Structural Characterization of Two Water-Soluble Polysaccharides

Article pubs.acs.org/JAFC

Structural Characterization of Two Water-Soluble Polysaccharides from Black Soybean (Glycine max (L.) Merr.) Jun Liu,* Xiao-yuan Wen, Juan Kan, and Chang-hai Jin* College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, Jiangsu, China ABSTRACT: Black soybeans (Glycine max (L.) Merr.) have been widely used as a health food and medicinal herb in oriental medicine. In the present study, the chemical structures of two water-soluble polysaccharides (black soybean polysaccharide 1 (BSPS-1) and black soybean polysaccharide 3 (BSPS-3)) isolated from black soybeans were characterized by high performance size-exclusion chromatography (HPSEC), methylation analysis, and 1D (1H, 13C) and 2D (COSY, TOCSY, HSQC, NOESY, and HMBC) NMR spectra. The molecular weights of BSPS-1 and BSPS-3 were 1.95 × 105 and 1.88 × 105 Da, respectively. Methylation analysis and NMR spectra indicate that BSPS-1 is composed of 1,6-α-D-glucopyranosyl residues. By contrast, BSPS-3 is mainly composed of a 1,3-β-D-galactopyranosyl residue backbone with side chains substituted at the O-6 position consisting of large content of T-α-L-Araf-(1→ residues, and small contents of →5)-α-L-Araf-(1→, →2)-α-L-Rhap-(1→, and 4-O-Me-β-DGlcAp-(1→ residues. Our results suggest that BSPS-1 is a linear (1→6)-α-D-glucan, whereas BSPS-3 is a type II arabinogalactan. The unique structures of BSPS-1 and BSPS-3 indicate that they might have wide applications in food and pharmaceutical industries. KEYWORDS: black soybean, NMR, structural characterization, polysaccharides

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INTRODUCTION Black soybeans (Glycine max (L.) Merr.) are soybeans with black seed coats, which have been widely used as a health food and medicinal herb in oriental medicine. The pigmentation on the black soybeans is due to the abundance of anthocyanins in the seed coat.1 Several studies suggest that black soybeans comprise various classes of bioactive compounds, including oil, isoflavones, anthocyanins, saponins, polysaccharides, and proteins.2−5 Among these compounds, anthocyanins have been demonstrated to have a wide range of beneficial health effects, such as antioxidant, anti-inflammatory, antiobesity, antidiabetic, and antimutagenicity properties.6−11 Although polysaccharides have been reported as one of the secondary metabolites of black soybeans, information on the structure characterization and biological activities of black soybean polysaccharides (BSPS) is still limited. Liao et al. revealed that (1,6)-α-D-glucan might be the major component of BSPS. The polysaccharide component could inhibit proliferation and induce differentiation in human leukemic U937 cells by activating the immune response of mononuclear cells.12 Moreover, BSPS could promote myelopoiesis activity in the bone marrow, stimulate production of various hematopoietic growth factors from spleen cells, and reconstitute bone marrow that has been myelosuppressed by irradiation and 5fluorouracil.13 Wu et al. reported that BSPS could stimulate granulocyte colony-stimulated factor production in peripheral blood mononuclear cells. 14 Recently, we reported the extraction, preliminary characterization, and in vitro antioxidant activity of BSPS.15 Two main water-soluble polysaccharide fractions (black soybean polysaccharide 1 (BSPS-1) and black soybean polysaccharide 3 (BSPS-3)) were obtained. These two fractions have been demonstrated to be composed of different monosaccharides and possess potential superoxide anion and DPPH radical scavenging activities. © XXXX American Chemical Society

In order to establish the structure−function relationships of BSPS and further facilitate its applications in food and pharmaceutical industries, the main objective of this study was to characterize the detailed structure features of BSPS-1 and BSPS-3 by high performance size-exclusion chromatography (HPSEC), methylation analysis, and nuclear magnetic resonance (NMR) spectra.

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MATERIALS AND METHODS

Materials and Reagents. Black soybean (G. max (L.) merr.) seeds were obtained from Heilongjiang Beizhen Food Co., Ltd. (Heilongjiang, China). Crude polysaccharides were isolated from black soybean flour and further purified on chromatography of DEAE-52 and Sepharose CL-4B.15 Two main purified fractions of BSPS-1 and BSPS-3 were used in this study. Deuterium oxide (D2O) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of analytical grade. Monosaccharide Composition Analysis. The monosaccharide compositions of BSPS-1 and BSPS-3 were determined as described previously.15 The trimethylsilyl derivatives of polysaccharide hydrolyzate were analyzed on a Trace GC Ultra gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) equipped with flame ionization detector (FID) and a CP-Sil5 capillary column (30 m × 0.25 mm × 0.25 μm). Nitrogen gas was used as the carrier gas. Temperature-programmed GC measurements were carried out from 100 °C (held for 5 min), to 150 °C (held for 5 min) at a rate of 5 °C/ min, and then to 240 °C (held for 2 min) at a rate of 5 °C/min. Determination of Purity and Molecular Weight. The purity and molecular weight (MW) of BSPS-1 and BSPS-3 were determined by HPSEC on an Agilent 1200 system (Agilent Technologies, Palo Alto, CA, USA) equipped with a TSK gel G4000 PWXL column (30 Received: October 27, 2014 Revised: December 13, 2014 Accepted: December 14, 2014

A

DOI: 10.1021/jf505172m J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry cm × 7.8 mm × 10 μm, Tosoh Corp., Tokyo, Japan) and an evaporative light scattering detector (ELSD). Sample solution (20 μL) was injected and eluted with distilled water at 50 °C with a flow rate of 0.7 mL/min. The molecular weights of polysaccharide samples were calculated from the calibration curve of T-series dextran standards (T500, T-200, T-100, T-50, and T-10). Methylation and GC−MS Analysis. For glycosyl linkage analysis, uronic acid residues of BSPS-1 and BSPS-3 were first reduced as described previously.16 Then, methylation of BSPS-1 and BSPS-3 was carried out according to the method of Ciucanu and Kerek.17 The complete methylation was confirmed by the lack of hydroxyl peak on a Varian 670 FT-IR spectrometer (Varian Inc., Palo Alto, CA, USA). The permethylated polysaccharide was then hydrolyzed with trifluoroacetic acid at 120 °C for 2 h, reduced with NaBH4, and acetylated with acetic anhydride. The resulting partially methylated alditol acetate derivatives were analyzed on a Thermo Trace DSQ II GC-MS (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a TR5MS capillary column (30 m × 0.25 mm × 0.25 μm). The initial column temperature was set at 100 °C, and programmed to 250 °C (held for 5 min) at 6 °C/min. Helium was used as the carrier gas at the rate of 1 mL/min. The partially methylated alditol acetates were identified by their retention times and electron ionization spectra. NMR Analysis. The fine chemical structures of BSPS-1 and BSPS-3 were analyzed by an AVANCE-600 NMR spectrometer (Bruker Inc., Rheinstetten, Germany). For NMR measurements, the dried sample (20 mg) was dissolved in 0.5 mL of D2O and kept at room temperature for 3 h. 1H (600 Hz) and 13C (150 Hz) NMR spectra were recorded at 25 °C. Then, the 2D NMR spectra including 1H/1H homonuclear correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple-bond coherence (HMBC), and nuclear Overhauser enhancement spectroscopy (NOESY) were recorded.

Figure 1. HPSEC elution profile of purified fractions BSPS-1 (a) and BSPS-3 (b).

curve of standards, the molecular weight of BSPS-1 was determined as 1.95 × 105 Da. As shown in Figure 2a, the 1H NMR spectrum of BSPS-1 shows only one dominant peak at δ 4.92 ppm in the anomeric region, indicating that these glucosyl residues are α-glycosidically linked. Combining the result of methylation analysis and literature data,18−22 this peak is assigned to the H-1 of →6)-αD-Glcp-(1→. The chemical shift of H-2 can be assigned from the COSY spectrum based on the principle that H-2 correlates with H-1 (Figure 2b). Similarly, H-3 to H-6 can also be assigned. Therefore, the chemical shifts at 3.53, 3.66, 3.45, 3.85, 3.70, and 3.93 ppm can be attributed to H-2, H-3, H-4, H-5, H6, and H-6′ of the glucosyl residues, respectively. All the proton chemical shifts can also be confirmed by TOCSY spectrum (Figure 2c). In the high field region of the 1H NMR spectrum, a small methyl signal at 1.24 ppm should be assigned to the H-6 of rhamnose residue. However, the signals of sugar residues such as arabinose, galactose, and mannose are not clearly detected in the 1H NMR spectrum of BSPS-1, which is probably due to the low contents of these residues. The 13C NMR spectrum of BSPS-1 is shown in Figure 3a. The chemical shift at 97.65 ppm is attributed to the anomeric carbon atoms of →6)-α-D-Glcp-(1→. The other carbon signals (C-2 to C-6) are identified from cross peaks in the HSQC spectrum (Figure 3b) as well as the literature data.18,19,22 Accordingly, the carbon chemical shifts at 71.29, 73.40, 69.53, 70.23, and 65.66 ppm can be assigned to C-2, C-3, C-4, C-5, and C-6 of glucosyl residue. However, no typical signal is observed for uronic acid groups, which is in agreement with the result of the m-hydroxydiphenyl colorimetric method.15 All the 1 H and 13C chemical shifts for BSPS-1 are listed in Table 2 and are consistent with previous reports.18−24 To confirm the sequence of glucosyl residues in the polysaccharide, HMBC and NOESY experiments were carried out. The cross peaks of H1− C6 and H6′−C1 are observed in the HMBC spectrum (Figure 3c). In the NOESY spectrum (Figure 3d), the cross peaks of H1−H6′ and H1−H6 are detected. These results all suggest that the glucosyl residues are linked by α-(1→6) glycosidic linkage.

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RESULTS AND DISCUSSION Structural Characterization of BSPS-1. Monosaccharide composition analysis showed that BSPS-1 was made up of arabinose, rhamnose, galactose, glucose, and mannose in the molar ratio of 1.79:1.00:2.59:26.54:1.01.15 Thus, BSPS-1 can be mainly regarded as a glucan. To determine the monosaccharide linkages, BSPS-1 was methylated and converted into the corresponding alditol acetates for GC−MS analysis. As shown in Table 1, the major derivative of BSPS-1 was 1,5,6-tri-OTable 1. Partial Methylated Alditol Acetates and Deduced Glycosidic Linkages of BSPS-1 and BSPS-3 molar ratio (%)

a b

partial methylated alditol acetates

deduced glycosidic linkages

BSPS-1

BSPS-3

2,3,5-Me3-Araa 2,3-Me2-Ara 3,4-Me2-Rha 2,4,6-Me3-Gal 2,4-Me2-Gal 2,3,4,6-Me4-GlcAp 2,3,4-Me3-Glc

T-Araf-(1→ →5)-Araf-(1→ →2)-Rhap(1→ →3)-Galp-(1→ →3,6)-Galp-(1→ T-GlcAp-(1→ →6)-Glcp-(1→

4.5 ndb 3.4 7.9 nd nd 84.2

16.9 6.7 3.8 38.3 16.6 8.4 9.3

2,3,5-Me3-Ara =2,3,5-tri-O-methyl-1,4-di-O-acetyl-arabinitol, etc. Not detected.

acetyl-2,3,4-tri-O-methyl-D-glucitol, indicating that BSPS-1 is mainly composed of (1→6)-linked glucopyranosyl residues. The purity and molecular weight of BSPS-1 was measured by HPSEC system. As shown in Figure 1a, BSPS-1 exhibited only one symmetrical sharp peak, indicating that this fraction was a homogeneous polysaccharide. According to the calibration B


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Figure 2. 1H NMR (a), 1H/1H-COSY (b), and TOCSY (c) spectra of BSPS-1. In 1H/1H-COSY and TOCSY spectra, H1−H2 represents correlation between H-1 and H-2 of →6)-α-D-Glcp-(1→ residue, etc.

On the basis of above results, BSPS-1 is determined as a linear (1→6)-α-D-glucan. Until now, only few plants including Ipomoea batatas, Cistanche deserticola, Angelica sinensis, Pueraria lobata, Panax ginseng, and Dimocarpus longan have been

reported to produce (1→6)-α-D-glucan. Zhao et al. reported that the (1→6)-α-D-glucan isolated from the root of Ipomoea batatas had strong immunostimulatory activity in a mouse model.24 Wu et al. found that three polysaccharides from the C


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Figure 3. continued

D


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Figure 3. 13C NMR (a), HSQC (b), HMBC (c), and NOESY (d) spectra of BSPS-1 in D2O at 25 °C. In the HSQC spectrum, H1−C1 represents the cross peak between H-1 and C-1 of →6)-α-D-Glcp-(1→ residue, etc. In the HMBC spectrum, H1−C6 represents the heteronuclear coherence between H-1 and C-6 of adjacent →6)-α-D-Glcp-(1→ residues. In the NOESY spectrum, H1−H6′ represents the NOE contacts between H-1 and H6′ of adjacent →6)-α-D-Glcp-(1→ residues.

Table 2. Summary of 1H and 13C Chemical Shifts for BSPS-1 glycosidic linkage →6)-α-D-Glcp(1→

H1/ C1

H2/ C2

H3/ C3

H4/ C4

H5/ C5

H6,H6′/ C6

4.92

3.53

3.66

3.45

3.85

97.65

71.29

73.40

69.53

70.23

3.70, 3.90 65.66

BSPS-3 exhibited one major symmetrical sharp peak and another two small peaks. The molecular weight of main peak in BSPS-3 was determined to be 1.88 × 105 Da. In the 1H NMR spectrum of BSPS-3 (Figure 4a), the chemical shift at 5.18 ppm should be assigned to an α-linked residue (residue A). The corresponding chemical shift of this residue at 109.26 ppm in 13C NMR spectrum (Figure 4b) can be confirmed by the HSQC spectrum (Figure 4c). According to the results of methylation analysis and literature data,25−28 this residue can be identified as T-α-L-Araf -(1→. From the TOCSY spectrum (Figure 5a), H-2 can be assigned at 4.16 ppm which correlates with H-1. H-3, H-4, H-5, and H-5′ can be assigned to the shifts at 3.88, 4.07, 3.80, and 3.67 ppm as shown in the COSY spectrum (Figure 5b). The corresponding 13C chemical shifts of T-α-L-Araf -(1→ can be assigned from the HSQC spectrum (Figure 4c) and are listed in Table 3. The assignments are consistent with the literature.25−31 From the 1H NMR spectrum of BSPS-3 (Figure 4a) and the literature,26−28 the H-1 signal at 5.02 ppm can be assigned as → 5)-α-L-Araf-(1→ (residue B). The assignment of H-2 for →5)α-L-Araf-(1→ at 4.05 ppm can be obtained by its correlation with H-1 in the TOCSY spectrum (Figure 5a). However, the other proton signals for →5)-α-L-Araf-(1→ are not detected in both TCOSY and COSY spectra, which is due to the low content of this residue. By comparing with literature data,26−28 all the 1H and 13C chemical shifts of →5)-α-L-Araf-(1→ are identified from the cross peaks in the HSQC spectrum (Figure 4c). As shown in Figure 4a, the H-1 signal at 4.62 ppm can be assigned to →3,6)-β-D-Galp-(1→ (residue C), which is consistent with literature.25,26,28,32 The assignment of H-2 (3.64 ppm) can be confirmed by its correlation with the H-1 signal in COSY and TOCSY spectra (Figure 5). The chemical shift of H-3 can be attributed to 3.83 ppm from its correlation with H-2. Comparing HSQC spectrum with the literature25,26,28,30,32 allows the signals of H-4, H-5, and H-6 to be

stem of Cistanche deserticola had a backbone of (1→6)-α-Dglucan with different molecular weights.21 Cao et al. characterized a (1→6)-α-D-glucan from the root of Angelica sinensis (Oliv.) Diels.18 Cui et al. extracted (1→6)-α-D-glucan from the root of Pueraria lobata (Willd.) Ohwi, and found that the sulfated derivative of glucan could significantly attenuate PC12 cell damage caused by hydrogen peroxide.19 Sun et al. reported the structural characterization and immunostimulatory activity of a linear (1→6)-α-D-glucan isolated from Panax ginseng C. A. Meyer.20 Recently, Zhu et al. isolated a (1→6)-αD-glucan from flesh tissues of Dimocarpus longan Lour and demonstrated that the polysaccharide had anticancer activity against the growth of HepG2 cells.22 In our previous study, BSPS-1 as a novel resource of (1→6)-α-D-glucan has been demonstrated to have potential superoxide anion and DPPH radical scavenging activities.15 From the above literature, it could be concluded that (1→6)-α-D-glucan could be a health beneficial polysaccharide and might have wide applications in food and pharmaceutical industries. Structural Characterization of BSPS-3. In our previous study, GC analysis showed that BSPS-3 was composed of arabinose, rhamnose, galactose, and mannose in the molar ratio of 16.80:3.60:33.66:1.00.15 This indicated that BSPS-3 was probably an arabinogalactan. Methylation analysis (Table 1) further showed that the dominant linkage types of BSPS-3 were T-α-L-Araf -(1→, →3,6)-β-D-Galp-(1→, and →3)-β-D-Galp(1→, accompanied by small amounts of →5)-α-L-Araf-(1→, GlcAp-(1→, →2)-α-L-Rhap-(1→, and →6)-α-D-Glcp-(1→. As shown in Figure 1b, the molecular weight determination of E


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Figure 4. 1H NMR (a), 13C NMR (b), and HSQC (c) spectra of BSPS-3 in D2O at 25 °C. A, B, C, D, E, F, and G represent the residues of α-L-Araf(1→, →5)-α-L-Araf-(1→, →3,6)-β-D-Galp-(1→, →3)-β-D-Galp-(1→, 4-O-Me-β-D-GlcAp-(1→, →2)-α-L-Rhap(1→, and →6)-α-D-Glcp-(1→, respectively. In the HSQC spectrum, A1 represents the cross peak between H-1 and C-1 of residue A, etc.

of this residue. The other 1H and 13C chemical shifts of 4-OMe-β-D-GlcAp are identified from the cross peaks in the COSY (Figure 5b) and HSQC spectra (Figure 4c). The chemical shifts identified are also consistent with literature data.33,34 In addition, the small anomeric signal at 5.21 ppm can be assigned to the H-1 of →2)-α-L-Rhap-(1→ (residue F) according to the literature.26,27,35,36 The signal at 1.24 ppm should be attributed to H-6 (exocyclic −CH3 group) of →2)-α1 13 L-Rhap-(1→in the H NMR spectrum (Figure 4a). In the C NMR spectrum, signals at 16.54 and 98.30 ppm belong to the −CH3 and C-1 of →2)-α-L-Rhap-(1→, respectively (Figure 4b). These chemical shifts can be confirmed by the HSQC spectrum (Figure 4c). Other chemical shifts are assigned acorrding to the 13C NMR spectrum and literature data.26,27,36 Notably, BSPS-3 also contains a small amount of →6)-α-DGlcp-(1→ (residue G) as confirmed by the 13C NMR and

assigned to chemical shifts at 4.18, 3.90, and 3.99 ppm, respectively. In addition, all the 13C chemical shifts of →3,6)-βD-Galp-(1→ can also be identified from the cross peaks of HSQC spectrum (Figure 4c). Similarly, the H-1 signal at 4.38 ppm can be assigned to →3)-β-D-Galp-(1→ (residue D). The 1 H and 13C chemical shifts of this residue can be identified from COSY, TOCSY, and HSQC spectra. The corresponding chemical shifts are listed in Table 3 and are also consistent with previous reports.29−32 The signal at δ 102.6 ppm in the 13C NMR spectrum can be attributed to C-1 of 4-O-Me-β-D-GlcAp-(1→ (residue E) according to the literature.33,34 A small peak observed at 175.30 ppm is a typical signal of carboxyl group from uronic acid. A cross peak at 3.40/59.90 ppm is shown in the HSQC spectrum (Figure 4c), which is assigned to the −OCH3 group F


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Figure 5. TOCSY (a), 1H/1H-COSY (b), and NOESY (c) spectra of BSPS-3 in D2O at 25 °C. In the TOCSY and 1H/1H-COSY spectra, A(1, 2) represents correlation between H-1 and H-2 of residue A, etc. In the NOESY spectrum, A1D3 represents the NOE contacts between H-1 of residue A and H-3 of residue D, etc. G


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Journal of Agricultural and Food Chemistry Table 3. Summary of 1H and 13C Chemical Shifts for BSPS-3 glycosidic linkage A: α-L-Araf-(1→ B: →5)-α-L-Araf-(1→ C: →3,6)-β-D-Galp-(1→ D: →3)-β-D-Galp-(1→ E: 4-O-Me-β-D-GlcAp-(1→ F: →2)-α-L-Rhap(1→ G: →6)-α-D-Glcp-(1→

H1/C1

H2/C2

H3/C3

H4/C4

H5/C5

H6,H6′/C6

−OCH3

5.18 109.26 5.02 107.50 4.62 103.70 4.38 103.63 4.40 102.60 5.21 98.3 4.92 97.65

4.16 81.48 4.05 81.39 3.64 69.89 3.45 69.85 3.31 72.75 4.09 77.6 3.53 71.29

3.88 76.56 3.92 76.57 3.83 81.43 3.65 80.18 3.42 75.21 3.87 69.30 3.66 73.40

4.07 83.95 4.15 82.07 4.18 68.48 4.06 68.48 3.24 82.02 3.34 73.70 3.45 69.53

3.80/3.67 61.09 3.85 67.92 3.90 73.42 3.64 75.16 3.60 75.34 3.78 69.80 3.85 70.23

3.99 69.41 3.73 61.09 − 175.30 1.24 16.54 3.70, 3.90 65.66

3.40 59.90

HSQC spectra. The chemical shifts of this residue are identical with that of BSPS-1. The sequence of glycosyl residues for BSPS-3 was determined by NOESY experiment. As shown in Figure 5c, H-1 of T-α-L-Araf -(1→ and →5)-α-L-Araf-(1→ have strong NOE contacts with H-6 of →3,6)-β-D-Galp-(1→, indicating that these two residues are linked to the O-6 position of →3,6)β-D-Galp-(1→. Similarly, T-α-L-Araf -(1→ is also linked to the O-5 position of →5)-α-L-Araf-(1→, →3,6)-β-D-Galp-(1→ is linked to the O-3 position of →3)-β-D-Galp-(1→, and →3)-βD-Galp-(1→ is linked to the O-3 position of →3,6)-β-D-Galp(1→ as assigned from the NOESY spectrum. However, →6)-αD-Glcp-(1→ has no significant NOE contact with other residues. This suggests that BSPS-1 is not absolutely separated from BSPS-3 in the purification step, thus, a small amount of BSPS-1 is contained in BSPS-3. The NOE contacts of →2)-α-LRhap-(1→ and 4-O-Me-β-D-GlcAp-(1→ with other residues are not observed in the NOESY spectrum due to their low contents. Combining all the information obtained above, the complete assignments of all the linkage patterns can be determined as listed in Table 3. The backbone of BSPS-3 is mainly composed of 1,3-β-D-galactopyranosyl residues with side chains substituted at the O-6 position consisting of large content of T-α-LAraf -(1→ residues, and lesser contents of →5)-α-L-Araf-(1→, →2)-α-L-Rhap-(1→ and 4-O-Me-β-D-GlcAp-(1→ residues. Therefore, BSPS-3 can be considered as a type II arabinogalactan. Till now, type II arabinogalactan has been isolated from various plants including Andrographis paniculata, Anogeissus latifolia, Chrysanthemum morifolium, Centella asiatica, Endopleura uchi, Plantago major, Stevia rebaudiana, Viscum album, etc., and demonstrated to possess many valuable bioactivities such as antitussive, antitumor, anticomplementary, antiviral, antioxidative, and immunological activities.16,28,30,31,37−41 In conclusion, our results suggest that BSPS-1 is a linear (1→ 6)-α-D-glucan, whereas BSPS-3 is a type II arabinogalactan. On the basis of methylation and NMR spectra analysis, the possible structural schemes for BSPS-1 and BSPS-3 are proposed in Figure 6. Notably, the unique structures of BSPS-1 and BSPS-3 indicated that they might possess many valuable bioactivities and have wide applications in food or pharmaceutical industries. Further works on the biological activities of these two polysaccharides are in progress.

Figure 6. Propsed stuctures of BSPS-1 and BSPS-3.

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AUTHOR INFORMATION

Corresponding Authors

*(J.L.) Tel: +86-514-87978158. Fax: +86-514-87313372. Email: [email protected]. *(C.-h.J.) Tel: +86-514-87978158. Fax: +86-514-87313372. Email: [email protected]. Funding

This work was partly supported by Grants-in-Aid for scientific research from the National Natural Science Foundation of China (No. 31101216), Natural Science Foundation of the Education Committee of Jiangsu province (No. 11KJB550006), Postgraduate Innovation Project of Jiangsu Province (No. SJLX_0610), Scientific and Technological Development Program of Jiangsu Province (No. BC2013408), and New Century Talents Project of Yangzhou University. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the testing center of Yangzhou University for NMR measurements. REFERENCES

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