analysis of the polysaccharide and the correction using the molar response factors.39 Steady Shear Rheometry. The poly1
3 solutions were prepared by mixing the dry polymers and deionized water for several hours. The concentration (weight percent) of the solutions used in this study was 40 wt %. The steady shear flows of the aqueous poly1
3 solutions were performed using a Brookfield cone and plate rotational viscometer (RVDV-IIþ Pro) at various temperatures. The cone diameter (CPE-40) was 24 mm, and the cone angle was 0.8°. Measurements were performed over a shear rate range from 2.25 to 1200 s
1. The testing temperatures were 10, 20, 25, and 30 °C, which were controlled using a constanttemperature bath. Fluorometric Assay of Lectin-Binding. Poly1 (entry 2), poly2 (entry 5), poly4 (run no. 1 in ref 24), and poly5 (run no. 3 in ref 23) were separated into their different molecular weights using preparative SEC performed in water, which were then used for the HPs-ConA conjugations; poly116200 (Mw,MALLS = 16 200 g 3 mol
1, Mw,MALLS/Mn,MALLS = 1.06, DB = 0.41), poly16400 (Mw,MALLS = 6400 g 3 mol
1, Mw,MALLS/Mn,MALLS = 1.01, DB = 0.41), poly13300 (Mw,MALLS = 3300 g 3 mol
1, Mw,MALLS/ Mn,MALLS = 1.04, DB = 0.41), poly217800 (Mw,MALLS = 17 800 g 3 mol
1, Mw,MALLS/Mn,MALLS = 1.08, DB = 0.42), poly27900 (Mw,MALLS = 7900 g 3 mol
1, Mw,MALLS/Mn,MALLS = 1.07, DB = 0.42), poly23900 (Mw,MALLS = 3900 g 3 mol
1, Mw,MALLS/Mn,MALLS = 1.10, DB = 0.42), poly46000 (Mw,MALLS = 6000 g 3 mol
1, Mw,MALLS/Mn,MALLS = 1.02, DB = 0.38), and poly517800 (Mw,MALLS = 17 800 g 3 mol
1, Mw,MALLS/Mn,MALLS = 1.08, DB = 0.43). The FL spectra were measured using a 10 mm quartz cell at 25 °C. A typical procedure is described as follows: aliquots (4 μL) of a stock solution (30 mM of terminal units for poly16400, poly13300, and poly46000; and 3 mM of terminal unit for poly116200, poly217800, poly27900, poly23900, and poly517800) in lectin buffer were added to a solution of FITC-ConA (2 mL, 1 μM). The FL measurements were excited at 495 nm, and the emission intensity at 518 nm was recorded. The binding constants were calculated based on the Scatchard equation as follows41 ½terminalF0 ½terminalF0 F0 ¼ þ ΔF ΔFmax ΔFmax Ka

ð2Þ

where [terminal], F0, and F are the concentrations of the terminal units of the HPs, the initial FL intensity of FITC-ConA, and the FL intensity, respectively.

Figure 1. Mark
Houwink
Sakurada plots of (a) poly1 (entry 1), (b) poly2 (entry 4), and (c) poly3 (entry 7) in 0.2 M NaNO3 aq.

’ RESULTS AND DISCUSSION Polymerization. The ring-opening polymerization of 1,6anhydro-β-D-glucofuranose (1), 1,6-anhydro-β-D-mannofuranose (2), and 1,6-anhydro-R-D-galactofuranose (3) was carried out using (S-2-butenyl)tetramethylenesulfonium hexafluoroantimonate (7) as a thermally induced cationic initiator, which is suitable for the polymerization above 120 °C, as shown in Scheme 1. The typical results were summarized in Table 1 in the Supporting Information. When propylene carbonate was used as a aprotic polar solvent with a high boiling point, the polymerizations of 1 and 2 heterogeneously proceeded, whereas the polymerization of 3 was similar to the polymerization of 1,6anhydro-β-D-glucopyranose (4), 1,6-anhydro-β-D-mannopyranose (5), and 1,6-anhydro-β-D-galactopyranose (6) reported in the literature,23,24 which homogeneously proceeded for the first 5 min; then, the reaction system became heterogeneous. The resulting polysaccharides, which were isolated by reprecipitation using water and methanol, were gel-free brownish white solids with the yield of ca. 60% and were soluble in water and dimethyl sulfoxide, slightly soluble in dimethylformamide, and insoluble in toluene, chloroform, acetone, and methanol. The weight-average molecular weights (Mw,MALLS) of poly1
3 measured by SEC equipped with multiangle laser light scattering (MALLS) increased with the increasing temperature; for instance, the Mw,MALLS of poly1 was (1.88 and 3.47)  104 g 3 mol
1 when the polymerizations of 1 were carried out at 130 and 150 °C, respectively (entries 1 and 2 of Table 1 in Supporting Information). The increase in the monomer concentration also led to an increase in Mw,MALLS, which was similar to the polymerizations of 4∼6.23,24 For the polymerization of 1
3 under the same conditions, the Mw,MALLS and the yield of poly2 was the highest value obtained, whereas the lowest value was that of poly3, meaning that the order of polymerizability was 2 > 1 > 3. This order was found to be similar to the polymerizations of 4∼6, that is, 5 > 4 > 6.23,24 The Mw,MALLS of poly1
3 varied in the range from (1.02 to 5.84)  104 g 3 mol
1 corresponding to the average degrees of polymerization (DP) from ca. 63 to 360. 1894

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Biomacromolecules

Figure 2. Steady shear viscosity as a function of the shear rate for poly1 (entry 2) in aqueous solution (40 wt %).

Figure 3. 13C NMR spectra of (a) poly4 (Mw,SLS = 33 00 g 3 mol
1), which was prepared from 1,6-anhydro-β-D-glucopyranose (run no. 1 in ref 24), and (b) poly1 (entry 1); symbols O and b correspond to R-Dand β-D-glucopyranosyl terminal units, respectively (T-p-glu), 4 and 2 correspond to R-D- and β-D-glucofuranosyl terminal units, respectively (T-f-glu), and 0 corresponds to (1f6)-linked glucopyranosyl unit ((1
6)-p-glu).

The polydispersities (Mw,SEC/Mn,SEC) of poly1
3 were found to be in the range of 1.5
8.5 and broader than those of poly4
6. In general, hyperbranched polymers are known to have spherical conformations in solution, and the Mw,SEC values of these polymers are often claimed to be lower than their absolute molecular weights because the hydrodynamic volumes of these polymers are smaller than the corresponding linear polymers used for calibration. To validate this fact, the weight-average molecular weights (Mw,SEC) of poly1
3 measured by SEC were compared with the absolute Mw,MALLS values. On the basis of these obtained results, the Mw,MALLS values were 2.5 to 4.3 times higher than the Mw,SEC values for poly1, 1.9 to 2.9 times that of poly2, and 2.3 to 3.4 that of poly3, indicating that poly1
3 have more compact forms in solution when compared with the linear polymers. The hyperbranched structures of the polymers were also confirmed by the viscosity measurements using an SEC equipped with a viscosity detector. The intrinsic viscosities

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([η]) of poly1∼3 were very low in the range from 4.9 to 7.4 mL 3 g
1, which were similar to those of poly4
6.23,24 The exponents (R) of the Mark
Houwink
Sakurada equations ([η] = KMR) of the polymers were 0.27 to 0.29 for poly1, 0.27 to 0.33 for poly2, and 0.20 to 0.24 for poly3 (Figure 1), which are