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Structural analysis of heteropolysaccharide from Saccharina japonica and its derived oligosaccharides
International Journal of Biological Macromolecules 62 (2013) 697–704
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Structural analysis of heteropolysaccharide from Saccharina japonica and its derived oligosaccharides Weihua Jin a,b,c , Wenjing Zhang a,b , Jing Wang a,c , Sumei Ren d , Ni Song d , Quanbin Zhang a,c,∗ a
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China c Nantong Branch, Institute of Oceanology, Chinese Academy of Sciences, Jiangsu 226006, PR China d College of Medicine and Pharmaceutics, Ocean University of China, Qingdao 266003, PR China b
a r t i c l e
i n f o
Article history: Received 5 September 2013 Received in revised form 1 October 2013 Accepted 11 October 2013 Available online 18 October 2013 Keywords: ESI-MS Oligosaccharides Saccharina japonica
a b s t r a c t Degraded fucoidan (F1) was desulfated by DMSO–MeOH. And anion exchange chromatography was performed to fractionate desulfated F1 (ds-F1) into five fractions. Electrospray ionization mass spectrometry (ESI-MS) showed that each fraction contained at least one set of neutral and/or sulfated fucooligosaccharides in the form of methyl glycosides. And the structures of oligomeric fragments were characterized by ESI-CID-MS/MS and ESI-CID-MS/MS/MS. In addition, more structural features were shown by NMR. Therefore, it was concluded that LF1 contained a backbone of (1 → 3)-linked fucopyranose residues sulfated at C-4 and branched at C-2 by fucopyranose residues and fucoglucuronomannan, fucoglucuronan, galactan and xylan were found in LF-5. Finally, it was concluded that F1 was the middle component, which contained the information of both F0.5 and F2, indicating that the differences between F1 and F0.5, F2 might be derived primarily from the different needs of algae itself. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, fucoidan extracted from brown algae increasingly attracts much attention since they had various biological activities, such as antitumor [1–3], anticoagulant activity [4–6] and immunomodulatory [6–8]. Though there are many reviews [9–11] on the relationships between fucoidan structure and biological activity, they are still poorly understood due to the complexity of fucoidan structures. For example, Teruya et al. [12] reported that oversulfated fucoidan increased anti-proliferative activity via caspase-3 and -7 activation-dependent pathway. And it was also reported by Cumashi et al. [13] that the content of sulfation was the main influence in the anti-angiogenic acitivity of MSPs. And it was shown [14] that the inhibiting activity of SK-MEL-28 depended on the presence of sulfates and (1 → 4)-linked ␣-l-Fucp residues in the main chain of fucoidan/oligosaccharides. In addition, previous study [4] showed that the ability of anticoagulant activities depended on the molecular weight and the molar ratio of fucose to galactose. Moreover, on the aspect of sulfated polysaccharides, there are at least three main kinds: (1) sulfated fucan consisted of
∗ Corresponding author. Tel.: +86 532 82898703/+86 13687638267; fax: +86 532 82898703. E-mail addresses: [email protected], [email protected] (Q. Zhang). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.017
fucose [15–17]. (2) Sulfated galactofucan contained galactose and fucose [18–21]; (3) sulfated heteropolysaccharides included fucose, galactose, glucuronic acid and mannose [22]. Mass spectroscopy (MS) with soft desorption/ionization and especially electrospray ionization (ESI-MS) are important tools for the analysis of heteropolysaccharides [23]. Its accuracy, sensitivity, selectivity and speed fit the requirements for the analysis of heteropolysaccharides. Recently, it was reported that it is possible to determine the sulfation pattern [24] and the type of linkage [25] by electrospray ionization mass spectrometry with collisioninduced dissociation tandem mass spectrometry (ESI-CID-MS/MS). Thus, MS studies [26–32] have shown bright prospects with regard to the analysis of the structures of heteropolysaccharides. However, the structures of heteropolysaccharides with high degree of substitution of sulfate and high molecular weight are not suitable by MS. Thus many methods, including degrading, desulfation and modification, have been developed. NMR spectroscopy, which overcomes some shortcomings of MS, is the most effective non-destructive method for the structural analysis of heteropolysaccharides. In our lab, the structure of fucogalactan sulfate was reported [33] using the traditional methods (Desulfation and NMR), which only showed some information. Later, the strutural features of heteropolysaccharide (F0.5) and sulfated fucan (F2) were elucidated [34,35]. F0.5 was degraded by mild acid hydrolysis before separation by anion exchange and gel filtration chromatography. Finally, the fractions were determined by ESI-MS and analyzed by
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Table 1 Chemical composition (%, dry weight) of the fractions of ds-F1 by anion exchange chromatography. Sample
LF-1 LF-2 LF-3 LF-4 LF-5 a b
Fuc (%)
78.64 56.02 27.27 14.44 9.61
U A (%)
1.47 11.42 35.52 49.05 53.04
SO4 (%)
4.34 18.97 11.57 9.86 14.56
Mw
Monosaccharides (molar ratio) Man
Rha
GlcA
Glc
Gal
Xyl
Fuc
0a 0.08a 0.47a 1.10a 0.81a
0 0.04 0.10 0 0
0b 0.08b 0.49b 1.14b 1.65b
0 0 0 0.12 0
0.03 0.12 0.21 0.29 0.52
0.02 0.07 0.21 0.48 0.56
1 1 1 1 1
1285 2508 4103 5969 8227
The molar ratio of Man to Fuc was underestimated. The molar ratio of GlcA to Fuc was underestimated.
ESI-CID-MS/MS or NMR. In addition, F2 was degraded by desulfation and methanolysis. Some oligosaccharides were separated by semi-preparative HPLC. And the fractions were also determined by ESI-MS and analyzed by ESI-CID-MS/MS or NMR. According to the previous study [35], F1 was a complex fraction. It had the highest content of fucose. However, the content of sulfate and uronic acid (UA) was between F0.5 and F2. Are there any relationships among them? Thus the present work was undertaken to reinvestigated the structure of F1 using a method that combines ESI-MS with NMR, which produced more structural information than would be obtained by either method alone.
2. Materials and methods 2.1. Materials Degraded fucoidan F1 was prepared according to the previous study [35]. And it was desulfated according to the method [34]. Briefly speaking, F1 (10 g) was dissolved in distilled water (1 L) and mixed with cationic resin (H+ -Na+ ) for 3 h. After filtration, the solution was neutralized with pyridinium and lyophilized. Solution was dissolved in dimethyl sulfoxide (DMSO): methanol (9:1; v/v, 20 mL). The mixture was heated at 80 ◦ C for 5 h, and the desulfated product (ds-F1) was dialyzed and lyophilized.
2.2. Preparation and purification of oligosaccharides from ds-F1 Ds-F1 (1.0 g) was separated by anion exchange chromatography on a DEAE-Bio Gel Agarose FF gel (2.6 cm × 30 cm) with elution by water (LF-1), 0.05 M NaCl (LF-2), 0.1 M NaCl (LF-3), 0.2 M NaCl (LF4) and 1 M NaCl (LF-5). And all of the fractions were dialyzed and lyophlilized.
2.3. Composition analysis The sulfated content was determined by ion chromatography on Shodex IC SI-52 4E column (4.0 × 250 mm) eluted with 3.6 mM Na2 CO3 at a flow rate of 0.8 mL min−1 at 45 ◦ C. The molar ratio of monosaccharide composition and the content of fucose (Fuc) were determined following Zhang et al. [36]. Briefly speaking, a solution of sample (10 mg mL−1 ) was hydrolyzed in 2 M trifluoroacetic acid in a 10 mL ampoule. The ampoule was sealed in a nitrogen atmosphere and hydrolyzed for 4 h at 110 ◦ C. Then the hydrolyzed mixture was neutralized to pH 7 with sodium hydroxide. Later the mixture was converted into its 1-phenyl-3-methyl-5-pyrazolone derivatives and separated by HPLC chromatography. Uronic acid (UA) was analyzed by a modified carbazole method [37]. Molecular weight was determined by GPC-HPLC on TSK gel PWxl 3000 column (7 m 7.8 × 300 mm) eluted with 0.2 M Na2 SO4 at a flow rate of 0.5 mL min−1 at 30 ◦ C.
2.4. MS analysis of oligosaccharides MS was performed on a LTQ ORBITRAR XL (Thermo Scientific). Samples were dissolved in CH3 CN–H2 O (1:1, v/v). The solution was centrifuged and the supernate was analyzed. Mass spectra were registered in the negative ion mode at a flow rate of 5 L min−1 . The capillary voltage was set to −3000 V, and the cone voltage was set at −50 V. The source temperature was 80 ◦ C, and the desolvation temperature was 150 ◦ C. The collision energy was optimized between 10 and 50 eV. All spectra were analyzed by Xcalibur. 2.5. NMR spectroscopy Polysaccharides (50 mg) were co-evaporated with deuterium oxide (99.9%) twice before dissolving in deuterium oxide (99.9%) containing 0.1 L deuterated acetone. NMR and two-dimensional spectra were recorded at a Bruker AVANCE III 600 MHz at 25 ◦ C. The chemical shifts were adjusted to the internal standard (deuterated acetone, 2.05 and 29.92 ppm, respectively). 3. Results and discussion 3.1. Isolation and purification According to the previous study [35], it was shown that F1 contained 54.84% Fuc, 7.3% UA, 32.26% sulfate and other monosacchrides, such as galactose (Gal), mannose (Man) and glucuronic acid (GlcA). And the molecular weight of F1 was 8436 Da. However, after desulfation, the content of sulfate decreased to 8.66%, while the content of Fuc and UA increased to 70.19% and 18.73%. In addition, the molecular weight of ds-F1 also decreased to 5785 Da. Moreover, ds-F1 still had many other monosaccharides. In order to elucidate the structural features of ds-F1, anion exchange chromatography was performed on a DEAE-Bio Gel Agarose FF gel with elution by water and various concentrations of NaCl. And their chemical constituents were determined as shown in Table 1. The highest content of Fuc and the lowest content of sulfate suggested LF-1 might be a fucan, which was formed because of the loss of the sulfate. The decreasing contents of Fuc of LF-1 to LF-5 indicated that sulfated fucan was partially desulfated under the solvolytic condition. On the other hand, the increasing contents of UA of LF-1 to LF-5 presumed that the UA relatively kept stable. In addition, the fluctuation of sulfate content was attributed to the following two reasons: (1) The sulfate substituted on not only Fuc but also Gal or Man, (2) the column retention specificity resulted from not only sulfate group but also carboxyl group. On the aspect of other monosaccharides, the ratio of GlcA, Gal and xylose (Xyl) to Fuc of LF-2 to LF-5 was increasing while the fluctuation of rhamnose (Rha) and glucose (Glc) did not take into account owe to the small amounts. Finally, the contents of Man of LF-2 to LF-4 were increasing while that of LF-5 was decreasing. According to the previous reports [35,38], it was shown that fucoidans contained
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Fig. 1. Negative-ion mode ESIMS spectrum of LF-3.
mannoglucuronan and glucuronan. The former one consisted of a backbone of alternating 4-linked GlcA and 2-linked Man, while the latter one contained 3-linked GlcA. Thus it was speculated that LF-1 to LF-4 contained mainly mannoglucuronan with less glucuronan while LF-5 contained not only mannoglucuronan but also glucuronan according to the ratio of Man to GlcA. MS is an important tool for the analysis of heteropolysaccharides because of its speed, sensitivity and accuracy. Thus we used ESI-MS to analyze the compositions of ds-F1 and its fractions. The spectrum (not shown) of ds-F1 only displayed parts of information because of the charge, molecular weight and solubility. Based on the MS spectra, it was showed that LF-1 (data not shown) corresponded to methy glycosides of fucooligosaccharides and sulfated fucooligosaccharides, while LF-2 (data not shown) was assigned to methyl glycosides of disulfated fucooligosaccharides with minor disulfated fucooligosaccharides and methyl glycosides of trisulfated fucooligosaccharides, which was consistent with the previous study [34]. In addition, LF-2 also contained other compositions, which were insoluble in CH3 CN–H2 O (1:1, v/v). It was shown in Fig. 1 that there were two series of triply charged ions: one was at m/z 381.739, 430.425, 479.110, 527.796, 576.482 and 625.168 corresponded to methyl glycosides of trisulfated fucooligosaccharides [Me Fucn (SO3 Na)3 -3Na]3− (n = 6–11), another was at m/z 474.439, 523.125, 571.810 and 620.496 corresponded to trisulfated fucooligosaccharides [Fucn (SO3 Na)3 -3Na]3− (n = 8–11). In addition, there were also some less intensity quadruply and quintuply charged ions (data not shown). On the aspect of other monosaccharides, it was not shown in MS spectrum. The molecular weights of LF-4 and LF-5 were 5969 and 8227, respectively. In addition, it was shown that LF-4 and LF-5 contained mainly UA with minor Fuc. Thus the spectra of LF-4 and LF-5 were complicated. However, it was shown (data not shown) that LF-4 contained quadruply charged ions at m/z 379.070, 415.585, 452.098, 488.613 and 525.129 corresponding to methyl glycosides of tetra-sulfated fucooligosaccharides [Me Fucn (SO3 Na)4 -4Na]4− (n = 8–12) while LF-5 also had quintuply charged ions at m/z 319.046, 348.257, 377.469 corresponding to methyl glycosides of penta-sulfated fucooligosaccharides [Me Fucn (SO3 Na)5 -5Na]5− (n = 8–10). In order to elucidate the structure of the above oligosaccharides, ESI-CID-MS/MS was performed. The fragmentation patterns (data not shown) for methyl fucooligosaccharides were similar with the previous study [34], suggesting that the structures of methyl fucooligosaccharides were methyl 3-linked fucooligomers. In addition, Fig. 2 showed the fragmentation pattern for the ion at m/z 1133.379, corresponding to the ion [Me Fuc7 SO3 Na–Na]− . According to the previous
Fig. 2. Negative-ion mode ESI-CID-MS/MS spectra of the ions [Me Fuc7 SO3 Na–Na]− at m/z 1133.379.
study [34], the ions at m/z 371.062, 517.119, 663.176, 809.232 and 955.290 (a loss of methyl fucopyranose residue (178 Da)) were assigned to be B -type ions, suggesting the sulfate was located at the non-reducing end. In addition, there was another set of fragment ions at m/z 549.145, 695.202, 841.258 and 987.315 (a loss of fucopyranose residue (164 Da)) corresponding to be Y-type ions, indicating that the sulfate was located at the reducing end. However, the ion at m/z 987.315 might also arise from the loss of the branch (fucose residue), which was confirmed by NMR. Because it was impossible to determine the branches’ positions, so the following discussion was based on the absence of the branched units. Thus it was speculated that Me Fuc7 SO3 Na is a mixture of the isomers Fuc(SO3 Na) → [3 Fuc 1→]5 → Fuc-OMe and Fuc → [3 Fuc 1→]5 → Fuc(SO3 Na)-OMe under the condition of that there was no branched units, which was different from the previous study [34]. The fragmentation for the doubly charged ion at m/z 387.077 (−2), corresponding to the ion [Me Fuc4 (SO3 Na)2 -2Na]2− , was performed in Fig. 3a. It showed four types of fragment ions contained: (1) Doubly charged fragment ions at m/z 225.006 (B2 ), 298.034 (B3 ) and 371.063 (B4 ) corresponded to [Fuc2 (SO3 Na)2 -2Na]2− , [Fuc3 (SO3 Na)2 -2Na]2− and [Fuc4 (SO3 Na)2 -2Na]2− . No fragment ion at m/z 152 [Fuc(SO3 Na)2 2Na]2− was observed, suggesting that one isomer of Me Fuc4 (SO3 Na)2 was Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc → Fuc-OMe. (2) A less intensity doubly charged fragment ion at m/z 314.047 (Y3 ) assigned to [Me Fuc3 (SO3 Na)2 -2Na]2− , arising probably from the glycosidic bond cleavage from the nonreducing terminus. Thus it was hypothesized that Me Fuc4 (SO3 Na)2 contained Fuc → Fuc → Fuc(SO3 Na) → Fuc(SO3 Na)-OMe. (3) Single charged fragment ions at m/z 225.006, 371.063 and 517.120 (namely B1 , B2 and B3 ) arised from the loss of methyl glycoside of sulfated fucose (257 Da) from the reducing terminus, suggesting that Me Fuc4 (SO3 Na)2 might consist of Fuc(SO3 Na) → Fuc → Fuc → Fuc(SO3 Na)-OMe or Fuc → Fuc → Fuc(SO3 Na) → Fuc(SO3 Na)-OMe. (4) Single charged fragment ion at m/z 549.146 (namely Y3 ) assigned to be [Me Fuc3 SO3 Na–Na]− , which arised from the loss of sulfated fucopyranose residue (225 Da) from the side chain or from the nonreducing terminus. And single charged fragment ion at 403.089 (namely Y2 ), corresponding to [Me Fuc2 SO3 Na–Na]− , derived from the loss of fucopyranose after the loss of the sulfated fucopyranose. Thus it was hypothesized that Me Fuc4 (SO3 Na)2 was made
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Fig. 3. Negative-ion mode ESI-CID-MS/MS spectra of the ions [Me Fuc4 (SO3 Na)2 -2Na]2− at m/z 387.077 (−2) (a), [Me Fuc10 (SO3 Na)2 -2Na]2− at m/z 825.250 (−2) (b), [Me Fuc9 SO3 Na–Na]− at m/z 1425.486 (c) and [Fuc10 (SO3 Na)2 -2Na]2− at m/z 818.242 (−2) (d).
up of Fuc(SO3 Na) → Fuc → Fuc → Fuc(SO3 Na)-OMe, Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc → Fuc-OMe and Fuc → Fuc → Fuc(SO3 Na) → Fuc(SO3 Na)-OMe. In order to confirm the above hypothesis, the fragmentation pattern for the doubly charged ion at m/z 825.250 (−2) assigned to the ion [Me Fuc10 (SO3 Na)2 -2Na]2− was performed in Fig. 3b. There were also four series of fragment ions included: (1) Doubly charged fragment ions at m/z 298.034, 371.063, 444.091, 517.112, 590.148, 663.177, 736.205 and 809.233 (a loss of MeOH (32 Da)) corresponded to be B-type ions, arising from the glycosidic bond cleavage from the reducing terminus. (2) Doubly charged fragment ions at m/z 533.132, 606.161, 679.189 and 752.218 (a loss of fucopyranose residue (146 Da)), corresponding to be Y -type ions, arised from the glycosidic bond cleavage from the nonreducing terminus. (3) Single charged fragment ions at m/z 955.290, 1101.347 and 1247.404 were observed. (4) There was a series of single charged fragment ions at m/z 841.259, 987.316, 1133.373, 1279.430 and 1425.486. And the fragment ion at m/z 1425.486 was characterized by ESI-CID-MS3 in Fig. 3c. One set of fragment ions at m/z 517.117, 663.177, 809.233, 955.290, 1101.348 and 1247.404 (a loss of methyl fucose (178 Da)) was assigned to be B-type ions arising from the glycosidic bond cleavage from the reducing end, suggesting the sulfate was located at the nonreducing end. Another set of fragment ions at m/z 695.203, 841.259, 987.316, 1133.373 and 1279.430 (a loss of fucopyranose residue
(146 Da)) arised from the glycosidic bond cleavage from the nonreducing end, suggesting the sulfate was located at the reducing end. Thus it was concluded that the ion [Me Fuc9 SO3 Na–Na]− at m/z 1425.486 was also a mixture of isomers Fuc(SO3 Na) → [3 Fuc 1→]7 → Fuc-OMe and Fuc → [3 Fuc 1→]7 → Fuc(SO3 Na)-OMe. In other words, the fragment ion at m/z 1425.486 derived from two ways. Thus it was concluded that the ion [Me Fuc10 (SO3 Na)2 -2Na]2− at m/z 825.250 (−2) was a mixture of isomers including: 1. Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]7 → FucOMe; 2. Fuc → [3 Fuc 1→]7 → Fuc(SO3 Na) → Fuc(SO3 Na)-OMe; 3. Fuc(SO3 Na) → [3 Fuc 1→]8 → Fuc(SO3 Na)-OMe. The fragmentation pattern (in Fig. 3d) for the doubly charged ion [Fuc10 (SO3 Na)2 -2Na]2− at m/z 818.242 (−2) was similar to the fragmentation pattern for the doubly charged ion at m/z [Me Fuc10 (SO3 Na)2 -2Na]2− at m/z 825.250 (−2). Thus it was concluded that the ion [Fuc10 (SO3 Na)2 -2Na]2− was a mixture of isomers including: 1. Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]7 → Fuc; 2. Fuc → [3 Fuc 1→]7 → Fuc(SO3 Na) → Fuc(SO3 Na); 3 Fuc(SO3 Na) → [3 Fuc 1→]8 → Fuc(SO3 Na). The fragmentation for the triply charged ion at m/z 576/482 (−3) was identified as [Me Fuc10 (SO3 Na)3 -3Na]3− in Fig. 4. (1) Single charged fragment ions at m/z 841.260, 987.317, 1133.374 and 1279.431 corresponded to Y -type ions. And the ion at m/z 1279.431 arised from the loss of two units of sulfated fucopyranose residues (450 Da) from the non-reducing end. In
W. Jin et al. / International Journal of Biological Macromolecules 62 (2013) 697–704
Fig. 4. Negative-ion mode ESI-CID-MS/MS/MS spectrum of the ion [Me Fuc10 (SO3 Na)3 -3Na]3− at m/z 576.482 (−3).
addition, the ion at m/z 1279.431 was also characterized by ESI-CID-MS3 (data not shown). The result was similar with the ion at m/z 1425.486 with two series of fragment ions—B-type ions and Y-type ions, suggesting that Me Fuc10 (SO3 Na)3 was Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]7 → Fuc(SO3 Na)-OMe and Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na) →[3 Fuc 1→]6 → Fuc-OMe. (2) Double charged fragment ions at m/z 225.006, 298.035, 371.149, 444.092, 517.120, 590.149, 663.177 and 752.218 was regarded as Y -type ions. From the first ion to seventh ion, two neighbouring ions differed only 146 Da, which was the fucopyranose residue. However, it was 178 Da between the ions at m/z 663.177 and 752.218, indicating that the loss of methyl fucose. In addition, the difference of the intensity of the ions at m/z 663.177 and 752.218 was apparent, presuming that there are more ways to generate the ion at m/z 752.218 than the ion at m/z 663.177. The fragment ion at m/z 752.218 came from the loss of sulfated fucopyranose residue from the non-reducing terminus. Thus it was hypothesized that the ion at m/z 752.218 came from a mixture of isomers: Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na) → 1→]6 → Fuc-OMe, Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc [3 Fuc 1→]7 → Fuc(SO3 Na)-OMe and Fuc(SO3 Na) → [3 Fuc 1→]7 Fuc(SO3 Na) → Fuc(SO3 Na)-OMe. In addition, the ion at m/z 663.177 might derive from the two ways: One was the loss of sulfated fucopyranose residue, followed by methyl fucose. Another was the loss of methyl glycoside of sulfated fucose, followed by fucopyranose residue. And these indicated that Me Fuc10 (SO3 Na)3 was a mixture of isomers: Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]6 → Fuc-OMe, Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]7 → Fuc(SO3 Na)-OMe, and Fuc(SO3 Na) → [3 Fuc 1→]7 Fuc(SO3 Na) → Fuc(SO3 Na)-OMe. (3) Triply charged fragment ions at m/z 225.006, 273.448, 322.377, 371.063, 419.749, 468.434 and 517.120 (a loss of methyl fucose (178 Da)) were identified as B-type ions. And no ion at m/z 176 (−3), corresponding to [Fuc2 (SO3 Na)3 -3Na]3− , was detected. It was concluded that Me Fuc10 (SO3 Na)3 was Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]6 → Fuc-OMe. (4) Triply charged fragment ions at m/z 527.795 (a loss of fucopyranose (146 Da)) was assigned to Y type ion, suggesting that Me Fuc10 (SO3 Na)3 was Fuc → [3 Fuc 1→]6 → Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na)-OMe. Therefore, it was concluded that Me Fuc10 (SO3 Na)3 was a mixture of four isomers: Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]6 → Fuc-OMe, Fuc → [3 Fuc 1→]6 → Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na)-OMe, Fuc(SO3 Na) → [3 Fuc 1→]7 Fuc(SO3 Na) →
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Fuc(SO3 Na)-OMe and Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]7 → Fuc(SO3 Na)-OMe. From the above results, it was hypothesized that the sulfate in the middle position of the backbone was more sensitive to lose than that in the ends (both the reducing terminus and nonreducing terminus), which was confirmed by the results (data not shown) of methyl glycosides of tetra-sulfated fucooligosaccharides and penta-sulfated fucooligosaccharides, which had similar results with the trisulfated fucooligosaccharides. In other words, it was more likely to identify and desulfate the sulfate at C-2, and then randomly degraded into oligosaccharides and desulfated at C-4 because the sulfate at C-2 was liable to lose when compared with the sulfate at C-4. Though many structural features of methyl glycosides of sulfated fucooligosaccharides and fucooligosaccharides were elucidated by ESI-MS and ESI-MS/MS, the information on the structural characteristics of glucuronic acid, galactose and mannose was not shown in the ESI-MS spectra. It might be arised from that the molecular weight of heteropolysaccharides, which contained glucuronic acid, galactose and mannose was too large to determine by ES-MS and the heteropolysaccharides were not soluble in 50% acetonitrile and water. Thus, LF-1 and LF-5 were determined by NMR. The yield of LF-1 is about 26.11%. Chemical analysis has already revealed that it was primarily composed of fucose with minor sulfate (The molar ration of fucose to sulfate was approximately 10.61) and other monosaccharides. And the molecular weight was about 1285 Da. For this above reason, it was possible to apply 2D spectroscopy for the assignment of resonances based on the both 1 H–NMR and DEPTQ (13 C) NMR spectra. And 1 H, 1 H-COSY, TOCSY, HMBC and HSQC (Fig. 5) demonstrated the presence of Fuc, Man, Glc, Gal and Xyl, which was consistent with chemical analysis (The molar ratio of Man, Glc, Gal and Xyl to Fuc was 0.02, 0.02, 0.03 and 0.02, respectively). Therefore, it was difficult to discuss the absolute structural characteristics of other monosaccharides except Fuc. The reducing terminal residues were observed as 3-linked methyl ␣ ()-fucopyranosides A␣ and A or 3-linked methyl sulfated ␣ ()-fucopyranosides R-4S␣ and R-4S, which was consistent with the MS results. The methyl group was formed with methanol under the solvolytic desulfation conditions, which was reported by Toida et al. [39] In addition, it was also confirmed by the positions of correlation peak H-OMe A␣ /C-1 A␣ , H-OMe A /C-1A , H-OMe R-G4S␣ /C-1R-G4S␣ , H-OMe R-G4S /C-1R-G4S in HMBC spectrum at 3.26/99.84, 3.42/103.93, 3.25/99.84 and 3.40/103.73 ppm, respectively. The position of correlation peaks H-1B /H-2B /H-3B /H-4B and H-5B /H-6B in TOCSY spectrum at 4.96/3.81/3.89/3.91 and 4.18/1.09 ppm, H-4B /H-5B in 1 H, 1 H-COSY spectrum at 3.91/4.18 ppm, H-1B /C-3B and H-3B /C-1B in HMBC spectrum at 4.96/75.37 and 3.89/95.91 ppm, H-1B /C-1B , H-2B /C-2B , H-3B /C-3B , H-4B /C-4B , H-5B /C-5B and H-6B /C-6B in HSQC spectrum at 4.96/95.93, 3.81/66.72, 3.89/75.37, 3.91/68.84, 4.18/66.93 and 1.09/15.77 ppm, respectively, confirmed the presence of 3-linked ␣-l-fucopyranose (residue B), which was also the main composition of LF-1. And the presence of branching signals was proven by the position of correlation peaks H-1C /C-1C , H-2C /C-2C , H-3C /C3C and H-4C /C-4C in HSQC spectrum at 5.23/92.15, 3.98/68.00, 4.08/72.26 and 3.98/68.00 ppm, respectively, which are typical of 2, 3-disubstituted ␣-l-fucose units in a (1 → 3)-linked fucan chain [40]. Besides, HMBC spectrum contained correlation peaks H-1D /C-3C , H-1E /C-2C , C-1E /H-2C and C-1C /H-3B at 5.05/72.26, 5.02/68.00, 95.74/3.98 and 92.15/3.89, suggesting the presence of 2, 3-disubstituted ␣-fucose. Moreover, the presence of residue E was further confirmed that the fucan had branches at position 2 in the form of ␣-l-fucopyranose residues. Finally, the non-reducing end residues of the backbone were identified as ␣-l-fucopyranose residues F and some N-4S, which was also not desulfated. Based on this result and those detailed above, it was concluded that LF-1
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Fig. 5. HSQC spectrum of LF-1.
Scheme 1. Structural features of LF-1.
was mainly consisting of (1 → 3)-linked ␣-l-fucan with minor branches (␣-l-fucose) at position 2 and its structural fragments were displayed in Scheme 1 and Table 2. The molar ratio of GlcA, Man, Gal and Xyl to Fuc was 1.65, 0.81, 0.52 and 0.56, respectively, suggesting that LF-5 had more complex constituents. And it was also confirmed by the presence of signals of monosaccharides in 1 H-NMR and DEPTQ (13 C) NMR spectra. The anomeric region of HSQC (Fig. 6) spectrum
mainly contained two signals belonging to Fuc residues, whose chemical shifts ranged from 4.90 to 5.00 ppm. The characteristic features of LF-5 were showed in Scheme 2 and Table 2. One main residue was M and another residue was N. The position of correlation peaks H-1M /C-3L and C-1M /H-3L in HMBC spectrum at 4.96/73.65 and 95.25/3.77 confirmed the correlations between anomeric protons of Man and Fuc. In addition, the chemical shifts of C-2, C-3 and C-4 of Fuc residues were at 68.51, 69.69 and
Table 2 13 C and 1 H NMR data for LF-1and LF-5. Chemical shifts (ppm)
Residue
A␣ A R-4S␣ R-4S B C D N-4S E F G H L M N O P Q R S T U V W
3)-␣ -l-Fucp-OMe 3)--l-Fucp-OMe →3)-␣-l-Fuc(4SO3 − )-OMe →3)--l-Fuc(4SO3 − )-OMe →3)-␣-l-Fuc-(1→ →2,3)-␣-l-Fuc-(1→ →3)-␣-l-Fuc-(1→ ␣-l-Fuc(4SO3 − )-(1→ ␣-l-Fuc-(1→ ␣-l-Fuc-(1→ →2)-␣-d-Manp-(1→ →4)--d-GlcAp-(1→ →2,3)-␣-d-Manp-(1→ ␣-l-Fucp-(1→ ␣-l-Fucp(4SO3 − )-(1→ →3)-␣-l-Fucp(4SO3 − )-(1→ →3)-␣-l-Fucp-(1→ →3)--d-GlcAp-(1→ →3,4)--d-GlcAp-(1→ ␣-l-Fucp-(1→ →6)- -d-Galp-(1→ →4,6)--d-Galp-(1→ -d-Galp-(1→ →4)- -d-Xylp-(1→
H-1/C-1
H-2/C-2
H-3/C-3
H-4/C-4
H-5/C-5
H-6/C-6
OMe
4.64/99.84 4.18/103.93 4.87/98.84 4.49/104.36 4.96/95.93 5.23/92.15 5.05/95.63 4.92/96.03 5.02/95.74 4.87/96.21 5.27/98.75 4.33/101.76 5.27/98.75 4.96/95.25 4.98/94.93 4.90/96.43 4.93/96.16 4.72/102.58 4.72/102.58 4.85/97.61 4.29/103.67 4.29/103.67 4.53/104.00 4.33/101.76
3.80/66.72 3.40/70.92 3.90/68.09 3.56/77.98 3.81/66.72 3.98/68.00 3.84/66.47 3.66/68.38 3.68/68.38 3.65/68.38 4.02/77.80 3.27/73.06 4.22/73.06 3.68/68.51 3.68/68.51 3.68/68.51 3.81/67.05 3.51/73.51 3.51/73.51 3.77/68.14 3.27/nd 3.31/nd 3.27/nd 3.17/73.06
3.80/75.29 3.56/78.00 4.18/nd 3.84/nd 3.89/75.37 4.08/72.26 3.80/75.29 3.81/69.83 3.62/70.14 3.71/70.16 3.68/69.92 3.51/76.43 3.77/73.65 3.81/69.69 3.95/69.69 4.02/77.80 3.91/nd 3.68/83.63 3.68/76.43 3.81/69.69 3.51/73.10 3.63/73.10 3.51/73.10 3.51/74.61
3.91/68.84 3.84/68.00 4.61/nd 4.18/nd 3.91/68.84 3.98/68.00 3.91/68.84 4.64/79.60 3.66/72.26 3.68/72.26 3.58/66.37 3.63/77.80 3.81/69.69 3.68/72.15 4.49/80.90 4.49/80.90 3.95/nd 3.51/70.60 3.81/81.7 3.68/72.15 3.77/69.69 3.91/78.50 3.77/69.69 3.63/76.71
3.89/66.53 3.66/70.30 nd/nd nd/nd 4.18/66.93 4.18/66.93 4.03/67.27 4.18/66.72 4.03/67.27 4.03/67.27 3.64/76.71 3.68/76.71 3.68/76.43 4.10/67.05 4.29/66.70 4.22/69.05 4.29/66.70 3.81/75.61 3.51/74.61 4.33/67.05 3.68/74.10 3.68/74.10 3.51/73.10 nd/nd
1.10/15.69 1.13/15.89 1.13/15.89 1.14/15.95 1.09/15.77 1.09/15.77 1.10/15.69 1.09/15.77 1.14/15.95 1.06/16.06 3.68/60.49 175.96 3.68/60.49 1.07/15.76 1.12/15.76 1.12/16.26 1.12/16.26 175.46 175.46 1.07/15.76 3.68,3.77/69.95 3.68,3.77/69.95 3.68/60.95
3.26/55.46 3.42/57.49 3.25/55.46 3.40/57.49
W. Jin et al. / International Journal of Biological Macromolecules 62 (2013) 697–704
703
Fig. 6. HSQC spectrum of LF-5.
Scheme 2. The structural features of LF-5.
72.15 ppm, respectively, from HSQC, 1 H, 1 H-COSY and TOCSY spectra, indicating the absence of any substituents. Therefore, the Fuc residues were present only as non-reducing terminal monosaccharides, which had a (1 → 3)-linkage with Man residues. Due to the incomplete cleavage of sulfate, there were still some sulfates substituted at Fuc residue. And the proportion of N was similar with that of M. The signal of H-4/C-4 in HSQC spectrum at 4.49/80.90 proved the presence of sulfate group at C-4, and the chemical shifts of H-3N /C-3N and H-3O /C-3O of Fuc residues were at 3.95/69.69 and 4.02/77.80, indicating residue N was ␣l-Fuc(4SO3 − )-(1→ and residue O was →3)-␣-l-Fuc(4SO3 − )-(1→. Besides, there was also a less intense anomeric signal of →3)␣-l-Fuc-(1→ at 4.93 ppm. Analysis of HSQC, 1 H, 1 H-COSY, HMBC and TOCSY (data not shown) revealed the residue P. According to the previous study [40], the position of correlation peaks H-1G /C1G and H-1L /C-1L in HSQC spectrum at 5.27/98.75, indicating the presence of 2-linked Man (residue G) and 2,3-linked Man (residue L). And analysis of 2D spectra confirmed the above results. GlcA was the predominant monosaccharide in LF-5. Analysis of NMR spectra of LF-5 allowed to speculate three kinds of GlcA H, Q and R, differing in substitution pattern. The corresponding correlation peaks in the anomeric region of HSQC spectrum were at 4.33/101.76, 4.72/102.58 and 4.72/102.58 ppm, respectively. HMBC spectra showed the correlation peak H-1G /C-4H , C-1G /H4H , H-1H /C-2L and C-1H /H-2L at 5.27/77.80, 98.75/3.63, 4.33/73.06 and 101.76/4.22 ppm, respectively, indicating the (1 → 2)-linkage between ␣-d-Man and -d-GlcAp and (1 → 4)-linkage between -d-GlcAp and ␣-d-Man, which had been reported by the previous studies [35,38]. Moreover, the correlation peak H-1M /C-3L in HMBC at 4.96/73.65, suggesting that Man residue sometimes had a 3-linked Fuc branch, which was consistent with the previous study [41]. Because the interpretation of residues Q/R and W/H
were complicated by overlapping of the corresponding anomeric proton signals with anomeric resonance of GlcA and Glcp/Xyl, therefore, the assignments of the signals were based on the 2D spectra and previous study [40]. And (1 → 3)-glucuronan was also reported by the previous study [35]. Like residue M, residue S was a (1 → 4)-linked branch substituented on residue R. Finally, the (1 → 6)-linked galactan with (1 → 4)-linked Gal as a branch, was confirmed by the position of correlation peaks H-1T and U /C-1T and U in HSQC spectra at 4.29/103.67 ppm, while the branch was proved by the signals of correlation peak H-4U /C-4U in HSQC spectrum at 3.91/78.50 ppm. The non-reducing end residues were presented by -d-Galp residue V based on the anomeric carbon resonance in 2D spectra. 4. Conclusion F1 was desulfated by DMSO–MeOH. Compared with the ESIMS spectra of ds-F0.5 (data not shown) and ds-F2 [34], it was suggested that all fractions (F0.5, F1 and F2) contained a backbone of 3-linked ␣-l-Fuc. The major difference was the degree of sulfation. This phenomenon might be attributed to the alga’s needs, which was to protect and survive. Then, anion exchange chromatography was performed to fractionate the desulfated mixture into five fractions. LF-1 was mainly t a fucan slightly sulfated at C-4 and branched at C-2 by fucopranose residues. Along with increasing content of UA and decreasing content of Fuc, the fractions became more complicated. Thus LF-5 was characterized by NMR. It was shown that it contained not only sulfated fucan but also fucoglucuronomannan, fucoglucuronan, galactan and xylan, which F0.5 also contained. Finally, it was concluded that F1 was the middle component, which contained the information of both F0.5 and F2, indicating that the differences between F1 and F0.5,
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F2 might be derived primarily from the different needs of algae itself. Acknowledgements This study was supported by the Science and Technology Project of Shandong Province (No. 2011GHY11529), Science and Technology program of applied basic research projects of Qingdao Municipal (NO. 12-1-4-8-(3)-jch) and the Prospective Joint Research Projects of Jiangsu Province (No. BY2011189). References [1] S. Koyanagi, N. Tanigawa, H. Nakagawa, S. Soeda, H. Shimeno, Biochem. Pharmacol. 65 (2003) 173–179. [2] O.S. Vishchuk, S.P. Ermakova, T.N. Zvyagintseva, Carbohydr. Res. 346 (2011) 2769–2776. [3] C. Zhuang, H. Itoh, T. Mizuno, H. Ito, Biosci. Biotechnol., Biochem. 59 (1995) 563–567. [4] W. Jin, Q. Zhang, J. Wang, W. Zhang, Carbohydr. Polym. 91 (2013) 1–6. [5] N.M. Mestechkina, V.D. Shcherbukhin, Appl. Biochem. Microbiol. 46 (2010) 267–273. [6] W.A.J.P. Wijesinghe, Y.-J. Jeon, Carbohydr. Polym. 88 (2012) 13–20. [7] H.R. Raghavendran, P. Srinivasan, S. Rekha, Int. Immunopharmacol. 11 (2011) 157–163. [8] M. Yang, C. Ma, J. Sun, Q. Shao, W. Gao, Y. Zhang, Z. Li, Q. Xie, Z. Dong, X. Qu, Int. Immunopharmacol. 8 (2008) 1754–1760. [9] G. Jiao, G. Yu, J. Zhang, H.S. Ewart, Mar. Drugs 9 (2011) 196–223. [10] V.H. Pomin, BBA—Gen. Subj. 1820 (2012) 1971–1979. [11] I. Wijesekara, R. Pangestuti, S.K. Kim, Carbohydr. Polym. 84 (2001) 14–21. [12] T. Teruya, T. Konishi, S. Uechi, H. Tamaki, M. Tako, Int. J. Biol. Macromol. 41 (2007) 221–226. [13] A. Cumashi, N.A. Ushakova, M.E. Preobrazhenskaya, A. D’Incecco, A. Piccoli, L. Totani, N. Tinari, G.E. Morozevich, A.E. Berman, M.I. Bilan, A.I. Usov, N.E. Ustyuzhanina, A.A. Grachev, C.J. Sanderson, M. Kelly, G.A. Rabinovich, S. Iacobelli, N.E. Nifantiev, Glycobiology 17 (2007) 541–552. [14] S.D. Anastyuk, N.M. Shevchenko, S.P. Ermakova, O.S. Vishchuk, E.L. Nazarenko, P.S. Dmitrenok, T.N. Zvyagintseva, Carbohydr. Polym. 87 (2012) 186–194. [15] U. Adhikari, C.G. Mateu, K. Chattopadhyay, C.A. Pujol, E.B. Damonte, B. Ray, Phytochemistry 67 (2006) 2474–2482. [16] O. Klarzynski, V. Descamps, B. Plesse, J.C. Yvin, B. Kloareg, B. Fritig, Mol. Plant 16 (2003) 115–122.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Structural analysis of heteropolysaccharide from Saccharina japonica and its derived oligosaccharides Weihua Jin a,b,c , Wenjing Zhang a,b , Jing Wang a,c , Sumei Ren d , Ni Song d , Quanbin Zhang a,c,∗ a
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China c Nantong Branch, Institute of Oceanology, Chinese Academy of Sciences, Jiangsu 226006, PR China d College of Medicine and Pharmaceutics, Ocean University of China, Qingdao 266003, PR China b
a r t i c l e
i n f o
Article history: Received 5 September 2013 Received in revised form 1 October 2013 Accepted 11 October 2013 Available online 18 October 2013 Keywords: ESI-MS Oligosaccharides Saccharina japonica
a b s t r a c t Degraded fucoidan (F1) was desulfated by DMSO–MeOH. And anion exchange chromatography was performed to fractionate desulfated F1 (ds-F1) into five fractions. Electrospray ionization mass spectrometry (ESI-MS) showed that each fraction contained at least one set of neutral and/or sulfated fucooligosaccharides in the form of methyl glycosides. And the structures of oligomeric fragments were characterized by ESI-CID-MS/MS and ESI-CID-MS/MS/MS. In addition, more structural features were shown by NMR. Therefore, it was concluded that LF1 contained a backbone of (1 → 3)-linked fucopyranose residues sulfated at C-4 and branched at C-2 by fucopyranose residues and fucoglucuronomannan, fucoglucuronan, galactan and xylan were found in LF-5. Finally, it was concluded that F1 was the middle component, which contained the information of both F0.5 and F2, indicating that the differences between F1 and F0.5, F2 might be derived primarily from the different needs of algae itself. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, fucoidan extracted from brown algae increasingly attracts much attention since they had various biological activities, such as antitumor [1–3], anticoagulant activity [4–6] and immunomodulatory [6–8]. Though there are many reviews [9–11] on the relationships between fucoidan structure and biological activity, they are still poorly understood due to the complexity of fucoidan structures. For example, Teruya et al. [12] reported that oversulfated fucoidan increased anti-proliferative activity via caspase-3 and -7 activation-dependent pathway. And it was also reported by Cumashi et al. [13] that the content of sulfation was the main influence in the anti-angiogenic acitivity of MSPs. And it was shown [14] that the inhibiting activity of SK-MEL-28 depended on the presence of sulfates and (1 → 4)-linked ␣-l-Fucp residues in the main chain of fucoidan/oligosaccharides. In addition, previous study [4] showed that the ability of anticoagulant activities depended on the molecular weight and the molar ratio of fucose to galactose. Moreover, on the aspect of sulfated polysaccharides, there are at least three main kinds: (1) sulfated fucan consisted of
∗ Corresponding author. Tel.: +86 532 82898703/+86 13687638267; fax: +86 532 82898703. E-mail addresses: [email protected], [email protected] (Q. Zhang). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.017
fucose [15–17]. (2) Sulfated galactofucan contained galactose and fucose [18–21]; (3) sulfated heteropolysaccharides included fucose, galactose, glucuronic acid and mannose [22]. Mass spectroscopy (MS) with soft desorption/ionization and especially electrospray ionization (ESI-MS) are important tools for the analysis of heteropolysaccharides [23]. Its accuracy, sensitivity, selectivity and speed fit the requirements for the analysis of heteropolysaccharides. Recently, it was reported that it is possible to determine the sulfation pattern [24] and the type of linkage [25] by electrospray ionization mass spectrometry with collisioninduced dissociation tandem mass spectrometry (ESI-CID-MS/MS). Thus, MS studies [26–32] have shown bright prospects with regard to the analysis of the structures of heteropolysaccharides. However, the structures of heteropolysaccharides with high degree of substitution of sulfate and high molecular weight are not suitable by MS. Thus many methods, including degrading, desulfation and modification, have been developed. NMR spectroscopy, which overcomes some shortcomings of MS, is the most effective non-destructive method for the structural analysis of heteropolysaccharides. In our lab, the structure of fucogalactan sulfate was reported [33] using the traditional methods (Desulfation and NMR), which only showed some information. Later, the strutural features of heteropolysaccharide (F0.5) and sulfated fucan (F2) were elucidated [34,35]. F0.5 was degraded by mild acid hydrolysis before separation by anion exchange and gel filtration chromatography. Finally, the fractions were determined by ESI-MS and analyzed by
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Table 1 Chemical composition (%, dry weight) of the fractions of ds-F1 by anion exchange chromatography. Sample
LF-1 LF-2 LF-3 LF-4 LF-5 a b
Fuc (%)
78.64 56.02 27.27 14.44 9.61
U A (%)
1.47 11.42 35.52 49.05 53.04
SO4 (%)
4.34 18.97 11.57 9.86 14.56
Mw
Monosaccharides (molar ratio) Man
Rha
GlcA
Glc
Gal
Xyl
Fuc
0a 0.08a 0.47a 1.10a 0.81a
0 0.04 0.10 0 0
0b 0.08b 0.49b 1.14b 1.65b
0 0 0 0.12 0
0.03 0.12 0.21 0.29 0.52
0.02 0.07 0.21 0.48 0.56
1 1 1 1 1
1285 2508 4103 5969 8227
The molar ratio of Man to Fuc was underestimated. The molar ratio of GlcA to Fuc was underestimated.
ESI-CID-MS/MS or NMR. In addition, F2 was degraded by desulfation and methanolysis. Some oligosaccharides were separated by semi-preparative HPLC. And the fractions were also determined by ESI-MS and analyzed by ESI-CID-MS/MS or NMR. According to the previous study [35], F1 was a complex fraction. It had the highest content of fucose. However, the content of sulfate and uronic acid (UA) was between F0.5 and F2. Are there any relationships among them? Thus the present work was undertaken to reinvestigated the structure of F1 using a method that combines ESI-MS with NMR, which produced more structural information than would be obtained by either method alone.
2. Materials and methods 2.1. Materials Degraded fucoidan F1 was prepared according to the previous study [35]. And it was desulfated according to the method [34]. Briefly speaking, F1 (10 g) was dissolved in distilled water (1 L) and mixed with cationic resin (H+ -Na+ ) for 3 h. After filtration, the solution was neutralized with pyridinium and lyophilized. Solution was dissolved in dimethyl sulfoxide (DMSO): methanol (9:1; v/v, 20 mL). The mixture was heated at 80 ◦ C for 5 h, and the desulfated product (ds-F1) was dialyzed and lyophilized.
2.2. Preparation and purification of oligosaccharides from ds-F1 Ds-F1 (1.0 g) was separated by anion exchange chromatography on a DEAE-Bio Gel Agarose FF gel (2.6 cm × 30 cm) with elution by water (LF-1), 0.05 M NaCl (LF-2), 0.1 M NaCl (LF-3), 0.2 M NaCl (LF4) and 1 M NaCl (LF-5). And all of the fractions were dialyzed and lyophlilized.
2.3. Composition analysis The sulfated content was determined by ion chromatography on Shodex IC SI-52 4E column (4.0 × 250 mm) eluted with 3.6 mM Na2 CO3 at a flow rate of 0.8 mL min−1 at 45 ◦ C. The molar ratio of monosaccharide composition and the content of fucose (Fuc) were determined following Zhang et al. [36]. Briefly speaking, a solution of sample (10 mg mL−1 ) was hydrolyzed in 2 M trifluoroacetic acid in a 10 mL ampoule. The ampoule was sealed in a nitrogen atmosphere and hydrolyzed for 4 h at 110 ◦ C. Then the hydrolyzed mixture was neutralized to pH 7 with sodium hydroxide. Later the mixture was converted into its 1-phenyl-3-methyl-5-pyrazolone derivatives and separated by HPLC chromatography. Uronic acid (UA) was analyzed by a modified carbazole method [37]. Molecular weight was determined by GPC-HPLC on TSK gel PWxl 3000 column (7 m 7.8 × 300 mm) eluted with 0.2 M Na2 SO4 at a flow rate of 0.5 mL min−1 at 30 ◦ C.
2.4. MS analysis of oligosaccharides MS was performed on a LTQ ORBITRAR XL (Thermo Scientific). Samples were dissolved in CH3 CN–H2 O (1:1, v/v). The solution was centrifuged and the supernate was analyzed. Mass spectra were registered in the negative ion mode at a flow rate of 5 L min−1 . The capillary voltage was set to −3000 V, and the cone voltage was set at −50 V. The source temperature was 80 ◦ C, and the desolvation temperature was 150 ◦ C. The collision energy was optimized between 10 and 50 eV. All spectra were analyzed by Xcalibur. 2.5. NMR spectroscopy Polysaccharides (50 mg) were co-evaporated with deuterium oxide (99.9%) twice before dissolving in deuterium oxide (99.9%) containing 0.1 L deuterated acetone. NMR and two-dimensional spectra were recorded at a Bruker AVANCE III 600 MHz at 25 ◦ C. The chemical shifts were adjusted to the internal standard (deuterated acetone, 2.05 and 29.92 ppm, respectively). 3. Results and discussion 3.1. Isolation and purification According to the previous study [35], it was shown that F1 contained 54.84% Fuc, 7.3% UA, 32.26% sulfate and other monosacchrides, such as galactose (Gal), mannose (Man) and glucuronic acid (GlcA). And the molecular weight of F1 was 8436 Da. However, after desulfation, the content of sulfate decreased to 8.66%, while the content of Fuc and UA increased to 70.19% and 18.73%. In addition, the molecular weight of ds-F1 also decreased to 5785 Da. Moreover, ds-F1 still had many other monosaccharides. In order to elucidate the structural features of ds-F1, anion exchange chromatography was performed on a DEAE-Bio Gel Agarose FF gel with elution by water and various concentrations of NaCl. And their chemical constituents were determined as shown in Table 1. The highest content of Fuc and the lowest content of sulfate suggested LF-1 might be a fucan, which was formed because of the loss of the sulfate. The decreasing contents of Fuc of LF-1 to LF-5 indicated that sulfated fucan was partially desulfated under the solvolytic condition. On the other hand, the increasing contents of UA of LF-1 to LF-5 presumed that the UA relatively kept stable. In addition, the fluctuation of sulfate content was attributed to the following two reasons: (1) The sulfate substituted on not only Fuc but also Gal or Man, (2) the column retention specificity resulted from not only sulfate group but also carboxyl group. On the aspect of other monosaccharides, the ratio of GlcA, Gal and xylose (Xyl) to Fuc of LF-2 to LF-5 was increasing while the fluctuation of rhamnose (Rha) and glucose (Glc) did not take into account owe to the small amounts. Finally, the contents of Man of LF-2 to LF-4 were increasing while that of LF-5 was decreasing. According to the previous reports [35,38], it was shown that fucoidans contained
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Fig. 1. Negative-ion mode ESIMS spectrum of LF-3.
mannoglucuronan and glucuronan. The former one consisted of a backbone of alternating 4-linked GlcA and 2-linked Man, while the latter one contained 3-linked GlcA. Thus it was speculated that LF-1 to LF-4 contained mainly mannoglucuronan with less glucuronan while LF-5 contained not only mannoglucuronan but also glucuronan according to the ratio of Man to GlcA. MS is an important tool for the analysis of heteropolysaccharides because of its speed, sensitivity and accuracy. Thus we used ESI-MS to analyze the compositions of ds-F1 and its fractions. The spectrum (not shown) of ds-F1 only displayed parts of information because of the charge, molecular weight and solubility. Based on the MS spectra, it was showed that LF-1 (data not shown) corresponded to methy glycosides of fucooligosaccharides and sulfated fucooligosaccharides, while LF-2 (data not shown) was assigned to methyl glycosides of disulfated fucooligosaccharides with minor disulfated fucooligosaccharides and methyl glycosides of trisulfated fucooligosaccharides, which was consistent with the previous study [34]. In addition, LF-2 also contained other compositions, which were insoluble in CH3 CN–H2 O (1:1, v/v). It was shown in Fig. 1 that there were two series of triply charged ions: one was at m/z 381.739, 430.425, 479.110, 527.796, 576.482 and 625.168 corresponded to methyl glycosides of trisulfated fucooligosaccharides [Me Fucn (SO3 Na)3 -3Na]3− (n = 6–11), another was at m/z 474.439, 523.125, 571.810 and 620.496 corresponded to trisulfated fucooligosaccharides [Fucn (SO3 Na)3 -3Na]3− (n = 8–11). In addition, there were also some less intensity quadruply and quintuply charged ions (data not shown). On the aspect of other monosaccharides, it was not shown in MS spectrum. The molecular weights of LF-4 and LF-5 were 5969 and 8227, respectively. In addition, it was shown that LF-4 and LF-5 contained mainly UA with minor Fuc. Thus the spectra of LF-4 and LF-5 were complicated. However, it was shown (data not shown) that LF-4 contained quadruply charged ions at m/z 379.070, 415.585, 452.098, 488.613 and 525.129 corresponding to methyl glycosides of tetra-sulfated fucooligosaccharides [Me Fucn (SO3 Na)4 -4Na]4− (n = 8–12) while LF-5 also had quintuply charged ions at m/z 319.046, 348.257, 377.469 corresponding to methyl glycosides of penta-sulfated fucooligosaccharides [Me Fucn (SO3 Na)5 -5Na]5− (n = 8–10). In order to elucidate the structure of the above oligosaccharides, ESI-CID-MS/MS was performed. The fragmentation patterns (data not shown) for methyl fucooligosaccharides were similar with the previous study [34], suggesting that the structures of methyl fucooligosaccharides were methyl 3-linked fucooligomers. In addition, Fig. 2 showed the fragmentation pattern for the ion at m/z 1133.379, corresponding to the ion [Me Fuc7 SO3 Na–Na]− . According to the previous
Fig. 2. Negative-ion mode ESI-CID-MS/MS spectra of the ions [Me Fuc7 SO3 Na–Na]− at m/z 1133.379.
study [34], the ions at m/z 371.062, 517.119, 663.176, 809.232 and 955.290 (a loss of methyl fucopyranose residue (178 Da)) were assigned to be B -type ions, suggesting the sulfate was located at the non-reducing end. In addition, there was another set of fragment ions at m/z 549.145, 695.202, 841.258 and 987.315 (a loss of fucopyranose residue (164 Da)) corresponding to be Y-type ions, indicating that the sulfate was located at the reducing end. However, the ion at m/z 987.315 might also arise from the loss of the branch (fucose residue), which was confirmed by NMR. Because it was impossible to determine the branches’ positions, so the following discussion was based on the absence of the branched units. Thus it was speculated that Me Fuc7 SO3 Na is a mixture of the isomers Fuc(SO3 Na) → [3 Fuc 1→]5 → Fuc-OMe and Fuc → [3 Fuc 1→]5 → Fuc(SO3 Na)-OMe under the condition of that there was no branched units, which was different from the previous study [34]. The fragmentation for the doubly charged ion at m/z 387.077 (−2), corresponding to the ion [Me Fuc4 (SO3 Na)2 -2Na]2− , was performed in Fig. 3a. It showed four types of fragment ions contained: (1) Doubly charged fragment ions at m/z 225.006 (B2 ), 298.034 (B3 ) and 371.063 (B4 ) corresponded to [Fuc2 (SO3 Na)2 -2Na]2− , [Fuc3 (SO3 Na)2 -2Na]2− and [Fuc4 (SO3 Na)2 -2Na]2− . No fragment ion at m/z 152 [Fuc(SO3 Na)2 2Na]2− was observed, suggesting that one isomer of Me Fuc4 (SO3 Na)2 was Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc → Fuc-OMe. (2) A less intensity doubly charged fragment ion at m/z 314.047 (Y3 ) assigned to [Me Fuc3 (SO3 Na)2 -2Na]2− , arising probably from the glycosidic bond cleavage from the nonreducing terminus. Thus it was hypothesized that Me Fuc4 (SO3 Na)2 contained Fuc → Fuc → Fuc(SO3 Na) → Fuc(SO3 Na)-OMe. (3) Single charged fragment ions at m/z 225.006, 371.063 and 517.120 (namely B1 , B2 and B3 ) arised from the loss of methyl glycoside of sulfated fucose (257 Da) from the reducing terminus, suggesting that Me Fuc4 (SO3 Na)2 might consist of Fuc(SO3 Na) → Fuc → Fuc → Fuc(SO3 Na)-OMe or Fuc → Fuc → Fuc(SO3 Na) → Fuc(SO3 Na)-OMe. (4) Single charged fragment ion at m/z 549.146 (namely Y3 ) assigned to be [Me Fuc3 SO3 Na–Na]− , which arised from the loss of sulfated fucopyranose residue (225 Da) from the side chain or from the nonreducing terminus. And single charged fragment ion at 403.089 (namely Y2 ), corresponding to [Me Fuc2 SO3 Na–Na]− , derived from the loss of fucopyranose after the loss of the sulfated fucopyranose. Thus it was hypothesized that Me Fuc4 (SO3 Na)2 was made
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Fig. 3. Negative-ion mode ESI-CID-MS/MS spectra of the ions [Me Fuc4 (SO3 Na)2 -2Na]2− at m/z 387.077 (−2) (a), [Me Fuc10 (SO3 Na)2 -2Na]2− at m/z 825.250 (−2) (b), [Me Fuc9 SO3 Na–Na]− at m/z 1425.486 (c) and [Fuc10 (SO3 Na)2 -2Na]2− at m/z 818.242 (−2) (d).
up of Fuc(SO3 Na) → Fuc → Fuc → Fuc(SO3 Na)-OMe, Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc → Fuc-OMe and Fuc → Fuc → Fuc(SO3 Na) → Fuc(SO3 Na)-OMe. In order to confirm the above hypothesis, the fragmentation pattern for the doubly charged ion at m/z 825.250 (−2) assigned to the ion [Me Fuc10 (SO3 Na)2 -2Na]2− was performed in Fig. 3b. There were also four series of fragment ions included: (1) Doubly charged fragment ions at m/z 298.034, 371.063, 444.091, 517.112, 590.148, 663.177, 736.205 and 809.233 (a loss of MeOH (32 Da)) corresponded to be B-type ions, arising from the glycosidic bond cleavage from the reducing terminus. (2) Doubly charged fragment ions at m/z 533.132, 606.161, 679.189 and 752.218 (a loss of fucopyranose residue (146 Da)), corresponding to be Y -type ions, arised from the glycosidic bond cleavage from the nonreducing terminus. (3) Single charged fragment ions at m/z 955.290, 1101.347 and 1247.404 were observed. (4) There was a series of single charged fragment ions at m/z 841.259, 987.316, 1133.373, 1279.430 and 1425.486. And the fragment ion at m/z 1425.486 was characterized by ESI-CID-MS3 in Fig. 3c. One set of fragment ions at m/z 517.117, 663.177, 809.233, 955.290, 1101.348 and 1247.404 (a loss of methyl fucose (178 Da)) was assigned to be B-type ions arising from the glycosidic bond cleavage from the reducing end, suggesting the sulfate was located at the nonreducing end. Another set of fragment ions at m/z 695.203, 841.259, 987.316, 1133.373 and 1279.430 (a loss of fucopyranose residue
(146 Da)) arised from the glycosidic bond cleavage from the nonreducing end, suggesting the sulfate was located at the reducing end. Thus it was concluded that the ion [Me Fuc9 SO3 Na–Na]− at m/z 1425.486 was also a mixture of isomers Fuc(SO3 Na) → [3 Fuc 1→]7 → Fuc-OMe and Fuc → [3 Fuc 1→]7 → Fuc(SO3 Na)-OMe. In other words, the fragment ion at m/z 1425.486 derived from two ways. Thus it was concluded that the ion [Me Fuc10 (SO3 Na)2 -2Na]2− at m/z 825.250 (−2) was a mixture of isomers including: 1. Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]7 → FucOMe; 2. Fuc → [3 Fuc 1→]7 → Fuc(SO3 Na) → Fuc(SO3 Na)-OMe; 3. Fuc(SO3 Na) → [3 Fuc 1→]8 → Fuc(SO3 Na)-OMe. The fragmentation pattern (in Fig. 3d) for the doubly charged ion [Fuc10 (SO3 Na)2 -2Na]2− at m/z 818.242 (−2) was similar to the fragmentation pattern for the doubly charged ion at m/z [Me Fuc10 (SO3 Na)2 -2Na]2− at m/z 825.250 (−2). Thus it was concluded that the ion [Fuc10 (SO3 Na)2 -2Na]2− was a mixture of isomers including: 1. Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]7 → Fuc; 2. Fuc → [3 Fuc 1→]7 → Fuc(SO3 Na) → Fuc(SO3 Na); 3 Fuc(SO3 Na) → [3 Fuc 1→]8 → Fuc(SO3 Na). The fragmentation for the triply charged ion at m/z 576/482 (−3) was identified as [Me Fuc10 (SO3 Na)3 -3Na]3− in Fig. 4. (1) Single charged fragment ions at m/z 841.260, 987.317, 1133.374 and 1279.431 corresponded to Y -type ions. And the ion at m/z 1279.431 arised from the loss of two units of sulfated fucopyranose residues (450 Da) from the non-reducing end. In
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Fig. 4. Negative-ion mode ESI-CID-MS/MS/MS spectrum of the ion [Me Fuc10 (SO3 Na)3 -3Na]3− at m/z 576.482 (−3).
addition, the ion at m/z 1279.431 was also characterized by ESI-CID-MS3 (data not shown). The result was similar with the ion at m/z 1425.486 with two series of fragment ions—B-type ions and Y-type ions, suggesting that Me Fuc10 (SO3 Na)3 was Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]7 → Fuc(SO3 Na)-OMe and Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na) →[3 Fuc 1→]6 → Fuc-OMe. (2) Double charged fragment ions at m/z 225.006, 298.035, 371.149, 444.092, 517.120, 590.149, 663.177 and 752.218 was regarded as Y -type ions. From the first ion to seventh ion, two neighbouring ions differed only 146 Da, which was the fucopyranose residue. However, it was 178 Da between the ions at m/z 663.177 and 752.218, indicating that the loss of methyl fucose. In addition, the difference of the intensity of the ions at m/z 663.177 and 752.218 was apparent, presuming that there are more ways to generate the ion at m/z 752.218 than the ion at m/z 663.177. The fragment ion at m/z 752.218 came from the loss of sulfated fucopyranose residue from the non-reducing terminus. Thus it was hypothesized that the ion at m/z 752.218 came from a mixture of isomers: Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na) → 1→]6 → Fuc-OMe, Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc [3 Fuc 1→]7 → Fuc(SO3 Na)-OMe and Fuc(SO3 Na) → [3 Fuc 1→]7 Fuc(SO3 Na) → Fuc(SO3 Na)-OMe. In addition, the ion at m/z 663.177 might derive from the two ways: One was the loss of sulfated fucopyranose residue, followed by methyl fucose. Another was the loss of methyl glycoside of sulfated fucose, followed by fucopyranose residue. And these indicated that Me Fuc10 (SO3 Na)3 was a mixture of isomers: Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]6 → Fuc-OMe, Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]7 → Fuc(SO3 Na)-OMe, and Fuc(SO3 Na) → [3 Fuc 1→]7 Fuc(SO3 Na) → Fuc(SO3 Na)-OMe. (3) Triply charged fragment ions at m/z 225.006, 273.448, 322.377, 371.063, 419.749, 468.434 and 517.120 (a loss of methyl fucose (178 Da)) were identified as B-type ions. And no ion at m/z 176 (−3), corresponding to [Fuc2 (SO3 Na)3 -3Na]3− , was detected. It was concluded that Me Fuc10 (SO3 Na)3 was Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]6 → Fuc-OMe. (4) Triply charged fragment ions at m/z 527.795 (a loss of fucopyranose (146 Da)) was assigned to Y type ion, suggesting that Me Fuc10 (SO3 Na)3 was Fuc → [3 Fuc 1→]6 → Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na)-OMe. Therefore, it was concluded that Me Fuc10 (SO3 Na)3 was a mixture of four isomers: Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]6 → Fuc-OMe, Fuc → [3 Fuc 1→]6 → Fuc(SO3 Na) → Fuc(SO3 Na) → Fuc(SO3 Na)-OMe, Fuc(SO3 Na) → [3 Fuc 1→]7 Fuc(SO3 Na) →
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Fuc(SO3 Na)-OMe and Fuc(SO3 Na) → Fuc(SO3 Na) → [3 Fuc 1→]7 → Fuc(SO3 Na)-OMe. From the above results, it was hypothesized that the sulfate in the middle position of the backbone was more sensitive to lose than that in the ends (both the reducing terminus and nonreducing terminus), which was confirmed by the results (data not shown) of methyl glycosides of tetra-sulfated fucooligosaccharides and penta-sulfated fucooligosaccharides, which had similar results with the trisulfated fucooligosaccharides. In other words, it was more likely to identify and desulfate the sulfate at C-2, and then randomly degraded into oligosaccharides and desulfated at C-4 because the sulfate at C-2 was liable to lose when compared with the sulfate at C-4. Though many structural features of methyl glycosides of sulfated fucooligosaccharides and fucooligosaccharides were elucidated by ESI-MS and ESI-MS/MS, the information on the structural characteristics of glucuronic acid, galactose and mannose was not shown in the ESI-MS spectra. It might be arised from that the molecular weight of heteropolysaccharides, which contained glucuronic acid, galactose and mannose was too large to determine by ES-MS and the heteropolysaccharides were not soluble in 50% acetonitrile and water. Thus, LF-1 and LF-5 were determined by NMR. The yield of LF-1 is about 26.11%. Chemical analysis has already revealed that it was primarily composed of fucose with minor sulfate (The molar ration of fucose to sulfate was approximately 10.61) and other monosaccharides. And the molecular weight was about 1285 Da. For this above reason, it was possible to apply 2D spectroscopy for the assignment of resonances based on the both 1 H–NMR and DEPTQ (13 C) NMR spectra. And 1 H, 1 H-COSY, TOCSY, HMBC and HSQC (Fig. 5) demonstrated the presence of Fuc, Man, Glc, Gal and Xyl, which was consistent with chemical analysis (The molar ratio of Man, Glc, Gal and Xyl to Fuc was 0.02, 0.02, 0.03 and 0.02, respectively). Therefore, it was difficult to discuss the absolute structural characteristics of other monosaccharides except Fuc. The reducing terminal residues were observed as 3-linked methyl ␣ ()-fucopyranosides A␣ and A or 3-linked methyl sulfated ␣ ()-fucopyranosides R-4S␣ and R-4S, which was consistent with the MS results. The methyl group was formed with methanol under the solvolytic desulfation conditions, which was reported by Toida et al. [39] In addition, it was also confirmed by the positions of correlation peak H-OMe A␣ /C-1 A␣ , H-OMe A /C-1A , H-OMe R-G4S␣ /C-1R-G4S␣ , H-OMe R-G4S /C-1R-G4S in HMBC spectrum at 3.26/99.84, 3.42/103.93, 3.25/99.84 and 3.40/103.73 ppm, respectively. The position of correlation peaks H-1B /H-2B /H-3B /H-4B and H-5B /H-6B in TOCSY spectrum at 4.96/3.81/3.89/3.91 and 4.18/1.09 ppm, H-4B /H-5B in 1 H, 1 H-COSY spectrum at 3.91/4.18 ppm, H-1B /C-3B and H-3B /C-1B in HMBC spectrum at 4.96/75.37 and 3.89/95.91 ppm, H-1B /C-1B , H-2B /C-2B , H-3B /C-3B , H-4B /C-4B , H-5B /C-5B and H-6B /C-6B in HSQC spectrum at 4.96/95.93, 3.81/66.72, 3.89/75.37, 3.91/68.84, 4.18/66.93 and 1.09/15.77 ppm, respectively, confirmed the presence of 3-linked ␣-l-fucopyranose (residue B), which was also the main composition of LF-1. And the presence of branching signals was proven by the position of correlation peaks H-1C /C-1C , H-2C /C-2C , H-3C /C3C and H-4C /C-4C in HSQC spectrum at 5.23/92.15, 3.98/68.00, 4.08/72.26 and 3.98/68.00 ppm, respectively, which are typical of 2, 3-disubstituted ␣-l-fucose units in a (1 → 3)-linked fucan chain [40]. Besides, HMBC spectrum contained correlation peaks H-1D /C-3C , H-1E /C-2C , C-1E /H-2C and C-1C /H-3B at 5.05/72.26, 5.02/68.00, 95.74/3.98 and 92.15/3.89, suggesting the presence of 2, 3-disubstituted ␣-fucose. Moreover, the presence of residue E was further confirmed that the fucan had branches at position 2 in the form of ␣-l-fucopyranose residues. Finally, the non-reducing end residues of the backbone were identified as ␣-l-fucopyranose residues F and some N-4S, which was also not desulfated. Based on this result and those detailed above, it was concluded that LF-1
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Fig. 5. HSQC spectrum of LF-1.
Scheme 1. Structural features of LF-1.
was mainly consisting of (1 → 3)-linked ␣-l-fucan with minor branches (␣-l-fucose) at position 2 and its structural fragments were displayed in Scheme 1 and Table 2. The molar ratio of GlcA, Man, Gal and Xyl to Fuc was 1.65, 0.81, 0.52 and 0.56, respectively, suggesting that LF-5 had more complex constituents. And it was also confirmed by the presence of signals of monosaccharides in 1 H-NMR and DEPTQ (13 C) NMR spectra. The anomeric region of HSQC (Fig. 6) spectrum
mainly contained two signals belonging to Fuc residues, whose chemical shifts ranged from 4.90 to 5.00 ppm. The characteristic features of LF-5 were showed in Scheme 2 and Table 2. One main residue was M and another residue was N. The position of correlation peaks H-1M /C-3L and C-1M /H-3L in HMBC spectrum at 4.96/73.65 and 95.25/3.77 confirmed the correlations between anomeric protons of Man and Fuc. In addition, the chemical shifts of C-2, C-3 and C-4 of Fuc residues were at 68.51, 69.69 and
Table 2 13 C and 1 H NMR data for LF-1and LF-5. Chemical shifts (ppm)
Residue
A␣ A R-4S␣ R-4S B C D N-4S E F G H L M N O P Q R S T U V W
3)-␣ -l-Fucp-OMe 3)--l-Fucp-OMe →3)-␣-l-Fuc(4SO3 − )-OMe →3)--l-Fuc(4SO3 − )-OMe →3)-␣-l-Fuc-(1→ →2,3)-␣-l-Fuc-(1→ →3)-␣-l-Fuc-(1→ ␣-l-Fuc(4SO3 − )-(1→ ␣-l-Fuc-(1→ ␣-l-Fuc-(1→ →2)-␣-d-Manp-(1→ →4)--d-GlcAp-(1→ →2,3)-␣-d-Manp-(1→ ␣-l-Fucp-(1→ ␣-l-Fucp(4SO3 − )-(1→ →3)-␣-l-Fucp(4SO3 − )-(1→ →3)-␣-l-Fucp-(1→ →3)--d-GlcAp-(1→ →3,4)--d-GlcAp-(1→ ␣-l-Fucp-(1→ →6)- -d-Galp-(1→ →4,6)--d-Galp-(1→ -d-Galp-(1→ →4)- -d-Xylp-(1→
H-1/C-1
H-2/C-2
H-3/C-3
H-4/C-4
H-5/C-5
H-6/C-6
OMe
4.64/99.84 4.18/103.93 4.87/98.84 4.49/104.36 4.96/95.93 5.23/92.15 5.05/95.63 4.92/96.03 5.02/95.74 4.87/96.21 5.27/98.75 4.33/101.76 5.27/98.75 4.96/95.25 4.98/94.93 4.90/96.43 4.93/96.16 4.72/102.58 4.72/102.58 4.85/97.61 4.29/103.67 4.29/103.67 4.53/104.00 4.33/101.76
3.80/66.72 3.40/70.92 3.90/68.09 3.56/77.98 3.81/66.72 3.98/68.00 3.84/66.47 3.66/68.38 3.68/68.38 3.65/68.38 4.02/77.80 3.27/73.06 4.22/73.06 3.68/68.51 3.68/68.51 3.68/68.51 3.81/67.05 3.51/73.51 3.51/73.51 3.77/68.14 3.27/nd 3.31/nd 3.27/nd 3.17/73.06
3.80/75.29 3.56/78.00 4.18/nd 3.84/nd 3.89/75.37 4.08/72.26 3.80/75.29 3.81/69.83 3.62/70.14 3.71/70.16 3.68/69.92 3.51/76.43 3.77/73.65 3.81/69.69 3.95/69.69 4.02/77.80 3.91/nd 3.68/83.63 3.68/76.43 3.81/69.69 3.51/73.10 3.63/73.10 3.51/73.10 3.51/74.61
3.91/68.84 3.84/68.00 4.61/nd 4.18/nd 3.91/68.84 3.98/68.00 3.91/68.84 4.64/79.60 3.66/72.26 3.68/72.26 3.58/66.37 3.63/77.80 3.81/69.69 3.68/72.15 4.49/80.90 4.49/80.90 3.95/nd 3.51/70.60 3.81/81.7 3.68/72.15 3.77/69.69 3.91/78.50 3.77/69.69 3.63/76.71
3.89/66.53 3.66/70.30 nd/nd nd/nd 4.18/66.93 4.18/66.93 4.03/67.27 4.18/66.72 4.03/67.27 4.03/67.27 3.64/76.71 3.68/76.71 3.68/76.43 4.10/67.05 4.29/66.70 4.22/69.05 4.29/66.70 3.81/75.61 3.51/74.61 4.33/67.05 3.68/74.10 3.68/74.10 3.51/73.10 nd/nd
1.10/15.69 1.13/15.89 1.13/15.89 1.14/15.95 1.09/15.77 1.09/15.77 1.10/15.69 1.09/15.77 1.14/15.95 1.06/16.06 3.68/60.49 175.96 3.68/60.49 1.07/15.76 1.12/15.76 1.12/16.26 1.12/16.26 175.46 175.46 1.07/15.76 3.68,3.77/69.95 3.68,3.77/69.95 3.68/60.95
3.26/55.46 3.42/57.49 3.25/55.46 3.40/57.49
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703
Fig. 6. HSQC spectrum of LF-5.
Scheme 2. The structural features of LF-5.
72.15 ppm, respectively, from HSQC, 1 H, 1 H-COSY and TOCSY spectra, indicating the absence of any substituents. Therefore, the Fuc residues were present only as non-reducing terminal monosaccharides, which had a (1 → 3)-linkage with Man residues. Due to the incomplete cleavage of sulfate, there were still some sulfates substituted at Fuc residue. And the proportion of N was similar with that of M. The signal of H-4/C-4 in HSQC spectrum at 4.49/80.90 proved the presence of sulfate group at C-4, and the chemical shifts of H-3N /C-3N and H-3O /C-3O of Fuc residues were at 3.95/69.69 and 4.02/77.80, indicating residue N was ␣l-Fuc(4SO3 − )-(1→ and residue O was →3)-␣-l-Fuc(4SO3 − )-(1→. Besides, there was also a less intense anomeric signal of →3)␣-l-Fuc-(1→ at 4.93 ppm. Analysis of HSQC, 1 H, 1 H-COSY, HMBC and TOCSY (data not shown) revealed the residue P. According to the previous study [40], the position of correlation peaks H-1G /C1G and H-1L /C-1L in HSQC spectrum at 5.27/98.75, indicating the presence of 2-linked Man (residue G) and 2,3-linked Man (residue L). And analysis of 2D spectra confirmed the above results. GlcA was the predominant monosaccharide in LF-5. Analysis of NMR spectra of LF-5 allowed to speculate three kinds of GlcA H, Q and R, differing in substitution pattern. The corresponding correlation peaks in the anomeric region of HSQC spectrum were at 4.33/101.76, 4.72/102.58 and 4.72/102.58 ppm, respectively. HMBC spectra showed the correlation peak H-1G /C-4H , C-1G /H4H , H-1H /C-2L and C-1H /H-2L at 5.27/77.80, 98.75/3.63, 4.33/73.06 and 101.76/4.22 ppm, respectively, indicating the (1 → 2)-linkage between ␣-d-Man and -d-GlcAp and (1 → 4)-linkage between -d-GlcAp and ␣-d-Man, which had been reported by the previous studies [35,38]. Moreover, the correlation peak H-1M /C-3L in HMBC at 4.96/73.65, suggesting that Man residue sometimes had a 3-linked Fuc branch, which was consistent with the previous study [41]. Because the interpretation of residues Q/R and W/H
were complicated by overlapping of the corresponding anomeric proton signals with anomeric resonance of GlcA and Glcp/Xyl, therefore, the assignments of the signals were based on the 2D spectra and previous study [40]. And (1 → 3)-glucuronan was also reported by the previous study [35]. Like residue M, residue S was a (1 → 4)-linked branch substituented on residue R. Finally, the (1 → 6)-linked galactan with (1 → 4)-linked Gal as a branch, was confirmed by the position of correlation peaks H-1T and U /C-1T and U in HSQC spectra at 4.29/103.67 ppm, while the branch was proved by the signals of correlation peak H-4U /C-4U in HSQC spectrum at 3.91/78.50 ppm. The non-reducing end residues were presented by -d-Galp residue V based on the anomeric carbon resonance in 2D spectra. 4. Conclusion F1 was desulfated by DMSO–MeOH. Compared with the ESIMS spectra of ds-F0.5 (data not shown) and ds-F2 [34], it was suggested that all fractions (F0.5, F1 and F2) contained a backbone of 3-linked ␣-l-Fuc. The major difference was the degree of sulfation. This phenomenon might be attributed to the alga’s needs, which was to protect and survive. Then, anion exchange chromatography was performed to fractionate the desulfated mixture into five fractions. LF-1 was mainly t a fucan slightly sulfated at C-4 and branched at C-2 by fucopranose residues. Along with increasing content of UA and decreasing content of Fuc, the fractions became more complicated. Thus LF-5 was characterized by NMR. It was shown that it contained not only sulfated fucan but also fucoglucuronomannan, fucoglucuronan, galactan and xylan, which F0.5 also contained. Finally, it was concluded that F1 was the middle component, which contained the information of both F0.5 and F2, indicating that the differences between F1 and F0.5,
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