Accepted Manuscript Structure of a sulfated xylofucan from the brown alga Punctaria plantaginea Maria I. Bilan, Alexander S. Shashkov, Anatolii I. Usov PII: DOI: Reference:
S0008-6215(14)00192-X http://dx.doi.org/10.1016/j.carres.2014.04.022 CAR 6739
To appear in:
Carbohydrate Research
Received Date: Revised Date: Accepted Date:
7 April 2014 29 April 2014 30 April 2014
Please cite this article as: Bilan, M.I., Shashkov, A.S., Usov, A.I., Structure of a sulfated xylofucan from the brown alga Punctaria plantaginea, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/j.carres.2014.04.022
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Structure of a sulfated xylofucan from the brown alga Punctaria plantaginea.*
Maria I. Bilan, Alexander S. Shashkov, Anatolii I. Usov
N.D.Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninskii prosp., 47, 119991 Moscow, Russian Federation
*
Polysaccharides of algae, Part 66. For Part 65, see Ref. 1. Corresponding author. Tel.: +7-499-137-6791; fax: +7-499-135-5328; e-mail: [email protected]
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Abstract – A polysaccharide composed of L-fucose, D-xylose and sulfate in a molar proportion of about 5:2:3 was isolated from the brown alga Punctaria plantaginea. Polysaccharide structure was elucidated by methylation analysis, Smith degradation, as well as by 1D and 2D NMR spectroscopy. The polysaccharide was shown to contain a backbone of 3-linked -L-fucopyranose residues, about two third of which are sulfated at O-2 forming trisaccharide repeating units →3)-L-Fucp2S-(13)--L-Fucp2S-(13)--L-Fucp-(1.
This structural regularity is masked by
random distribution of non-sulfated β-D-Xylp residues attached to positions 4 of the backbone. The polysaccharide is a new representative of a complex “fucoidan” family of sulfated polysaccharides of brown seaweeds.
Keywords: Fucoidan; Sulfated xylofucan; NMR spectroscopy; Brown algae; Punctaria plantaginea
2
1. Introduction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Sulfated polysaccharides of brown algae, usually named fucoidans, represent a heterogeneous group of biopolymers, which structures may vary considerably depending on the algal species.2-5 The simplest fucoidans obtained from several representatives of Laminariales and Fucales contain L-fucose, sulfate and acetate and are characterized by uneven distribution of branching points and non-carbohydrate substituents along the linear backbones built up of 3-linked or alternating 3- and 4-linked α-L-fucopyranose residues. More complex polysaccharides may additionally contain several other monosaccharides, such as galactose, mannose, xylose, and uronic acids. In particular, an unusual polysaccharide having a linear backbone of 3-linked α-Lfucopyranose residues with sulfate groups at O-4 and branches containing α-L-fucofuranose and αD-glucopyranosyluronic
acid residues at position 2 of the main chain was isolated from the Pacific
brown alga Chordaria flagelliformis, a representative of the family Chordariaceae (the order Ectocarpales).6 The present paper is devoted to the structural analysis of a sulfated polysaccharide containing fucose and xylose, which was isolated from the Pacific brown alga Punctaria plantaginea (Roth) Greville, belonging to the same family Chordariaceae. In our previous paper1 the algal biomass was shown to be rich in fucose-containing polysaccharides, and hence, the alga was regarded as a good practical source of fucoidan.
2. Results and discussion
2.1 Isolation of a sulfated xylofucan.
Water-soluble polysaccharides were isolated from the defatted algal biomass by extraction with dilute aqueous calcium chloride at 85C. The resulting extract was treated with
3
cetyltrimethylammonium bromide to precipitate the sulfated polysaccharide, which was then 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
transformed into a water-soluble sodium salt (preparation PPF) by a conventional procedure.7 The yield of PPF was 19.2% of defatted algal biomass, [α]D24 −128 (c 0.4; water), its percentage composition is given in Table 1. The molar ratio of the main components of PPF, L-Fuc : D-Xyl : SO3Na, was calculated as 49:21:30. The absolute configurations of monosaccharides were determined by GLC of acetylated S-(+)-sec-butyl glycosides.8 Several minor monosaccharides, such as galactose and uronic acids, were also detected in PPF, but their structural significance is obscure.
2.2. Preliminary characterization and desulfation of PPF.
Both 13C (Fig. 1a) and 1H (Fig. 2a) NMR spectra of PPF, similar to other non-regular polysaccharides, were rather complex, but they were resolved enough to apply 2D spectroscopy for the assignment of the most intense resonances. Analysis of COSY, TOCSY and HSQC spectra revealed the presence of 2,3-di-, 3,4-di- and 2,3,4-trisubstituted residues of α-fucopyranose together with unsubstituted non-reducing terminal β-xylopyranose. However, the absence of well-resolved and interpretable ROESY and HMBC spectra of the native polysaccharide did not allow determination of the type of linkages in the backbone and location of sulfate and xylose residues. Therefore, several chemical modifications of PPF were made for further structural analysis. The polysaccharide was desulfated by a solvolytic procedure7 to afford the desulfated preparation PPFdeS in a yield of 37.7% (Table 1), containing Fuc, Xyl and SO3Na in a molar ratio of 69 : 29 : 2. The native and desulfated polysaccharides were used for methylation analysis, Smith degradation and further investigation by NMR spectroscopy.
2.3. Methylation analysis.
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
PPF and PPFdeS were treated with methyl iodide in the presence of sodium hydroxide in dimethyl sulfoxide, the negatively charged PPF being first converted into the PyH+-salt to enhance its solubility in DMSO. The methylated polysaccharides were hydrolyzed, and the resulting mixtures of partially methylated monosaccharides were analyzed as alditol acetates by GLC-MS. It is evident from the results of methylation (Table 2) that PPFdeS is built up mainly of 3-linked and 3,4-linked fucopyranose and non-reducing terminal xylopyranose residues in a molar ratio of about 1:1:1. The methylation results of PPF differed from those of PPFdeS by the presence of derivatives of 2,3-di- and 2,3,4-tri-linked fucose instead of 3-linked fucose, evidently due to sulfation of both xylosylated and non-xylosylated fucose residues at position 2. A minor amount of sulfate groups may occupy also positions 4. This structural feature is probably responsible for some residual sulfate in PPFdeS, since similar retention of sulfate at C-4 under solvolytic desulfation conditions was observed previously for fucoidans from Fucus evanescens9 and Saccharina latissima.10
2.4. Periodate oxidation
According to the methylation analysis data, it was hypothesized that PPFdeS contains a linear backbone built up of 3-linked α-L-Fucp, about half of which are xylosylated at position 4. To verify this assumption, the Smith degradation procedure (periodate oxidation followed by borohydride reduction and partial acid hydrolysis) was applied to PPFdeS. A preparation PPFdeSOR obtained after oxidation and reduction of PPFdeS contained a negligible amount of xylose (Table 1). Its mild hydrolysis gave rise to a preparation PPFdeS-Sm, which was insoluble in water. According to NMR spectra (Fig 1c and Fig. 2c) registered in DMSO-d6 solution in the presence of 10% LiCl and to methylation analysis results (Table 2), PPFdeS-Sm was a linear (1→3)-α-L-
5
fucopyranan with an average chain length of about 17 residues, similar to a Smith-degraded 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
polysaccharide prepared from a fucoidan of the brown alga Analipus japonicus.11 The native polysaccharide PPF was also subjected to Smith degradation. The oxidizedreduced preparation PPF-OR contained only traces of xylose (Table 1). Its partial hydrolysis under standard conditions (1% AcOH, 100 ºC, 2.25 h) gave rise unexpectedly to a mixture of oligomeric fragments PPF-Sm1 containing a disulfated trisaccharide as the main component. According to the NMR spectral data, this trisaccharide had the structure α-L-Fucp2S-(1→3)-α-L-Fucp2S-(1→3)-α,βL-Fuc-OH.
Its formation may be explained by a specific influence of partial sulfation at positions 2
of (1→3)-linked fucan backbone on the direction of its degradation, stabilizing the glycosidic bonds of sulfated residues, but facilitating hydrolysis of the neighboring residues devoid of sulfate. A similar selective hydrolysis leading to oligosaccharides and directed by an appropriate distribution of sulfate groups has been reported previously for several other sulfated fucans.12,13 Smith degradation of PPF was repeated using milder hydrolysis conditions (AcOH, pH 2.9, 100 ºC, 30 min). The polymeric product of Smith degradation PPF-Sm2, [α]D24 −200 (c 1.0; water), was isolated by GPS and used for NMR spectroscopic analysis.
2.5. NMR spectroscopy.
Both 13C (Fig. 1b) and 1H (Fig. 2b) NMR spectra of desulfated polysaccharide PPFdeS were rather complex, but analysis of COSY, TOCSY, ROESY, HSQC (Fig. 3) and HMBC spectra showed that the polymer consists of only three types of residues, namely, 3-linked and 3,4-linked fucopyranose and terminal β-xylopyranose. Moreover, ROESY and HMBC spectra revealed exclusively (1→3)-linkages between α-L-Fucp and (1→4)-linkages between terminal β-D-Xylp and 3-linked α-L-Fucp residues. In addition, a small amount of residual sulfate at O-4 was observed by two correlations of very low intensity in HSQC spectrum (not visible in Fig. 3) at 4.55/80.8 (non-
6
reducing teminal Fucp4S) and 4.66/78.3 (3-linked Fucp4S). The complex character of the spectra, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
such as multiple signals of C-6 of -fucose (Fig. 1b), may be accounted for by uneven distribution of xylosyl substituents along the backbone and by a different long-range influence of xylosylated and non-xylosylated residues on the position of these signals. In accordance with this explanation, the 13C NMR spectrum of PPFdeS-Sm devoid of xylose consisted of only six sharp signals of 3linked α-L-Fucp (Fig. 1c). Smith degradation of the native polysaccharide PPF resulted in complete elimination of xylose residues and formation of disulfated trisaccharide PPF-Sm1 or sulfated fucan PPF-Sm2, depending on the conditions of mild acid hydrolysis. 1D 13C (Fig. 1d) and 1H (Fig. 2d) NMR spectra of PPF-Sm2 were assigned (Table 3) using 2D COSY, TOCSY, ROESY (Fig. 4), HSQC (Fig. 5) and HMBC (Fig. 6) experiments. The NMR data revealed a polymeric molecule with 3linked L-Fucp at the reducing end, 2-sulfated α-L-Fucp at the non-reducing end, and a regular chain built up of trisaccharide repeating units of the following structure: F-nr
→
[
A
→
→
B
C
]n
→
F-r
α-L-Fucp2S-(1→[3)-α-L-Fucp2S-(1→3)-α-L-Fucp-(1→3)-α-L-Fucp2S-(1→]n3)-α,β-L-Fuc-OH,
where n was about 5 according to the relative integrated intensities of 1H and 13C signals belonging to the terminal (F-nr, F-r) and inner (A, B, C) residues (Fig. 1d, 2d, 5 and Table 3). The regular →(A→B→C)n→ sequence for the inner part of PPF-Sm2 was proved by the ROESY spectrum (Fig. 4), where distinct inter-residue correlation peaks 1A/3,4B, 1B/3,4C and 1C/3,4A were observed. The 1→3 linkage between residues was confirmed by the HMBC spectrum (Fig. 6) containing inter-residue correlation peaks 1A/3B, 1B/3C and 1C/3A (here Arabic numerals before slash denote protons and those after slash denote carbon atoms in the residues A, B and C).
2.6. IR spectroscopy and conformation of heavily substituted Fucp.
7
IR spectrum of PPF (Fig. 7a) contained two intense absorption bands of sulfate groups at 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
1259 and 847 cm–1. The latter signal might be regarded as an indication on the axial position of sulfate,14 but this conclusion is controversial to the evidence on the preferential sulfation of Fucp at position 2 given above. An unusual position of this band may alternatively be explained by conformational distortion of 3-linked 2-sulfated fucose residues xylosylated at axial position 4, rendering the formally equatorial sulfate to adopt a pseudo-axial position. This opinion was supported by IR spectrum of PPF-Sm2 devoid of xylose residues (Fig. 7b), where the corresponding sulfate absorption band was shifted to 837 cm–1. The notion of conformational distortion of fucose residues overcrowded by different substituents may also help to understand the multicomponent appearance of the Fuc C-6 signal in the 13C NMR spectrum of PPF (Fig. 1a), which is markedly more complex than that in the spectra of several other fucoidans.9,11,15 Xylosylation of the axial hydroxyl group at C-4 should result in conformational distortion of the fucosyl ring moving this heavy substituent into pseudo-equatorial position. As a result, some of Fuc C-6 signals in 13C NMR spectrum are shifted downfield to a position, which is more typical of rhamnose or quinovose residues16 having an equatorial substituent at C-4. A similar low-field shift of the C-6 signal of 3linked Fuc due to additional fucosylation of O-4 has been observed previously in the spectrum of a desulfated and deacetylated fucoidan from A. japonicus.11 Hence, according to these observations, IR-spectra should be used with great care for the determination of sulfate positions in heavily substituted fucoidans.
3. Conclusion
According to our preliminary data1, the Pacific brown alga Punctaria plantaginea has a peculiar polysaccharide composition. It contains a linear (1→6)-β-D-glucan as a storage polysaccharide (instead of traditional laminaran), an alginate with prevalence of guluronic acid
8
residues (M/G 0.5) and a sulfated polysaccharide composed of fucose, xylose and negligible 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
amounts of other monosaccharides. The structure of this new representative of the fucoidan family was elucidated in the present work. The polysaccharide was shown to have a linear backbone of 3linked α-L-fucopyranose residues, two third of which are sulfated at position 2 giving rise to trisaccharide repeating unit →3)-α-L-Fucp2S-(1→3)-α-L-Fucp-(1→3)-α-L-Fucp2S-(1→. This structural regularity is masked by random substitution of about one half of the fucose residues at position 4 by single non-sulfated β-D-xylopyranose residues. A minor amount of sulfate may be also placed at O-4 of the backbone. Brown algal fucoidans often contain some xylose, usually as a minor monosaccharide constituent. There is no reliable structural information about these minor xylosyl residues, since chemical and spectral methods usually reveal only some 4-linked β-D-Xylp without any evidence on their connections with other polysaccharide components.10,11,15 More appreciable amounts of xylose have been found in preparations from Ascophyllum nodosum,17 Spatoglossum schröederi,18 Padina gymnospora19 and Sargassum fusiforme.20 The sulfated xyloglucuronofucan known as ascophyllan is present in A. nodosum together with a sulfated fucan, and these two polysaccharides could be separated one from another.21,22 Ascophyllan is composed of fucose, xylose, glucuronic acid and sulfate in nearly equimolar amounts. It contains a backbone of glucuronic acid residues and side chains built up of fucose and xylose.23 The 3-O-β-D-Xylp-L-Fuc disaccharide was isolated from partial hydrolysis products of ascophyllan.24 Several interesting observations on the specific differences in biological activity of ascophyllan and fucan sulfate were made by comparison of these two polysaccharides isolated from the same alga.21,22,25-28 Evidently different biological action cannot be explained only by the presence of xylosyl residues in ascophyllan, since it has many other appreciable structural differences from the fucan sulfate. The biological role of xylosyl residues may probably be more clearly demonstrated by comparison of a xylofucan sulfate from Punctaria plantaginea with its Smith degradation product devoid of xylose.
9
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
4. Experimental 4.1. General methods. Quantitative determination of monosaccharides by gas-liquid chromatography of alditol acetates and turbidimetric determination of sulfate were carried out as described previously.9,11 Uronic acids were estimated colorimetrically with 3,5-dimethylphenol using sodium alginate as the standard.29 The absolute configurations of fucose and xylose were established by GLC analysis of the corresponding acetylated (S)-(+)-sec-butyl glycosides.8 Optical rotations was measured using a digital polarimeter PU-07 (Russia) for aqueous solutions of polysaccharides. GPC was carried out on a column (78 x 2.6 cm) containing TSK HW-40(S) Toyopearl gel (Toyo Soda Manufacturing, Japan), which was eluted with water at a rate of 1 mL/min, a differential refractometer (Knauer, Germany) being used as detector. The IR spectra of polysaccharides were recorded with a Bruker Alpha-T spectrometer in KBr pellets.
4.2. NMR spectroscopy. Samples were freeze-dried twice from 99.9 % D2O and dissolved in 99.96 % D2O. 1H and 13
C NMR spectra were recorded on Bruker Avance II 600 MHz spectrometer at 303 and/or 333
K. Spectra of PPFdeS-Sm were recorded for 2% solution of a polysaccharide in 99.95% DMSOd6 containing 10% LiCl at 333 K. Chemical shifts are reported with internal sodium 3(trimethylsilyl)propanoate-2,2,3,3-d4 (H 0.0 and C −1.6) as internal standard. The NMR spectra were assigned by 2D 1H, 1H COSY, TOCSY, ROESY, H-detected 1H,13C HSQC, HSQC-TOCSY and HMBC experiments, which were performed and analysed using standard Bruker software. Mixing time of 100 ms was used in both TOCSY and HSQC-TOCSY experiments. The ROESY spectrum was acquired with 150 ms duration of spin-lock time. The HMBC experiment was optimized for coupling constant JH,C 8 Hz.
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
4.3. Isolation of a xylofucan sulfate. A sample of the alga P. plantaginea was collected from the littoral of the Possjet Bay of the Sea of Japan in September 2006, dried in air and milled (particles about 0.25 mm). The algal biomass was treated subsequently with methanol and acetone in Soxhlet apparatus to remove lipids and colored matter and vacuum dried. 20 g of defatted material and 2% aqueous CaCl2 solution (5 200 mL) were mechanically stirred at 85 C for 2 h. The combined extracts were treated with 10% aqueous cetyltrimethylammonium bromide solution (100 mL). The precipitate was centrifuged, washed with water and solubilised in 200 mL of 4 M CaCl2 by moderate heating up to 65 ºC. Ethanol (600 mL) was added to this solution with vigorous stirring, the precipitate was centrifuged, dissolved in 2% aqueous CaCl2, dialyzed and centrifuged to discard a small amount of insoluble material. The solution was treated with NaBH4 (750 mg) overnight, then NaOH (12 g) and additional NaBH4 (3 g) were added, the mixture was heated at 80 ºC for 8 h, cooled, centrifuged, dialysed, concentrated and lyophilized to afford PPF (3.9 g).
4.3. Chemical modifications of polysaccharides. Solvolytic desulfation of PPF (as pyridinium salt) was carried out as described earlier,7,11 giving rise to desulfated polysaccharide PPFdeS with the yield of 37.7%. Methylation of PPF (as pyridinium salt), PPFdeS and PPFdeS-Sm followed by hydrolysis and GLC-MS of partially methylated alditol acetates was performed as previously described.11,30 Smith degradation of PPFdeS: an aqueous solution of NaIO4 (0.021M, 44 mL) was added to a solution of PPFdeS (108 mg, 0.226 mM of Xyl) in water (44 mL), and the mixture was left in the dark for 24 h at room temperature (consumption of the oxidant monitored by a decrease in the optical density of the solution at 305 nm was 0.378 mM). Ethylene glycol (0.5 mL) was added to the reaction mixture, which was then dialyzed and concentrated to about 10 mL, NaBH4 (150 mg)
11
was added, and the mixture was left overnight. The solution was neutralized with HOAc, dialyzed, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
and lyophilized to give oxidized and reduced polysaccharide PPFdeS-OR (94 mg, 87%). This preparation (90 mg) was dissolved in 1% HOAc (6 mL), the solution was heated for 2 h at 100°C, the mixture was cooled, centrifuged, the precipitate was washed with water, suspended in water and lyophilized to give PPFdeS-Sm (30 mg, 33.3%). Additional amount of PPFdeS-Sm of similar structure (more soluble fraction, MW >7 kDa, 20 mg) was isolated from the acid mother liquor by GPC. Smith degradation of PPF: an aqueous solution of NaIO4 (0.021M, 112 mL) was added to a solution of PPF (200 mg, 0.607 mM of Fuc, 0.259 mM of Xyl) in water (112 mL), and the mixture was left in the dark for 24 h at room temperature. Consumption of periodate was 0.63 mM. The mixture was treated as above to give rise to PPF-OR (165 mg, 82.5%). Partial hydrolysis of this material was carried out in 1% AcOH in a boiling water bath for 2 h 15 min. According to GPC data, the hydrolyzate contained two approximately equivalent fractions (about 50 mg each), one of them being essentially a disulfated trisaccharide PPF-Sm1, whereas another one was a mixture of the same trisaccharide with higher molecular weight fragments. The Smith degradation procedure was repeated using shorter time of partial hydrolysis (30 min at 100 ºC). The product was eluted from TSK-40 column in a void volume and lyophilized to give PPF-Sm2 (30 mg from 124 mg of PPF, 24.2 %).
Acknowledgments The authors are grateful to Prof. V. E. Vaskovsky (G.B.Elyakov Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Vladivostok, Russia) for collection of the alga and to Dr. D. N. Platonov (N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia) for GLC-MS analysis of methylation products.
12
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
1. Bilan, M. I.; Smirnova, G. P.; Shashkov, A. S.; Usov, A. I. Russ. Chem. Bull., Int. Ed. 2014, in press. 2. Berteau, O.; Mulloy, B. Glycobiology 2003, 13, 29R-40R. 3. Usov, A. I.; Bilan, M. I. Russ. Chem. Rev. 2009, 78, 785-799. 4. Wijesinghe, W. A. J. P.; Jeon, Y. J. Carbohydr. Polym. 2012, 88, 13-20. 5. Ale, M. T.; Meyer, A. S. RSC Adv. 2013, 3, 8131-8141. 6. Bilan, M. I.; Vinogradova, E. V.; Tsvetkova, E. A.; Grachev, A. A.; Shashkov, A. S.; Nifantiev, N. E.; Usov, A. I. Carbohydr. Res. 2008, 343, 2605-2612. 7. Usov, A.I.; Smirnova, G.P.; Bilan, M.I.; Shashkov, A.S. Bioorg. Khim. 1998, 24, 437-445 (Russ. J. Bioorg. Chem. 1998, 24, 382-389, English translation). 8. Gerwig, G.J.; Kamerling, J.P.; Vliegenthart, J.F.G. Carbohydr. Res. 1978, 62, 349-357. 9. Bilan, M.I.; Grachev, A.A.; Ustuzhanina, N.E.; Shashkov, A.S.; Nifantiev, N.E.; Usov, A.I. Carbohydr. Res. 2002, 337, 719-730. 10. Bilan, M. I.; Grachev, A. A.; Shashkov, A. S.; Kelly, M.; Sanderson, C. J.; Nifantiev, N. E.; Usov, A. I. Carbohydr. Res. 2010, 345, 2038-2047. 11. Bilan, M.I.; Zakharova, A.N.; Grachev, A.A.; Shashkov, A.S.; Nifantiev, N.E.; Usov, A.I. Bioorg. Khim. 2007, 33, 44-53 (Russ. J. Bioorg. Chem. 2007, 33, 38-46, English translation). 12. Pomin, V. H.; Pereira, M. S.; Valente, A.-P.; Tollefsen, D. M.; Pavão, M. S. G.; Mourão, P. A. S. Glycobiology 2005, 15, 369-381. 13. Pomin, V. H.; Valente, A. P.; Pereira, M. S.; Mourão, P. A. S. Glycobiology 2005, 15, 13761385. 14. Anderson, N. S.; Dolan, T. C. S.; Penman, A.; Rees, D. A.; Mueller, G. P.; Stancioff, D. J.; Stanley, N. F. J. Chem. Soc. C 1968, 602-606.
13
15. Bilan, M. I.; Grachev, A. A.; Shashkov, A. S.; Nifantiev, N. E.; Usov, A. I. Carbohydr. Res. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
2006, 341, 238-245. 16. Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. Biochem. 1983, 41, 27-66. 17. Foley, S. A.; Szegezdi, E.; Mulloy, B.; Samali, A.; Tuohy, M. G. J. Nat. Prod. 2011, 74, 1851-1861. 18. Leite, E. L.; Medeiros, M. G. L.; Rocha, H. A. O.; Farias, G. G. M.; da Silva, L. F.; Chavante, S. F.; de Abreu, L. D.; Dietrich, C. P.; Nader, H. B. Plant Sci. 1998, 132, 215228. 19. Silva, T. M. A.; Alves, L. G.; de Queiroz, K. C. S.; Santos, M. G. L.; Marques, C. T.; Chavante, S. F.; Rocha, H. A. O.; Leite, E. L. Brazilian J. Med. Biol. Res. 2005, 38, 523533. 20. Li, B.; Wei, X.-J.; Sun, J.-L.; Xu, S.-Y. Carbohydr. Res. 2006, 341, 1135-1146. 21. Nakayasu, S.; Soegima, R.; Yamaguchi, K.; Oda, T. Biosci. Biotechnol. Biochem. 2009, 73, 961-964. 22. Jiang, Z.; Okimura, T.; Yokose, T.; Yamasaki, Y.; Yamaguchi, K.; Oda, T. J. Biosci. Bioeng. 2010, 110, 113-117. 23. Larsen, B.; Haug, A.; Painter, T. J. Acta Chem. Scand. 1966, 20, 219-230. 24. Larsen, B. Acta Chem. Scand. 1967, 21, 1395-1396. 25. Jiang, Z.; Okimura, T.; Yamaguchi, K.; Oda, T. Nitric Oxide 2011, 25, 407-415. 26. Nakano, K.; Kim, D.; Jiang, Z.; Ueno, M.; Okimura, T.; Yamaguchi, K.; Oda, T. Biosci. Biotechnol. Biochem. 2012, 76, 1573-1576. 27. Abu, R.; Jiang, Z.; Ueno, M.; Okimura, T.; Yamaguchi, K.; Oda, T. Int. J. Biol. Macromol. 2013, 59, 305-312. 28. Jiang, Z.; Ueno, M.; Nishiguchi, T.; Abu, R.; Isaka, S.; Okimura, T.; Yamaguchi, K.; Oda, T. Carbohydr. Res. 2013, 380, 124-129.
14
29. Usov, A.I.; Bilan, M.I.; Klochkova, N.G. Bot. Mar. 1995, 38, 43-51. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
30. Chizhov, A.O.; Dell, A.; Morris, H.R.; Haslam, S.M.; McDowell, R.A.; Shashkov, A.S.; Nifant’ev, N.E.; Khatuntseva, E.A.; Usov, A.I. Carbohydr. Res. 1999, 320, 108-119.
15
Table 1. Composition of the native polysaccharide PPF and some of its modification products. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Fraction
Neutral monosaccharides, %
SO3Na,
Uronic
Fuc
Xyl
Man
Glc
Gal
%
acids, %
PPF
44.3
17.1
tr.
tr.
2.6
19.2
2.3
PPFdeS
63.7
24.1
1.3
2.3
4.3
1.1
n.d.
PPFdeS-OR
58.1
1.7
tr.
tr.
2.4
4.5
n.d.
PPF-OR
44.7
1.3
tr.
tr.
2.0
20.8
n.d.
tr. - amounts lesser than 1% n.d. – not determined
16
Table 2. Methylation analysis of the native sulfated xylofucan PPF, its desulfated preparation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
PPFdeS, and Smith-degraded polysaccharide PPFdeS-Sm (M % of partially methylated fucitol and xylitol acetates). Position of
Deduced positions
O-methyl
of substitution:
PPFdeS
PPF
PPFdeS-Sm
groups in: Fucitol 2,3,4
Fucp-(1→
2
tr.
5
2,3
→4)-Fucp-(1→
4
tr.
-
2,4
→3)-Fucp-(1→
33
3
91
2
→3,4)-Fucp-(1→
31
26
1
4
→2,3)-Fucp-(1→
-
24
3
Fuc
→2,3,4)-Fucp-(1→
1
18
-
Xylp-(1→
29
29
-
Xylitol 2,3,4
17
Table 3. 13C and 1H NMR data for Smith-degraded polysaccharide PPF-Sm2. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
F-nr
A
B
C
F-r
α-L-Fucp2S-(1→[3)-α-L-Fucp2S-(1→3)-α-L-Fucp-(1→3)-α-L-Fucp2S-(1→]n3)-α,β-L-Fuc-OH
Residue
F-nr α-L-Fucp2S-(1→3)-
A
B
C
C-1
C-2
H-1
H-2
H-3
H-4
H-5
H-6
96.18
76.50
68.59
73.39
68.26
16.50
5.34
4.46
4.12
3.91
4.22
1.24
74.37
75.72
70.76
67.74
16.67
5.42
4.59
4.17
4.12
4.33
1.27
97.88
67.74
76.50
69.64
67.98
16.50
5.09
3.98
4.01
4.08
4.42
1.27
74.27
74.37
70.64
67.98
16.77
5.36
4.57
4.16
4.11
4.48
1.28
97.47
71.28
80.02
69.23
71.89
16.88
4.61
3.62
3.70
4.00
3.82
1.28
→3)-α-L-Fucp2S-(1→3)- 95.06
→3)-α-L-Fucp-(1→3)-
→3)-α-L-Fucp2S-(1→3)- 95.86
F-r →3)-β-L-Fucp
Chemical shifts, ppm C-3 C-4 C-5
18
C-6
Captions for figures 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Fig. 1. 13C NMR spectra of polysaccharides: PPF (a), PPFdeS (b), PPFdeS-Sm (c), and PPFSm2 (d). Abbreviations of residues: F – fucose, X – xylose, F-x – xylosylated fucose, nr – nonreducing terminal, r – reducing terminal; A, B and C as in Table 3. Fig. 2. 1H NMR spectra of polysaccharides: PPF (a), PPFdeS (b), PPFdeS-Sm (c), and PPFSm2 (d). Fig. 3. 2D HSQC spectrum of PPFdeS (abbreviations as in Fig. 1). Fig. 4. A part of 2D ROESY spectrum of PPF-Sm2 (abbreviations as in Fig. 1). Fig. 5. 2D HSQC spectrum of PPF-Sm2 (abbreviations as in Fig. 1). Fig. 6. A part of 2D HMBC spectrum of PPF-Sm2 (abbreviations as in Table 3). Fig. 7. IR spectra of PPF (a) and PPF-Sm2 (b).
19
6F 2A 3,2C 2F-nr
1F-rb 1A 1B 1C
d
4A,C 4B
3A
1F-nr
3,5F-nr
4F-rb
4F-nr
3F-rb
1F-ra
5B,C
2B 5A
6F 3F
1F
4F 2F 5F
c 4X 3X
6F
2X 2,5F,F-x
1X
5X
4F 1F 4F-x-nr
b
3F-x 4F-x 3F
4X
3X
6F 5X
2X 1X 1F
a 105
100
95
90
85
80
75
70
ppm
20
18
16
ppm
d
c
b
a
ppm
ppm á-OMe â-OMe
60 5X
5X
5F
2F,F-õ
5F-õ
4X
65
1.4 75 3F,F-õ
3X
80
4F-x
85 90 1F-x
95 100
1F 1X
5.0
4.8
4.6
105 4.4
4.2
6F,F-x
4.0
3.8
3.6
18
70
4F 2X
5.2
16
3.4
ppm
1.2
ppm
ppm
5.00 1B/3C 1B/4C
1,2B
5.05 5.10 5.15 5.20 5.25
1,2C
1,2F-nr
5.30
1C/3A 1C/4A
5.35 5.40 1A/4B
1A/3B
5.45
1,2A
5.50
4.7
4.6
4.5
4.4
4.3
4.2
4.1
4.0
5.55 3.9 ppm
ppm
ppm
65 5C 5B
5A
5F-nr 3F-nr 4B
2B 4F-rb 5 F-rb
4A 4C 2A 2C
14 2 F-rb70
4F-nr
3C
18 75
2F-nr
1.5
3A 3B 3 F-rb
80
85
90 1A
95
1F-nr
1F-rb
1B
1C
5.4
5.2
5.0
4.8
4.6
4.4
16
4.2
4.0
3.8
ppm
ppm
ppm
68 1A/5A
1C/5C
1B/5B
69 70 71 72 73
1A/2A
1B/3C
1C/3C
74 75
1A/3A
76 1C/3A
77 1A/3B
5.45
1B/3B
5.40
5.35
5.30
5.25
5.20
5.15
5.10
78 ppm
(a)
(b) (b)
Structure of a sulfated xylofucan from the brown alga Punctaria plantaginea Maria I. Bilan, Alexander S. Shashkov, Anatolii I. Usov
R ↓ 4
R ↓ 4
R ↓ 4
[→3)-α-L-Fucp-(1→3)-α-L-Fucp-(1→3)-α-L-Fucp-(1→]n 2 ↑
SO3Na
2 ↑
SO3Na
where R is β-D-Xylp or H
A sulfated xylofucan was isolated from the brown alga Punctaria plantaginea.
Polysaccharide structure was elucidated by chemical and spectral methods.
The polysaccharide backbone is built up of 3-linked α-L-Fucp.
Two of every three Fuc are sulfated at O-2 forming trisaccharide repeating units.
The backbone regularity is masked by random substitution by β-D-Xylp at Fuc O-4.
S0008-6215(14)00192-X http://dx.doi.org/10.1016/j.carres.2014.04.022 CAR 6739
To appear in:
Carbohydrate Research
Received Date: Revised Date: Accepted Date:
7 April 2014 29 April 2014 30 April 2014
Please cite this article as: Bilan, M.I., Shashkov, A.S., Usov, A.I., Structure of a sulfated xylofucan from the brown alga Punctaria plantaginea, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/j.carres.2014.04.022
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Structure of a sulfated xylofucan from the brown alga Punctaria plantaginea.*
Maria I. Bilan, Alexander S. Shashkov, Anatolii I. Usov
N.D.Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninskii prosp., 47, 119991 Moscow, Russian Federation
*
Polysaccharides of algae, Part 66. For Part 65, see Ref. 1. Corresponding author. Tel.: +7-499-137-6791; fax: +7-499-135-5328; e-mail: [email protected]
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Abstract – A polysaccharide composed of L-fucose, D-xylose and sulfate in a molar proportion of about 5:2:3 was isolated from the brown alga Punctaria plantaginea. Polysaccharide structure was elucidated by methylation analysis, Smith degradation, as well as by 1D and 2D NMR spectroscopy. The polysaccharide was shown to contain a backbone of 3-linked -L-fucopyranose residues, about two third of which are sulfated at O-2 forming trisaccharide repeating units →3)-L-Fucp2S-(13)--L-Fucp2S-(13)--L-Fucp-(1.
This structural regularity is masked by
random distribution of non-sulfated β-D-Xylp residues attached to positions 4 of the backbone. The polysaccharide is a new representative of a complex “fucoidan” family of sulfated polysaccharides of brown seaweeds.
Keywords: Fucoidan; Sulfated xylofucan; NMR spectroscopy; Brown algae; Punctaria plantaginea
2
1. Introduction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Sulfated polysaccharides of brown algae, usually named fucoidans, represent a heterogeneous group of biopolymers, which structures may vary considerably depending on the algal species.2-5 The simplest fucoidans obtained from several representatives of Laminariales and Fucales contain L-fucose, sulfate and acetate and are characterized by uneven distribution of branching points and non-carbohydrate substituents along the linear backbones built up of 3-linked or alternating 3- and 4-linked α-L-fucopyranose residues. More complex polysaccharides may additionally contain several other monosaccharides, such as galactose, mannose, xylose, and uronic acids. In particular, an unusual polysaccharide having a linear backbone of 3-linked α-Lfucopyranose residues with sulfate groups at O-4 and branches containing α-L-fucofuranose and αD-glucopyranosyluronic
acid residues at position 2 of the main chain was isolated from the Pacific
brown alga Chordaria flagelliformis, a representative of the family Chordariaceae (the order Ectocarpales).6 The present paper is devoted to the structural analysis of a sulfated polysaccharide containing fucose and xylose, which was isolated from the Pacific brown alga Punctaria plantaginea (Roth) Greville, belonging to the same family Chordariaceae. In our previous paper1 the algal biomass was shown to be rich in fucose-containing polysaccharides, and hence, the alga was regarded as a good practical source of fucoidan.
2. Results and discussion
2.1 Isolation of a sulfated xylofucan.
Water-soluble polysaccharides were isolated from the defatted algal biomass by extraction with dilute aqueous calcium chloride at 85C. The resulting extract was treated with
3
cetyltrimethylammonium bromide to precipitate the sulfated polysaccharide, which was then 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
transformed into a water-soluble sodium salt (preparation PPF) by a conventional procedure.7 The yield of PPF was 19.2% of defatted algal biomass, [α]D24 −128 (c 0.4; water), its percentage composition is given in Table 1. The molar ratio of the main components of PPF, L-Fuc : D-Xyl : SO3Na, was calculated as 49:21:30. The absolute configurations of monosaccharides were determined by GLC of acetylated S-(+)-sec-butyl glycosides.8 Several minor monosaccharides, such as galactose and uronic acids, were also detected in PPF, but their structural significance is obscure.
2.2. Preliminary characterization and desulfation of PPF.
Both 13C (Fig. 1a) and 1H (Fig. 2a) NMR spectra of PPF, similar to other non-regular polysaccharides, were rather complex, but they were resolved enough to apply 2D spectroscopy for the assignment of the most intense resonances. Analysis of COSY, TOCSY and HSQC spectra revealed the presence of 2,3-di-, 3,4-di- and 2,3,4-trisubstituted residues of α-fucopyranose together with unsubstituted non-reducing terminal β-xylopyranose. However, the absence of well-resolved and interpretable ROESY and HMBC spectra of the native polysaccharide did not allow determination of the type of linkages in the backbone and location of sulfate and xylose residues. Therefore, several chemical modifications of PPF were made for further structural analysis. The polysaccharide was desulfated by a solvolytic procedure7 to afford the desulfated preparation PPFdeS in a yield of 37.7% (Table 1), containing Fuc, Xyl and SO3Na in a molar ratio of 69 : 29 : 2. The native and desulfated polysaccharides were used for methylation analysis, Smith degradation and further investigation by NMR spectroscopy.
2.3. Methylation analysis.
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
PPF and PPFdeS were treated with methyl iodide in the presence of sodium hydroxide in dimethyl sulfoxide, the negatively charged PPF being first converted into the PyH+-salt to enhance its solubility in DMSO. The methylated polysaccharides were hydrolyzed, and the resulting mixtures of partially methylated monosaccharides were analyzed as alditol acetates by GLC-MS. It is evident from the results of methylation (Table 2) that PPFdeS is built up mainly of 3-linked and 3,4-linked fucopyranose and non-reducing terminal xylopyranose residues in a molar ratio of about 1:1:1. The methylation results of PPF differed from those of PPFdeS by the presence of derivatives of 2,3-di- and 2,3,4-tri-linked fucose instead of 3-linked fucose, evidently due to sulfation of both xylosylated and non-xylosylated fucose residues at position 2. A minor amount of sulfate groups may occupy also positions 4. This structural feature is probably responsible for some residual sulfate in PPFdeS, since similar retention of sulfate at C-4 under solvolytic desulfation conditions was observed previously for fucoidans from Fucus evanescens9 and Saccharina latissima.10
2.4. Periodate oxidation
According to the methylation analysis data, it was hypothesized that PPFdeS contains a linear backbone built up of 3-linked α-L-Fucp, about half of which are xylosylated at position 4. To verify this assumption, the Smith degradation procedure (periodate oxidation followed by borohydride reduction and partial acid hydrolysis) was applied to PPFdeS. A preparation PPFdeSOR obtained after oxidation and reduction of PPFdeS contained a negligible amount of xylose (Table 1). Its mild hydrolysis gave rise to a preparation PPFdeS-Sm, which was insoluble in water. According to NMR spectra (Fig 1c and Fig. 2c) registered in DMSO-d6 solution in the presence of 10% LiCl and to methylation analysis results (Table 2), PPFdeS-Sm was a linear (1→3)-α-L-
5
fucopyranan with an average chain length of about 17 residues, similar to a Smith-degraded 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
polysaccharide prepared from a fucoidan of the brown alga Analipus japonicus.11 The native polysaccharide PPF was also subjected to Smith degradation. The oxidizedreduced preparation PPF-OR contained only traces of xylose (Table 1). Its partial hydrolysis under standard conditions (1% AcOH, 100 ºC, 2.25 h) gave rise unexpectedly to a mixture of oligomeric fragments PPF-Sm1 containing a disulfated trisaccharide as the main component. According to the NMR spectral data, this trisaccharide had the structure α-L-Fucp2S-(1→3)-α-L-Fucp2S-(1→3)-α,βL-Fuc-OH.
Its formation may be explained by a specific influence of partial sulfation at positions 2
of (1→3)-linked fucan backbone on the direction of its degradation, stabilizing the glycosidic bonds of sulfated residues, but facilitating hydrolysis of the neighboring residues devoid of sulfate. A similar selective hydrolysis leading to oligosaccharides and directed by an appropriate distribution of sulfate groups has been reported previously for several other sulfated fucans.12,13 Smith degradation of PPF was repeated using milder hydrolysis conditions (AcOH, pH 2.9, 100 ºC, 30 min). The polymeric product of Smith degradation PPF-Sm2, [α]D24 −200 (c 1.0; water), was isolated by GPS and used for NMR spectroscopic analysis.
2.5. NMR spectroscopy.
Both 13C (Fig. 1b) and 1H (Fig. 2b) NMR spectra of desulfated polysaccharide PPFdeS were rather complex, but analysis of COSY, TOCSY, ROESY, HSQC (Fig. 3) and HMBC spectra showed that the polymer consists of only three types of residues, namely, 3-linked and 3,4-linked fucopyranose and terminal β-xylopyranose. Moreover, ROESY and HMBC spectra revealed exclusively (1→3)-linkages between α-L-Fucp and (1→4)-linkages between terminal β-D-Xylp and 3-linked α-L-Fucp residues. In addition, a small amount of residual sulfate at O-4 was observed by two correlations of very low intensity in HSQC spectrum (not visible in Fig. 3) at 4.55/80.8 (non-
6
reducing teminal Fucp4S) and 4.66/78.3 (3-linked Fucp4S). The complex character of the spectra, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
such as multiple signals of C-6 of -fucose (Fig. 1b), may be accounted for by uneven distribution of xylosyl substituents along the backbone and by a different long-range influence of xylosylated and non-xylosylated residues on the position of these signals. In accordance with this explanation, the 13C NMR spectrum of PPFdeS-Sm devoid of xylose consisted of only six sharp signals of 3linked α-L-Fucp (Fig. 1c). Smith degradation of the native polysaccharide PPF resulted in complete elimination of xylose residues and formation of disulfated trisaccharide PPF-Sm1 or sulfated fucan PPF-Sm2, depending on the conditions of mild acid hydrolysis. 1D 13C (Fig. 1d) and 1H (Fig. 2d) NMR spectra of PPF-Sm2 were assigned (Table 3) using 2D COSY, TOCSY, ROESY (Fig. 4), HSQC (Fig. 5) and HMBC (Fig. 6) experiments. The NMR data revealed a polymeric molecule with 3linked L-Fucp at the reducing end, 2-sulfated α-L-Fucp at the non-reducing end, and a regular chain built up of trisaccharide repeating units of the following structure: F-nr
→
[
A
→
→
B
C
]n
→
F-r
α-L-Fucp2S-(1→[3)-α-L-Fucp2S-(1→3)-α-L-Fucp-(1→3)-α-L-Fucp2S-(1→]n3)-α,β-L-Fuc-OH,
where n was about 5 according to the relative integrated intensities of 1H and 13C signals belonging to the terminal (F-nr, F-r) and inner (A, B, C) residues (Fig. 1d, 2d, 5 and Table 3). The regular →(A→B→C)n→ sequence for the inner part of PPF-Sm2 was proved by the ROESY spectrum (Fig. 4), where distinct inter-residue correlation peaks 1A/3,4B, 1B/3,4C and 1C/3,4A were observed. The 1→3 linkage between residues was confirmed by the HMBC spectrum (Fig. 6) containing inter-residue correlation peaks 1A/3B, 1B/3C and 1C/3A (here Arabic numerals before slash denote protons and those after slash denote carbon atoms in the residues A, B and C).
2.6. IR spectroscopy and conformation of heavily substituted Fucp.
7
IR spectrum of PPF (Fig. 7a) contained two intense absorption bands of sulfate groups at 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
1259 and 847 cm–1. The latter signal might be regarded as an indication on the axial position of sulfate,14 but this conclusion is controversial to the evidence on the preferential sulfation of Fucp at position 2 given above. An unusual position of this band may alternatively be explained by conformational distortion of 3-linked 2-sulfated fucose residues xylosylated at axial position 4, rendering the formally equatorial sulfate to adopt a pseudo-axial position. This opinion was supported by IR spectrum of PPF-Sm2 devoid of xylose residues (Fig. 7b), where the corresponding sulfate absorption band was shifted to 837 cm–1. The notion of conformational distortion of fucose residues overcrowded by different substituents may also help to understand the multicomponent appearance of the Fuc C-6 signal in the 13C NMR spectrum of PPF (Fig. 1a), which is markedly more complex than that in the spectra of several other fucoidans.9,11,15 Xylosylation of the axial hydroxyl group at C-4 should result in conformational distortion of the fucosyl ring moving this heavy substituent into pseudo-equatorial position. As a result, some of Fuc C-6 signals in 13C NMR spectrum are shifted downfield to a position, which is more typical of rhamnose or quinovose residues16 having an equatorial substituent at C-4. A similar low-field shift of the C-6 signal of 3linked Fuc due to additional fucosylation of O-4 has been observed previously in the spectrum of a desulfated and deacetylated fucoidan from A. japonicus.11 Hence, according to these observations, IR-spectra should be used with great care for the determination of sulfate positions in heavily substituted fucoidans.
3. Conclusion
According to our preliminary data1, the Pacific brown alga Punctaria plantaginea has a peculiar polysaccharide composition. It contains a linear (1→6)-β-D-glucan as a storage polysaccharide (instead of traditional laminaran), an alginate with prevalence of guluronic acid
8
residues (M/G 0.5) and a sulfated polysaccharide composed of fucose, xylose and negligible 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
amounts of other monosaccharides. The structure of this new representative of the fucoidan family was elucidated in the present work. The polysaccharide was shown to have a linear backbone of 3linked α-L-fucopyranose residues, two third of which are sulfated at position 2 giving rise to trisaccharide repeating unit →3)-α-L-Fucp2S-(1→3)-α-L-Fucp-(1→3)-α-L-Fucp2S-(1→. This structural regularity is masked by random substitution of about one half of the fucose residues at position 4 by single non-sulfated β-D-xylopyranose residues. A minor amount of sulfate may be also placed at O-4 of the backbone. Brown algal fucoidans often contain some xylose, usually as a minor monosaccharide constituent. There is no reliable structural information about these minor xylosyl residues, since chemical and spectral methods usually reveal only some 4-linked β-D-Xylp without any evidence on their connections with other polysaccharide components.10,11,15 More appreciable amounts of xylose have been found in preparations from Ascophyllum nodosum,17 Spatoglossum schröederi,18 Padina gymnospora19 and Sargassum fusiforme.20 The sulfated xyloglucuronofucan known as ascophyllan is present in A. nodosum together with a sulfated fucan, and these two polysaccharides could be separated one from another.21,22 Ascophyllan is composed of fucose, xylose, glucuronic acid and sulfate in nearly equimolar amounts. It contains a backbone of glucuronic acid residues and side chains built up of fucose and xylose.23 The 3-O-β-D-Xylp-L-Fuc disaccharide was isolated from partial hydrolysis products of ascophyllan.24 Several interesting observations on the specific differences in biological activity of ascophyllan and fucan sulfate were made by comparison of these two polysaccharides isolated from the same alga.21,22,25-28 Evidently different biological action cannot be explained only by the presence of xylosyl residues in ascophyllan, since it has many other appreciable structural differences from the fucan sulfate. The biological role of xylosyl residues may probably be more clearly demonstrated by comparison of a xylofucan sulfate from Punctaria plantaginea with its Smith degradation product devoid of xylose.
9
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
4. Experimental 4.1. General methods. Quantitative determination of monosaccharides by gas-liquid chromatography of alditol acetates and turbidimetric determination of sulfate were carried out as described previously.9,11 Uronic acids were estimated colorimetrically with 3,5-dimethylphenol using sodium alginate as the standard.29 The absolute configurations of fucose and xylose were established by GLC analysis of the corresponding acetylated (S)-(+)-sec-butyl glycosides.8 Optical rotations was measured using a digital polarimeter PU-07 (Russia) for aqueous solutions of polysaccharides. GPC was carried out on a column (78 x 2.6 cm) containing TSK HW-40(S) Toyopearl gel (Toyo Soda Manufacturing, Japan), which was eluted with water at a rate of 1 mL/min, a differential refractometer (Knauer, Germany) being used as detector. The IR spectra of polysaccharides were recorded with a Bruker Alpha-T spectrometer in KBr pellets.
4.2. NMR spectroscopy. Samples were freeze-dried twice from 99.9 % D2O and dissolved in 99.96 % D2O. 1H and 13
C NMR spectra were recorded on Bruker Avance II 600 MHz spectrometer at 303 and/or 333
K. Spectra of PPFdeS-Sm were recorded for 2% solution of a polysaccharide in 99.95% DMSOd6 containing 10% LiCl at 333 K. Chemical shifts are reported with internal sodium 3(trimethylsilyl)propanoate-2,2,3,3-d4 (H 0.0 and C −1.6) as internal standard. The NMR spectra were assigned by 2D 1H, 1H COSY, TOCSY, ROESY, H-detected 1H,13C HSQC, HSQC-TOCSY and HMBC experiments, which were performed and analysed using standard Bruker software. Mixing time of 100 ms was used in both TOCSY and HSQC-TOCSY experiments. The ROESY spectrum was acquired with 150 ms duration of spin-lock time. The HMBC experiment was optimized for coupling constant JH,C 8 Hz.
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
4.3. Isolation of a xylofucan sulfate. A sample of the alga P. plantaginea was collected from the littoral of the Possjet Bay of the Sea of Japan in September 2006, dried in air and milled (particles about 0.25 mm). The algal biomass was treated subsequently with methanol and acetone in Soxhlet apparatus to remove lipids and colored matter and vacuum dried. 20 g of defatted material and 2% aqueous CaCl2 solution (5 200 mL) were mechanically stirred at 85 C for 2 h. The combined extracts were treated with 10% aqueous cetyltrimethylammonium bromide solution (100 mL). The precipitate was centrifuged, washed with water and solubilised in 200 mL of 4 M CaCl2 by moderate heating up to 65 ºC. Ethanol (600 mL) was added to this solution with vigorous stirring, the precipitate was centrifuged, dissolved in 2% aqueous CaCl2, dialyzed and centrifuged to discard a small amount of insoluble material. The solution was treated with NaBH4 (750 mg) overnight, then NaOH (12 g) and additional NaBH4 (3 g) were added, the mixture was heated at 80 ºC for 8 h, cooled, centrifuged, dialysed, concentrated and lyophilized to afford PPF (3.9 g).
4.3. Chemical modifications of polysaccharides. Solvolytic desulfation of PPF (as pyridinium salt) was carried out as described earlier,7,11 giving rise to desulfated polysaccharide PPFdeS with the yield of 37.7%. Methylation of PPF (as pyridinium salt), PPFdeS and PPFdeS-Sm followed by hydrolysis and GLC-MS of partially methylated alditol acetates was performed as previously described.11,30 Smith degradation of PPFdeS: an aqueous solution of NaIO4 (0.021M, 44 mL) was added to a solution of PPFdeS (108 mg, 0.226 mM of Xyl) in water (44 mL), and the mixture was left in the dark for 24 h at room temperature (consumption of the oxidant monitored by a decrease in the optical density of the solution at 305 nm was 0.378 mM). Ethylene glycol (0.5 mL) was added to the reaction mixture, which was then dialyzed and concentrated to about 10 mL, NaBH4 (150 mg)
11
was added, and the mixture was left overnight. The solution was neutralized with HOAc, dialyzed, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
and lyophilized to give oxidized and reduced polysaccharide PPFdeS-OR (94 mg, 87%). This preparation (90 mg) was dissolved in 1% HOAc (6 mL), the solution was heated for 2 h at 100°C, the mixture was cooled, centrifuged, the precipitate was washed with water, suspended in water and lyophilized to give PPFdeS-Sm (30 mg, 33.3%). Additional amount of PPFdeS-Sm of similar structure (more soluble fraction, MW >7 kDa, 20 mg) was isolated from the acid mother liquor by GPC. Smith degradation of PPF: an aqueous solution of NaIO4 (0.021M, 112 mL) was added to a solution of PPF (200 mg, 0.607 mM of Fuc, 0.259 mM of Xyl) in water (112 mL), and the mixture was left in the dark for 24 h at room temperature. Consumption of periodate was 0.63 mM. The mixture was treated as above to give rise to PPF-OR (165 mg, 82.5%). Partial hydrolysis of this material was carried out in 1% AcOH in a boiling water bath for 2 h 15 min. According to GPC data, the hydrolyzate contained two approximately equivalent fractions (about 50 mg each), one of them being essentially a disulfated trisaccharide PPF-Sm1, whereas another one was a mixture of the same trisaccharide with higher molecular weight fragments. The Smith degradation procedure was repeated using shorter time of partial hydrolysis (30 min at 100 ºC). The product was eluted from TSK-40 column in a void volume and lyophilized to give PPF-Sm2 (30 mg from 124 mg of PPF, 24.2 %).
Acknowledgments The authors are grateful to Prof. V. E. Vaskovsky (G.B.Elyakov Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Vladivostok, Russia) for collection of the alga and to Dr. D. N. Platonov (N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia) for GLC-MS analysis of methylation products.
12
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
1. Bilan, M. I.; Smirnova, G. P.; Shashkov, A. S.; Usov, A. I. Russ. Chem. Bull., Int. Ed. 2014, in press. 2. Berteau, O.; Mulloy, B. Glycobiology 2003, 13, 29R-40R. 3. Usov, A. I.; Bilan, M. I. Russ. Chem. Rev. 2009, 78, 785-799. 4. Wijesinghe, W. A. J. P.; Jeon, Y. J. Carbohydr. Polym. 2012, 88, 13-20. 5. Ale, M. T.; Meyer, A. S. RSC Adv. 2013, 3, 8131-8141. 6. Bilan, M. I.; Vinogradova, E. V.; Tsvetkova, E. A.; Grachev, A. A.; Shashkov, A. S.; Nifantiev, N. E.; Usov, A. I. Carbohydr. Res. 2008, 343, 2605-2612. 7. Usov, A.I.; Smirnova, G.P.; Bilan, M.I.; Shashkov, A.S. Bioorg. Khim. 1998, 24, 437-445 (Russ. J. Bioorg. Chem. 1998, 24, 382-389, English translation). 8. Gerwig, G.J.; Kamerling, J.P.; Vliegenthart, J.F.G. Carbohydr. Res. 1978, 62, 349-357. 9. Bilan, M.I.; Grachev, A.A.; Ustuzhanina, N.E.; Shashkov, A.S.; Nifantiev, N.E.; Usov, A.I. Carbohydr. Res. 2002, 337, 719-730. 10. Bilan, M. I.; Grachev, A. A.; Shashkov, A. S.; Kelly, M.; Sanderson, C. J.; Nifantiev, N. E.; Usov, A. I. Carbohydr. Res. 2010, 345, 2038-2047. 11. Bilan, M.I.; Zakharova, A.N.; Grachev, A.A.; Shashkov, A.S.; Nifantiev, N.E.; Usov, A.I. Bioorg. Khim. 2007, 33, 44-53 (Russ. J. Bioorg. Chem. 2007, 33, 38-46, English translation). 12. Pomin, V. H.; Pereira, M. S.; Valente, A.-P.; Tollefsen, D. M.; Pavão, M. S. G.; Mourão, P. A. S. Glycobiology 2005, 15, 369-381. 13. Pomin, V. H.; Valente, A. P.; Pereira, M. S.; Mourão, P. A. S. Glycobiology 2005, 15, 13761385. 14. Anderson, N. S.; Dolan, T. C. S.; Penman, A.; Rees, D. A.; Mueller, G. P.; Stancioff, D. J.; Stanley, N. F. J. Chem. Soc. C 1968, 602-606.
13
15. Bilan, M. I.; Grachev, A. A.; Shashkov, A. S.; Nifantiev, N. E.; Usov, A. I. Carbohydr. Res. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
2006, 341, 238-245. 16. Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. Biochem. 1983, 41, 27-66. 17. Foley, S. A.; Szegezdi, E.; Mulloy, B.; Samali, A.; Tuohy, M. G. J. Nat. Prod. 2011, 74, 1851-1861. 18. Leite, E. L.; Medeiros, M. G. L.; Rocha, H. A. O.; Farias, G. G. M.; da Silva, L. F.; Chavante, S. F.; de Abreu, L. D.; Dietrich, C. P.; Nader, H. B. Plant Sci. 1998, 132, 215228. 19. Silva, T. M. A.; Alves, L. G.; de Queiroz, K. C. S.; Santos, M. G. L.; Marques, C. T.; Chavante, S. F.; Rocha, H. A. O.; Leite, E. L. Brazilian J. Med. Biol. Res. 2005, 38, 523533. 20. Li, B.; Wei, X.-J.; Sun, J.-L.; Xu, S.-Y. Carbohydr. Res. 2006, 341, 1135-1146. 21. Nakayasu, S.; Soegima, R.; Yamaguchi, K.; Oda, T. Biosci. Biotechnol. Biochem. 2009, 73, 961-964. 22. Jiang, Z.; Okimura, T.; Yokose, T.; Yamasaki, Y.; Yamaguchi, K.; Oda, T. J. Biosci. Bioeng. 2010, 110, 113-117. 23. Larsen, B.; Haug, A.; Painter, T. J. Acta Chem. Scand. 1966, 20, 219-230. 24. Larsen, B. Acta Chem. Scand. 1967, 21, 1395-1396. 25. Jiang, Z.; Okimura, T.; Yamaguchi, K.; Oda, T. Nitric Oxide 2011, 25, 407-415. 26. Nakano, K.; Kim, D.; Jiang, Z.; Ueno, M.; Okimura, T.; Yamaguchi, K.; Oda, T. Biosci. Biotechnol. Biochem. 2012, 76, 1573-1576. 27. Abu, R.; Jiang, Z.; Ueno, M.; Okimura, T.; Yamaguchi, K.; Oda, T. Int. J. Biol. Macromol. 2013, 59, 305-312. 28. Jiang, Z.; Ueno, M.; Nishiguchi, T.; Abu, R.; Isaka, S.; Okimura, T.; Yamaguchi, K.; Oda, T. Carbohydr. Res. 2013, 380, 124-129.
14
29. Usov, A.I.; Bilan, M.I.; Klochkova, N.G. Bot. Mar. 1995, 38, 43-51. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
30. Chizhov, A.O.; Dell, A.; Morris, H.R.; Haslam, S.M.; McDowell, R.A.; Shashkov, A.S.; Nifant’ev, N.E.; Khatuntseva, E.A.; Usov, A.I. Carbohydr. Res. 1999, 320, 108-119.
15
Table 1. Composition of the native polysaccharide PPF and some of its modification products. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Fraction
Neutral monosaccharides, %
SO3Na,
Uronic
Fuc
Xyl
Man
Glc
Gal
%
acids, %
PPF
44.3
17.1
tr.
tr.
2.6
19.2
2.3
PPFdeS
63.7
24.1
1.3
2.3
4.3
1.1
n.d.
PPFdeS-OR
58.1
1.7
tr.
tr.
2.4
4.5
n.d.
PPF-OR
44.7
1.3
tr.
tr.
2.0
20.8
n.d.
tr. - amounts lesser than 1% n.d. – not determined
16
Table 2. Methylation analysis of the native sulfated xylofucan PPF, its desulfated preparation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
PPFdeS, and Smith-degraded polysaccharide PPFdeS-Sm (M % of partially methylated fucitol and xylitol acetates). Position of
Deduced positions
O-methyl
of substitution:
PPFdeS
PPF
PPFdeS-Sm
groups in: Fucitol 2,3,4
Fucp-(1→
2
tr.
5
2,3
→4)-Fucp-(1→
4
tr.
-
2,4
→3)-Fucp-(1→
33
3
91
2
→3,4)-Fucp-(1→
31
26
1
4
→2,3)-Fucp-(1→
-
24
3
Fuc
→2,3,4)-Fucp-(1→
1
18
-
Xylp-(1→
29
29
-
Xylitol 2,3,4
17
Table 3. 13C and 1H NMR data for Smith-degraded polysaccharide PPF-Sm2. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
F-nr
A
B
C
F-r
α-L-Fucp2S-(1→[3)-α-L-Fucp2S-(1→3)-α-L-Fucp-(1→3)-α-L-Fucp2S-(1→]n3)-α,β-L-Fuc-OH
Residue
F-nr α-L-Fucp2S-(1→3)-
A
B
C
C-1
C-2
H-1
H-2
H-3
H-4
H-5
H-6
96.18
76.50
68.59
73.39
68.26
16.50
5.34
4.46
4.12
3.91
4.22
1.24
74.37
75.72
70.76
67.74
16.67
5.42
4.59
4.17
4.12
4.33
1.27
97.88
67.74
76.50
69.64
67.98
16.50
5.09
3.98
4.01
4.08
4.42
1.27
74.27
74.37
70.64
67.98
16.77
5.36
4.57
4.16
4.11
4.48
1.28
97.47
71.28
80.02
69.23
71.89
16.88
4.61
3.62
3.70
4.00
3.82
1.28
→3)-α-L-Fucp2S-(1→3)- 95.06
→3)-α-L-Fucp-(1→3)-
→3)-α-L-Fucp2S-(1→3)- 95.86
F-r →3)-β-L-Fucp
Chemical shifts, ppm C-3 C-4 C-5
18
C-6
Captions for figures 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Fig. 1. 13C NMR spectra of polysaccharides: PPF (a), PPFdeS (b), PPFdeS-Sm (c), and PPFSm2 (d). Abbreviations of residues: F – fucose, X – xylose, F-x – xylosylated fucose, nr – nonreducing terminal, r – reducing terminal; A, B and C as in Table 3. Fig. 2. 1H NMR spectra of polysaccharides: PPF (a), PPFdeS (b), PPFdeS-Sm (c), and PPFSm2 (d). Fig. 3. 2D HSQC spectrum of PPFdeS (abbreviations as in Fig. 1). Fig. 4. A part of 2D ROESY spectrum of PPF-Sm2 (abbreviations as in Fig. 1). Fig. 5. 2D HSQC spectrum of PPF-Sm2 (abbreviations as in Fig. 1). Fig. 6. A part of 2D HMBC spectrum of PPF-Sm2 (abbreviations as in Table 3). Fig. 7. IR spectra of PPF (a) and PPF-Sm2 (b).
19
6F 2A 3,2C 2F-nr
1F-rb 1A 1B 1C
d
4A,C 4B
3A
1F-nr
3,5F-nr
4F-rb
4F-nr
3F-rb
1F-ra
5B,C
2B 5A
6F 3F
1F
4F 2F 5F
c 4X 3X
6F
2X 2,5F,F-x
1X
5X
4F 1F 4F-x-nr
b
3F-x 4F-x 3F
4X
3X
6F 5X
2X 1X 1F
a 105
100
95
90
85
80
75
70
ppm
20
18
16
ppm
d
c
b
a
ppm
ppm á-OMe â-OMe
60 5X
5X
5F
2F,F-õ
5F-õ
4X
65
1.4 75 3F,F-õ
3X
80
4F-x
85 90 1F-x
95 100
1F 1X
5.0
4.8
4.6
105 4.4
4.2
6F,F-x
4.0
3.8
3.6
18
70
4F 2X
5.2
16
3.4
ppm
1.2
ppm
ppm
5.00 1B/3C 1B/4C
1,2B
5.05 5.10 5.15 5.20 5.25
1,2C
1,2F-nr
5.30
1C/3A 1C/4A
5.35 5.40 1A/4B
1A/3B
5.45
1,2A
5.50
4.7
4.6
4.5
4.4
4.3
4.2
4.1
4.0
5.55 3.9 ppm
ppm
ppm
65 5C 5B
5A
5F-nr 3F-nr 4B
2B 4F-rb 5 F-rb
4A 4C 2A 2C
14 2 F-rb70
4F-nr
3C
18 75
2F-nr
1.5
3A 3B 3 F-rb
80
85
90 1A
95
1F-nr
1F-rb
1B
1C
5.4
5.2
5.0
4.8
4.6
4.4
16
4.2
4.0
3.8
ppm
ppm
ppm
68 1A/5A
1C/5C
1B/5B
69 70 71 72 73
1A/2A
1B/3C
1C/3C
74 75
1A/3A
76 1C/3A
77 1A/3B
5.45
1B/3B
5.40
5.35
5.30
5.25
5.20
5.15
5.10
78 ppm
(a)
(b) (b)
Structure of a sulfated xylofucan from the brown alga Punctaria plantaginea Maria I. Bilan, Alexander S. Shashkov, Anatolii I. Usov
R ↓ 4
R ↓ 4
R ↓ 4
[→3)-α-L-Fucp-(1→3)-α-L-Fucp-(1→3)-α-L-Fucp-(1→]n 2 ↑
SO3Na
2 ↑
SO3Na
where R is β-D-Xylp or H
A sulfated xylofucan was isolated from the brown alga Punctaria plantaginea.
Polysaccharide structure was elucidated by chemical and spectral methods.
The polysaccharide backbone is built up of 3-linked α-L-Fucp.
Two of every three Fuc are sulfated at O-2 forming trisaccharide repeating units.
The backbone regularity is masked by random substitution by β-D-Xylp at Fuc O-4.
