Accepted Manuscript Structural investigation of an uronic acid-containing polysaccharide from abalone by graded acid hydrolysis followed by PMP-HPLC-MSn and NMR analysis Hong-xu Wang, Jun Zhao, Dong-mei Li, Shuang Song, Liang Song, Ying-huan Fu, Li-peng Zhang PII: DOI: Reference:

S0008-6215(14)00372-3 http://dx.doi.org/10.1016/j.carres.2014.10.010 CAR 6858

To appear in:

Carbohydrate Research

Received Date: Revised Date: Accepted Date:

24 June 2014 16 October 2014 18 October 2014

Please cite this article as: Wang, H-x., Zhao, J., Li, D-m., Song, S., Song, L., Fu, Y-h., Zhang, L-p., Structural investigation of an uronic acid-containing polysaccharide from abalone by graded acid hydrolysis followed by PMPHPLC-MSn and NMR analysis, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/j.carres.2014.10.010

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Structural investigation of an uronic acid-containing polysaccharide from abalone by graded acid hydrolysis followed by PMP-HPLC-MSn and NMR analysis

Hong-xu Wanga, Jun Zhaob,c, Dong-mei Lia,b, Shuang Songa,b,∗, Liang Songa,b, Ying-huan Fub,c, Li-peng Zhanga

a

School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, P. R. China

b

National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian 116034, P.

R. China c

School of Light Industry and Chemical Engineering, Dalian Polytechnic University, Dalian 116034, P.

R. China

∗ To whom correspondence should be addressed. Tel: 86-0411-86323262. Fax: 0411-86323262. E-mail: [email protected].

ABSTRACT: A new strategy was applied to elucidate the structure of a polysaccharide from abalone gonad (AGSP). It was hydrolyzed by 0.05 M, 0.2 M, 0.5 M, and 2.0 M TFA at 100 °C for 1 h, sequentially. Every hydrolysate was ultrafiltrated (3000 Da) to collect oligo- and monosaccharides, and the final retentate was further hydrolyzed with 2.0 M TFA at 110 °C and 121 °C for 2 h, respectively. 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization followed by HPLC-MSn analysis was applied to detect the sugar residues in these hydrolysates, which allowed proposing their location in the polysaccharide structure. The retentate after 0.5 M TFA hydrolysis was confirmed as the polysaccharide backbone, and it was further analyzed by 1D and 2D NMR spectroscopy. Thus, the structural

elucidation

of

AGSP

was

accomplished,

and

it

has

a

backbone

of

→4)-β-GlcA(1→2)-α-Man(1→ repeating unit with Fuc, Xyl and Gal in the branch. The analytical strategy demonstrated was useful to characterize the structure of polysaccharides.

Keywords: Acid hydrolysis, Abalone, PMP derivatization, Polysaccharide backbone, Disaccharide

1. Introduction It was difficult to elucidate the structure of polysaccharides on account of their complicated chemical composition and large molecular weight. Structural characterization of polysaccharides is achieved usually using a combination of different techniques including NMR, MS, chemical reactions and so on.1-3 Polysaccharides were often hydrolyzed into oligosaccharides by acid, and detailed characterization of these oligosaccharides would help to elucidate the original polysaccharide structure4-7. Moreover, acid hydrolysis could also remove the branches of polysaccharides. Jia et al8 prepared the backbone of a heteropolysaccharide (HEP-1) by treatment with 0.05 M and 0.1 M TFA, and analysis on the hydrolysates revealed that HEP-1 had a (1→6)-linked α-D-galactan backbone with branches composed of rhamnose and glucose. Therefore, partial acid hydrolysis of polysaccharides was very useful in structural elucidation of polysaccharides. Pacific abalone, Haliotis discus hannai Ino, is a highly valued marine mollusk. Several polysaccharides have been isolated from the abalone. Zhu et al9 reported a heteroglycan from abalone viscera, which had a backbone consisting of 1,3-linked rhamnose and 1,3,6-linked galactose, with glucose, fucose, xylose and galactose of different linkage types distributing in branch chains. Li et al10 identified a glycosaminoglycan-like polysaccharide with the backbone chain of a repeated disaccharide unit containing galactosamine and glucuronic acid, and the sulfated-fucose and galactose distributing in the branch. Others11,12 reported the monosaccharide composition of polysaccharides prepared from the abalone, but these investigations didn’t provide enough information to accomplish the structural elucidation. Polysaccharides from abalones possess antioxidant, antitumour, and immunomodulating, anticoagulant, cholecystokinin-releasing activities and so on.9-14 AGSP was a polysaccharide isolated from abalone gonads that showed osteogenic activity.15 In the present work, the structure of AGSP was studied by graded acid hydrolysis followed by PMP-HPLC-MSn and NMR analysis.

2. Results and discussion The analytical strategy described in Scheme 1 was applied to elucidate the structure of AGSP. AGSP was subjected to multi-step acid hydrolysis, and the hydrolysates were characterized using NMR and HPLC-MSn. 2.1. Analysis of acid hydrolysates

TFA at concentrations of 0.05 M, 0.1 M and 0.5 M was reported to hydrolyze the polysaccharides partially and to remove branch chains, while 2.0 M TFA could almost totally degrade the polysaccharides into monosaccharides at 110°C16,17. Therefore, four graded TFA concentrations (0.05 M, 0.2 M, 0.5 M, and 2.0 M, respectively) were applied to degrade the polysaccharide progressively in this study. The pore size of ultrafiltration membranes is another critical parameter. The molecular weight cutoff (MWCO) of commercial ultrafiltration membranes is usually calibrated based on spherically shaped analytes, such as globular proteins not linear, rod-like oligosaccharides. Fu et al18 investigated heparin oligosaccharides using commercial membranes with various MWCO, and the results indicated only monosaccharides, disaccharides and tetrasaccharides could pass through a 3000 Da MW cut-off membrane. A similar result was found in the present study, and only these saccharide residues were observed in the filtrates of hydrolysates. These mono- or oligosaccharides were analyzed by PMP (1-phenyl-3-methyl-5-pyrazolone) derivatization followed by HPLC-PAD-MSn. TFA is known to suppress the ESI signals of analytes due to its ability to form gas-phase ion pairs with positively-charged analyte ions19. In this experiment, TFA was removed by drying the solution repeatedly, and the result indicates HPLC-MS is more sensitive than HPLC-UV at 245 nm. The present method had a lower limit of detection (LOD) as 0.6 mg for the analysis of major sugar components. As shown in Figure1, Fuc was the main monosaccharide released in the first hydrolysis, and its amount decreased in the follow-up experiments, which indicated Fuc was possibly at the terminal position of the branch chain as the polysaccharide found by Li et al10 in abalone pleopod. Xyl and Gal were also detected in AGSP-0.05, AGSP-0.2 and AGSP-0.5, and their absence in AGSP-2 suggested that, the two monosaccharides were not in the backbone but in the branch of AGSP. The portion of Man increased alone with the increase of TFA concentration, and it was the only monosaccharide in AGSP-2, which indicated Man was in the core of AGSP structure. No disaccharides or tetrasaccharides were found in AGSP-0.05. Two disaccharides (DS1 and DS2) and a tetrasaccharide (TS1) were observed in AGSP-0.2 and AGSP-0.5. PMP derivatives of DS1 and DS2 both gave a pseudomolecular ion at m/z 687, which yielded a product ion at m/z 511 (Supplementary data). These MS data indicated they were aldobiuronic acids that composed of a hexose and a hexuronic acid with a linkage from the anomeric hydroxyl of the hexuronic acid. The pseudomolecular ion of PMP-labeled TS1 at m/z 1025 gave a product ion at m/z 849, 687 and 511 in

MS2 spectrum, and the ion at m/z 687 further yielded a fragment ion at m/z 511 in MS3 spectrum (Supplementary data). Considering the coexistence of TS1 and DS2 in AGSP-2, TS1 was proposed as a dimmer of DS2. The final ultrafiltration retentate was then hydrolyzed at 110°C and 121°C, respectively. As shown in Figure 2, DS2 was the most abundant component, and TS1 was observable in JL-110; whereas DS2 decreased and TS1 disappeared in JL-121. In contrast, the amounts of Man and GlcA increased obviously. Thus comparison of JL-110 and JL-121 revealed that TS1 and DS2 degraded to produce Man and GlcA when temperature increased. On the basis of above results, DS2 was deduced as GlcA→Man, and TS1 as GlcA→Man→GlcA→Man. In addition, as seen from the analysis results of AGSP-2, JL-110 and JL-121, the retentate of 0.5 M TFA hydrolysate contained only DS2 unit, so it was identified as the polysaccharide backbone. The composition of the original AGSP was analyzed by acid hydrolysis at 121°C with 2 M TFA followed by PMP-HPLC-MSn . As shown in Figure 3, the only disaccharide was observed as DS2, while monosaccharides, including Man, Gal, GlcA, Xyl, Fuc and Rha were detected. As DS2 and Man were the main components in the AGSP hydrolysate, the DS2 unit was in a major content in AGSP. 2.2. NMR analysis Detailed structure of AGSP backbone was completed by 1D NMR and 2D NMR spectroscopy. Two signals appeared in the anomeric region in the 1H spectrum (Supplementary data), indicating the presence of two monosaccharide residues, namely, Man and GlcA, respectively. The doublet at δ 4.52 could only be assigned to the anomeric proton of GlcA for its large coupling constant J1,2 of about 7.5 Hz, which suggested GlcA had a β-configuration. Then the brand singlet at δ 5.40 was assigned to anomeric proton of Man. 12 signals were observed in the 13C spectrum (Supplementary data), and characteristic signals at 172.9 and 60.2 ppm were due to C-6 of GlcA and Man, respectively. 1H-1 H COSY spectrum (Figure 4) showed clear vicinal coupling correlations of H-G1 (H-1 of GlcA) with H-G2, H-G2 with H-G3, H-G4 with H-G5, H-M1 (H-1 of Man) with H-M2, H-M2 with H-M3, and H-M5 with H2-M6. Then the directly bonded carbons of these protons were attributed by HSQC spectrum (Supplementary data). Long range correlations observed in the HMBC spectrum (Figure 5) were also useful for the assignment of the 1H and 13C resonances (Table 1). The linkages between the residues were established by HMBC correlations from H-G4 to C-M1 and from H-M2 to C-G1, which was confirmed by the cross peaks between H-M1 and H-G4, and between H-G1 and H-M2 in the

1

H-NOESY spectrum (Figure 6). In addition, 2D J-resolved spectroscopy (Supplementary data) showed

J1,2 of Man was about 2.0 Hz suggesting an α-configuration. Then, its 1H and 13C resonances were similar to those of →2)-α-Man(1→ and →4)-β-GlcA(1→ reported by others20. Thus, the repeating disaccharide unit in the backbone of AGSP was elucidated as →4)-β-GlcA(1→2)-α-Man(1→ (Figure 7). 1

H and 13C NMR spectra of the original AGSP were shown in Figure 8. In the 1H-NMR spectrum,

the signals at 1.50 ppm and 1.24 ppm were assigned to the methyl protons of Rha and Fuc, respectively, and the minor signals between 2.01 ppm and 2.17 ppm accounted for the methyl protons (CH3CO) of acetyl group in N-acetylamino sugars. However, it was impossible to identify other proton signals. In the 13C NMR spectrum, a signal at 15.3 ppm was assigned to the methyl group of Fuc. The signal at around 174.8 ppm was due to carbonyl group of GlcA, and its intensity suggests AGSP had a high ratio of GlcA. Two strong anomeric carbon signals at 100.1 ppm and 98.4 ppm were likely corresponded to anomeric carbons of the two most monosaccharides, Man and GlcA in AGSP. However, it was difficult to assign the crowded signal between 55 ppm and 85 ppm. To sum up, although NMR spectra of the original polysaccharide could give a total profile of its composition, these spectra were usually too complicated to analyze. After removing the branches, the polysaccharide structure became simple, and their spectra were clear for unambiguous signal assignment and structure elucidation. Therefore, partial acid hydrolysis combined with ultrafiltration was a useful method in the investigation of polysaccharide structure. In the present study, the polysaccharide backbone was prepared by 0.5 M TFA at 100 °C for 1 h, but the treatment parameters may vary for other polysaccharides depending on the resistance of their glycosidic linkages to acid hydrolysis.21 It is necessary to analyze constituent sugar residues released by graded acid hydrolysis to make sure all the branches to be deleted.

3. Conclusions A novel strategy to characterize the structure of polysaccharides was demonstrated. PMP-HPLC-MSn analysis on ultrafiltrates of the graded acid hydrolysates determined the distribution of the mono- or oligosaccharide residues in the polysaccharide. Then the polysaccharide backbone obtained by acid hydrolysis was structurally elucidated by 1D and 2D NMR spectra. Therefore, the polysaccharide from abalone gonad (AGSP) was deemed to an uronic acid-containing polysaccharide, with the backbone chain of →4)-β-GlcA(1→2)-α-Man(1→ repeating unit, and Fuc, Xyl and Gal in the

branch.

4. Experimental 4.1. Materials AGSP was previously isolated from abalone (Haliotis discus hannai Ino) gonad in our laboratory.15 Standard monosaccharides were purchased from Pharmacia Co. (Uppsala, Sweden). TFA and ammonium acetate were obtained from Guangfu precise chemical institute. (Tianjin, China). PMP was acquired from Sinopharm Chemical Reagent Co. (Beijing, China). Acetonitrile (Fisher, Pittsburgh, PA, USA) was of HPLC grade, and ultrapure water was used for HPLC-MS analysis. 4.2. Partial acid hydrolysis AGSP (200 mg) was infiltrated with 0.05 M TFA in a sealed cube and kept at 100 °C for 1 h. After the cube was cooled to room temperature, it was opened and evaporated to dryness by rotary evaporation at 45°C to remove TFA. Then 6 ml H2O was added and the solution was filtrated using a 2 ml ultrafiltration centrifuge tube with a MW cutoff of 3000 Da. Every time about 2 ml solution was added into the same tube and filtrated, and after three times the ultrafiltration was completed. The filtrate was lyophilized and labelled AGSP-0.05 (60 mg), while the retentate (113 mg) was collected for further acid hydrolysis using 0.2 M TFA. In the same way, AGSP-0.2 (19 mg) and the retentate (90 mg) were acquired using 0.2 M TFA, AGSP-0.5 (27 mg) and the retentate (57 mg) using 0.5 M TFA, and AGSP-2 (39 mg) and the retentate (9 mg) using 2.0 M TFA. Then the final retentate after 4-step hydrolysis was collected and dissolved in 2.0 M TFA, and it was divided into two fractions and heated at 110°Cand 121°C for 2h, respectively. And then, the two hydrolysates were evaporated to dryness by rotary evaporation, and labeled as JL-110 and JL-121, respectively. In addition, another 200 mg AGSP was treated as above procedure using 0.05 M, 0.2 M, and 0.5M TFA successively, and the final ultrafiltration retentate was collected for NMR analysis. 4 mg of AGSP was dissolved in 1 mL trifluoroacetic acid (TFA, 2 M), and subsequently hydrolyzed at 121 °C for 2 h. Then the total AGSP hydrolysate was obtained for PMP-HPLC-MSn analysis. 4.3. PMP derivatization procedure The derivatization was carried out as previously described.14 Briefly, the sample (1~4 mg) was dissolved in 2 mL ammonia, and 200 µL of the solution was collected to mixed with 200 µL 0.3 M PMP methanolic solution. The mixture was allowed to react for 30 min at 70°C. After the reaction

mixture was cooled to room temperature, 1.5 ml H2O was added, and then the resultant solution was dried with rotary evaporation and repeated twice until the NH3 was fully evaporated. Water and chloroform (1.0 ml each) were added, and the mixture was shaken vigorously. The chloroform layer was discarded, and the extraction process was repeated three times. The aqueous layer was filtered through a 0.22 µm pore membrane filter for HPLC-PAD-MSn analysis. AGSP-0.05, AGSP-0.2, AGSP-0.5, AGSP-2, JL-121, JL-110 and the total AGSP hydrolysate were derivatized with PMP as above described. 11 monosaccharide standards, including GlcN, Man, GalN, Rib, Rha, GlcA, GalA, Glc, Gal, Xyl, and Fuc, were used as references, and they were converted to their PMP derivatives following identical protocol. 4.4. HPLC-PAD-MSn analysis The HPLC-PAD-MSn analysis were performed on a LXQ linear ion trap mass spectrometer equipped with an electrospray ion source (ESI) and a photodiode array detector (PAD), controlled by XCalibur software (all Thermo Fisher Scientific, Basel, Switzerland). The ESI-MS settings were as follows: spray voltage: 4.5 kV, capillary temperature: 275 °C, capillary voltage: 37 V, sheath gas: 40 arbitrary units (AU), auxiliary gas: 10 AU. All the data were acquired in positive mode, and the scan range was set from m/z 100 to 2000 am. A Silgreen ODS C18 (250×4.6 mm, 5 µm) was used and kept at 30 °C, and the mobile phase consisted of 20 mM ammonium acetate–acetonitrile (78:22, v/v) with a flow rate of 1.0 ml min-1. Five PMP-monosaccharide ions at m/z 481.2, 495.2, 510.2, 511.2, and 525.2, two PMP-disaccharide ions at m/z 686.3 and 687.3, and a PMP-tetrasaccharide ion at m/z 1025.5 were extracted from total ion chromatographs to obtain extracted-ion chromatograms. 4.5. NMR spectroscopic analysis The ultrafiltration retentate of the hydrolysate after 0.05 M, 0.2 M, and 0.5 M TFA hydrolysis was subjected to NMR analysis. The sample was dissolved in 0.5 mL of D2O (99.9%), and NMR spectra were recorded on a Bruker Acsend 400 spectrometer operating at 400 MHz (1H) and 100 MHz (13C). The water signal at 4.78 ppm in 1H NMR was suppressed. COSY and J-resolved spectrum were both acquired with collecting data points of 2048 using a sweep width of 3201.024 Hz, acquisition time of 0.32 s with relaxation delay of 2 s. The acquisition parameters used for HSQC spectrum were 0.16 s acquisition time, 2.0 s delay time, 3201.024 Hz sweep width. For acquiring HMBC spectrum, acquisition time of 0.64 s, sweep width of 3201.024 Hz with a relaxation delay of 1.5 s. However, 1H

NOESY spectrum was recorded on a Bruker AV-500 spectrometer with collecting data points of 1024 ×256 using a sweep width of 1602 Hz, acquisition time of 0.32 s with relaxation delay of 2 s. The 1H and 13C NMR spectra of AGSP were recorded on a Bruker Acsend 400 spectrometer following the above procedure. Acknowledgements The work was funded by National Natural Science Foundation of China (Nos. 31171635, 31301431), National High Technology Research and Development Program 863 (No. 2014AA093602), China Torch Program (No. 2012GH560223), and Universities Innovation Ability Promotion Plan—Collaborative Innovation Center for Green Processing and Safety Control of Food in Beijing and Tianjin Area. Supplementary data Supplementary data associated with this article can be found as Supporting Information. References 1.

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Li, B.; Wei, X. J.; Sun, J. L.; Xu, S. Y. Carbohydr. Res. 2006, 341, 1135-1146.

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Gloaguen, V.; Plancke, Y.; Strecker, G.; Vebret, L.; Hoffmann, L.; Morvan, H. Int. J. Biol. Macromol. 1997, 21, 73-79.

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Jin, W.; Wang, J.; Ren, S.; Song, N.; Zhang, Q. Mar. Drugs. 2012, 10, 2138-2152.

7.

Gloaguen, V.; Morvan, H.; Hoffmann, L.; Plancke, Y.; Wieruszeski, J. M.; Lippens, G.; Strecker, G. Eur. J. Biochem. 1999, 266, 762-770.

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Jia, L. M.; Liu, L.; Dong, Q.; Fang, J. N. Carbohydr.Res. 2004, 339, 2667-2671.

9.

Zhu, B. W.; Li, D. M.; Zhou, D. Y.; Han, S.; Yang, J. F.; Li, T.; Ye, W. X.; Greeley, G. H. Food Chem. 2011, 125, 1273-1278.

10. Li, G. Y.; Chen, S. G.; Wang, Y. M.; Xue, Y.; Chang, Y. G.; Li, Z. J.; Wang, J. F.; Xue, C. H. Int. J. Biol. Macromol. 2011, 49, 1160-1166. 11. Zhu, B. W.; Zhou, D. Y.; Li, T.; Yan, S.; Yang, J. F.; Li, D. M.; Dong, X. P.; Murata, Y. Food Chem. 2010, 121, 712-718. 12. She, Z. G.; Hu, G. P.; Wu, Y. W.; Lin, Y. C. Chin. J. Org. Chem. 2002, 22, 367-370.

13. Sun, L. M.; Zhu, B. W.; Li, D. M.; Wang, L. S.; Dong, X. P.; Murata, Y. Food Agr. Immunol. 2011, 21, 15-26. 14. Zhu, B. W.; Wang, L. S.; Zhou, D. Y.; Li, D. M.; Sun, L. M.; Yang, J. F. Eur. Food. Res. Technol. 2008, 227, 1663-1668. 15. Song, S.; Huang, L.; Li, D. M.; Wang, H. X.; Pan, J. F.; Qi, H.; Sun, M. N.; Zhu, B. W. Osteogenic bioactivity of a sulfated polysaccharide from abalone gonad. (submitted). 16. Jia, L. M.; Liu, L.; Dong, Q.; Fang, J. N. Carbohydr. Res. 2004, 339, 2667-2671. 17. Han, X. Q.; Chung, Lap, Chan, B.; Dong, C. X.; Yang, Y. H.; Ko, C. H.; Gar-Lee, Yue, G.; Chen, D.; Wong, C. K.; Bik-San, Lau, C.; Tu, P. F.; Shaw, P. C.; Fung, K. P.; Leung, P. C.; Hsiao, W. L.; Han, Q. B. J. Agric. Food. Chem. 2012, 60, 4276-4281. 18. Fu, L.; Zhang, F.; Li, G; Onishi, A.; Bhaskar, U.; Sun, P.; Linhardt, R. J. J. Pharm. Sci. 2014, 103, 1375-1383. 19. Shou, W. Z.; Weng, N. D. J. Chromatogr. B. 2005, 825, 186-192. 20. Li, B.; Wei, X.Z.; Sun, J. L.; Xu, S. Y. Carbohydr. Res. 2006, 341, 1135-1146. 21. De Ruiter, G. A.; Schols, H. A.; Voragen, A. G. J.; Rombouts, F. M. Anal. Biochem. 1992, 207, 176-185.

Table 1 1H and 13C NMR chemical shifts for AGSP backbone Residue

H1/C1

H2/C2

H3/C3

H4/C4

H5/C5

H6/C6

GlcA

4.52/104.1

3.41/75.0

3.71/78.1

3.83/79.6

4.04/76.6

-/175.1

Man

5.40/101.5

4.16/80.1

3.79/72.0

3.74/68.7

3.58/75.6

3.82;3.74/62.4

Scheme 1. Analytical procedure.

Figure 1. Chromatograms of PMP-labelled with monosaccharides extracted ions at m/z 481.2, 495.2, 510.2, 511.2, and 525.2 (A; C; E and G) and oligosaccharides with extracted ions at m/z 686.3, 687.3, and 1025.5 (B; D; F and H) in AGSP-0.05, AGSP-0.2, AGSP-0.5, and AGSP-2, respectively.

Figure 2. Extracted-ion chromatograms of JL-110 (A) and JL-121 (B) after PMP derivatization.

Figure 3. Chromatograms of PMP-labelled oligosaccharides with extracted ions at m/z 481.2, 495.2, 510.2, 511.2, and 525.2 (A) and oligosaccharides with extracted ions at m/z 686.3, 687.3, and 1025.5 (B) in AGSP hydrolysate.

Figure 4. 1H-1H COSY spectrum of AGSP backbone

Figure 5. HMBC spectrum of AGSP backbone

Figure 6. 1H NOESY spectrum of AGSP backbone

OH

O O O HO

O

HO HO

OH O OH

OH

O N

HO

O HO

NH

HO O

HO

OH OH

CH3 O

HO H3C

O

A

O

HN N

B

Figure 7. Structures of AGSP backbone (A) and PMP-labelled DS2 (B)

Figure. 8. 1H-NMR and 13C NMR spectra of AGSP

Graphical Abstract Structural investigation of an uronic acid-containing polysaccharide from abalone by graded acid hydrolysis followed by PMP-HPLC-MSn and NMR analysis

Hong-xu Wanga, Jun Zhaob,c, Dong-mei Lia,b, Shuang Songa,b,∗, Liang Songa,b, Ying-huan Fub,c, Li-peng Zhanga OH

O O O HO

O

HO HO

OH O OH

OH

O N

HO

O HO

NH

HO O

HO

OH OH

CH3 O

HO H3C

O

A

O

HN N

B

∗ To whom correspondence should be addressed. Tel: 86-0411-86323262. Fax: 0411-86323262. E-mail: [email protected].

Highlights A new strategy was demonstrated to characterize a polysaccharide (AGSP). The

composition was analyzed

by graded

acid

hydrolysis followed

by

PMP-HPLC-MSn. The backbone of AGSP was elucidated by 1D and 2D NMR spectra. The

backbone

consisted

of

repeating

→4)-β-GlcA(1→2)-α-Man(1→. Fuc, Xyl, and Gal were in the branch chains.

disaccharide

units

of

Structural investigation of a uronic acid-containing polysaccharide from abalone by graded acid hydrolysis followed by PMP-HPLC-MSn and NMR analysis.

A new strategy was applied to elucidate the structure of a polysaccharide from abalone gonad (AGSP). It was hydrolyzed by 0.05 M, 0.2 M, 0.5 M, and 2...
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