Phytochemistry, Vol. 30, No. 2, pp. 535-539, 1991 Printedin Great Britain.

AN ANTICOAGULANT

TAKASHI NISHINO,

0

0031-9422/91 $3.00+0.00 1991 PergamonPressplc

FUCOIDAN FROM THE BROWN SEAWEED ECKLONIA KUROME

HIROAKI KIYOHARA,*

HARUKI YAMADA*

and

TERUKAZU

NAGUMO

Department of Biophysics, School of Hygienic Sciences, Kitasato University, 1-15-l Kitasato, Sagamihara-shi, Kanagawa 228, Japan; *Oriental Medicine Research Center of Kitasato Institute, 5-9-l Shirokane, Minato-ku, Tokyo 108, Japan (Received in revised form 23 July 1990)

Key Word Index--Ecklonia kurome; brown seaweed; fucoidan; sulphated polysaccharide; anticoagulant activity; structural study.

Abstract-The structure of a a+fucose-rich, sulphated polysaccharide (C-I) with a potent anticoagulant activity, which was isolated from the brown seaweed Ecklonia kurome, has been studied. Methylation analysis showed that C-I consisted mainly of 3-linked and 3,4-disubstituted fucopyranosyl residues in addition to non-reducing terminal fucofuranosyl and fucopyranosyl residues, 2,3-di- and 2,3,4-t&substituted fucopyranosyl residues and galactopyranosyl residues with various glycosidic linkages. Methanolysis of C-I gave neutral di-, tri-, tetra- and highly polymerized-oligosaccharide fractions. GC-MS and methylation analysis indicated that di- and trisaccharide fractions consisted mainly of Fuc-( 1+3)-Fuc and Fuc-(l+3)-Fuc-( l-3)-Fuc, respectively, in addition to small amounts of Fuc(l-*4)-Fuc, Fuc-( l-+4)-Gal and Fuc-( 1+3)-[Fuc-(l+2)-]Fuc. When methylated C-I was subjected to methanolysis for desulphation followed by remethylation with deuterated methyl iodide, most of deuteriomethyl groups substituted to position 4 of 3-linked Fuc.

INTRODUCTION

We have purified four fucose-containing, sulphated polysaccharides (B-I, B-II, C-I and C-II) from the brown seaweed Ecklonia kurome [l]. C-I and C-II possess remarkably high anticoagulant activity (N 81 and 85% for activity of heparin, respectively) with respect to the activated partial thromboplastin time [l]. C-I and C-II consisted a fucose, galactose, glucuronic acid and sulphate in the molar ratios of 1.0:0.03:0.03: 1.61 and 1.0:0.19:0.07: 1.48, respectively [l]. Although the M, (32000) of C-I differed from that (21000) of C-II, C-I and C-II were shown to be very similar in composition to each other except for their galactose content. We now deal with the structural characterization of C-I. RESULTS AND

DI!XUS!SION

Methylation analysis (Table 1) showed that C-I contained mainly 3-linked and 3,4-disubstituted fucopyranosyl residues in addition to small proportions of terminal fucofuranosyl and fucopyranosyl residues, 4-linked, 2,3di- and 2,3,4-tri-substituted fucopyranosyl residues, and galactosyl residues with various glycosidic linkages. C-I also contained a trace of 4-O-methyl-1,2,3,5,6-penta-Oacetylglucitol-6,6-&f, indicating the presence of 2,3-disubstituted glucuronic acid residues in C-I. The ‘H NMR spectrum of C-I in D,O showed the methyl protons of 6deoxy-sugars at 6 1.2-1.5, acetyl protons at 6 -2.2 (0.2% by the method of McComb and McCready [Z]) and anomeric protons due to the a-configuration at 6 N 5-5.6. The 13C NMR spectrum of C-I showed two major anomeric carbon signals at 6 99.16 and 95.17, which were assigned to C-I of 3-linked a+fucopyranosyl residues (cf. 699.1

for C-l of 3-linked a-D-fucopyranosyl residues [S]) and 3,4-disubstituted fucopyranosyl residues, respectively. These results indicate that C-I consists of a-L-fucosyl residues. C-I was subjected to methanolysis in order to remove sulphate groups [4, 51. After the carbohydrate in methanolysate was desalted by Sephadex G-25 chromatography, the neutral fraction (major) and a trace amount of acidic fraction were fractionated on an anion-exchange resin. The neutral fraction was further fractionated into large amounts of neutral oligosaccharide fractions (POF and oligo I-IV) and a monosaccharide fraction. Fucose was only detected (GC) in the monosaccharide fraction. The acidic fraction was not further investigated because of the very small amount available. These results suggest that methanolysis of C-I may cause partial cleavage of fucosyl glycosidic linkages in C-I together with desulphation of the polysaccharide. Neutral oligosaccharides, oligo II-IV and POF were composed of fucose and galactose in the molar ratios of 21.6:1.0, 17.4: 1.0, 33.3: 1.0 and 63.0: 1.0, respectively. Methylation analysis (Table 2) showed that oligo II consisted mainly of 3-linked fucosyl residues and 2- and 3-linked galactosyl residues, whereas POF, oligo III and IV comprised mainly 3-linked fucosyl and 2,3-disubstituted fucosyl residues. After oligosaccharides in oligo I and II were converted into oligosaccharide-alditols by reduction with NaBD4, the oligosaccharide-alditols were methylated by the method of Hakomri [6]. The resulting methylated oligosaccharide-alditols, which were partially purified by HPLC (data not shown), were analysed by GC-CIMS and GC-EIMS. The CI mass spectrum showed that oligo I contained three fragments [a (major), b (minor) and c (minor)]. Fragments a and b gave ions at

535

T. NISHINO et al.

536

Table

1. Methylation

analysis

of C-I

Methylated sugars

Mol. %

Major mass spectral fragment ions (m/z)

Linkages*

2,3,5-Me,-Fuc 2,3,4-Me,-Fuc 2,3-Me,-Fuc 2,4-Me,-Fuc

6.2 4.1 5.7 25.2

175, 175, 203, 233,

Fucf-l-. Fucp-( 1-+ -+4)-Fuc-(l -v -+ 3)-Fuc-( 1 -+

2-Me-Fuc

27.6

275, 173, 129, 117, 113,99,

4-Me-Fuc

5.1

261, 201, 131, 127, 89

-$Fuc+

Fuc

3.2

231,201,

-+2)\Fuc-(l-r -3)’ I

117, 161, 143, 173,

101, 131, 117, 159,

59 117, 101, 89 101 131, 117, 101, 89

+3)‘Fuc-(1 -4)’

87

187, 170, 157, 145, 128, 115, 103

-+

T t 2,4,6-Me&al 2,3,4-Me,-Gal

4.0 2.5

233, 161, 129, 117, 101, 87,45 233, 189, 161, 129, 117, 101,99,

4,6-Me,-Gal

4.3

261,201,

3,6-Me,-Gal

5.4

233, 189, 129, 113, 99, 87, 45

Gal-(l+

2,4-Me,-Gal

2.2

233, 189, 129, 117, 87

-‘3)‘Gal-(l-+ -6)’

3-Me-Gal

1.4

261,201,

‘2)1Gal-(l-+ -4)’ A 6 t

4-Me-Glc-6,6-d,d *Fucf=

Table

trace

fucofuranosyl

2. Methylation

+3)-Gal-(l-+ +6)-Gal-(l-+

87

-‘2)\Gal-(l-+ -3)’

161, 129, 127, 101,45

189, 129, 127, 99, 87

-+2)\GlcA-(1+ -3)’

261, 201, 191, 159, 131

residue.

Fucp = fucopyranosyl

analysis

of neutral

residue.

ohgosaccharide

fractions

from C-I by methanolysis

Mol. % Methylated sugars

____Oligo II

III

IV

POF

Linkages*

2,3,5-Me,-Fuc 2,3,4-Me,-Fuc 2,3-Me,-Fuc 2,4-Me,-Fuc

2.0 4.8 2.3 42.9

2.8 7.6 2.2 63.2

1.7 3.2 1.1 58.6

2.8 7.9 trace 78.0

Fuc.4 1 -+ Fucp-(l-. -~4)-Fuc-(1 + -+3)-Fuc-(l-+

2-Me-Fuc

2.3

trace

3.7

2.0

4-Me-Fuc

6.3

13.3

20.1

7.0

~~;)Fuc-(l-+

5.3 11.7 14.6 7.9

trace 4.7 4.9 1.4

trace 3.8 4.6 3.0

trace trace trace trace

Galp-( 1 + -+2)-Gal-(l -+ -+3)-Gal_(l-+ -+6)-Gal-(l-+

n.d.

n.d.

2.2

-*4) ‘Gal-(1 -+6)’

2,3,4,6-Me,-Gal 3,4,6-Me,-Gal 2,4,6-Me,-Gal 2,3,4-Me,-Gal 2,3-Me,-Gal *Fucf= fucofuranosyl tNot detected.

n.d.t residue.

-.._____

Fucp = fucopyranosyl

m/z 412 due to [M+H]+, 206 to aJ, and 189 to bA,, fragment c at 442 to [M+H]+, 236 to aJ, and 189 to bA,, suggesting fragments a and b possessed a fucosyl+fucitol-l-d unit and fragment c a fucosyl+galactitol-l-d unit. The EI mass spectrum of these fragments showed the specific fragment ions of the ald series at m/z 352, 308, 276 and 103 in fragment a, identifying it as fucosyl-( 1+ 3)-fucitol-l-d, fragment b (m/z 352, 308, 277, 245 and 134) as fucosyl-(l-+4)-fucitol-l-d and fragment c (m/z 352,308,276,134 and 89) as fucosyl-

‘3)‘Fuc-(l+ -4)’

+

residue.

(1+4)-galactitol-l-d. The CI mass spectrum showed that oligo II contained two fragments [d (minor) and e (major)]. Fragments d and e gave [M +H]+ at m/z 586, indicating fucosyl trisaccharide-alditols-l-d. Fragment e also gave fragment ions at m/z 189 (CA,), 363 (cbA,), 398 (abJ,OHi) and 224 (aJ,OHi), indicating it to be fucosyl -+fucosyl -r fucitol- l-d. Fragment d was suggested to be fucosyl+[fucosyl-,]fucitol-l-d, owing to the presence of fragment ions at 189 (CA,) and 398 (abJ,OH:) and the absence of fragment ions at m/z 363 (cbA,) and

Fucoidan

from Ecklonia kurome

224 (aJ,OH:). Because specific fragment ions of the ald series on EI mass spectrometry were not observed in fragments d and e , glycosidic linkages of fragments d and e could not be deduced. However, as methylation analysis (Table 2) showed that oligo II consisted mainly of 3linked fucosyl residues, fragment e was suggested to be fucosyl-(l+3)-fucosyl-(1+3)-fucitol-l-d. Oligo II also contained small amounts of 2,3- and 3,4-branched fucosyl residues, thus fragment d was assumed to be either fucosyl-(l-+3)-[fucosyl-(l-+2)-]fucitol-l-d or fucosyl(1 -+ 3)-[fucosyl-( l-+4)-lfucitol-l-d. Methylation analysis (Table 2) also indicated that the content of 2,3disubstituted fucosyl residues was higher in oligosaccharides larger than trisaccharide, comparing with that of 3,4disubstituted fucosyl residues. Therefore, it was assumed that fragment d was fucosyl-(1+3)-[fucosyl-( l-+2)-]fucitol-l-d. These results suggested that C-I consisted of a (l-+3)-linked fucosyl backbone. It was also suggested that the fucosyl side-chain was mainly linked to position 2 of the 3-linked fucosyl backbone, because of the presence of 2,3disubstituted fucosyl residues in oligosaccharides larger than trisaccharide (Table 2) and fucosyl-(l+3)[fucosyl-( l-+2)-lfucose. To determine the positions of sulphate groups in C-I, methylated C-I was subjected to methanolysis followed by remethylation (CD,I) because it is expected that the Odeuteriomethylated position of sugars indicates the Osulphated position. However, the present methanolysis may cause not only desulphation but also partial degradation of glycosidic linkages as described above. Therefore, the deuterated product was fractionated into the fractions of high (HMF) and low M, (LMF), and the Osulphated position of C-I estimated by the O-deuteriomethylated position of HMF. Methylation analysis (Table 3) showed that both HMF and LMF contained mainly 1,3,5-tri-0-acetyl-2,4-di-O-methyl-, 1,3,5-tri-Oacetyl-4-0-deuteriomethyl-2-O-methyland 1,2,3,5-tetra0-acetyl-4-0-methyl-fucitols. These results suggest that sulphate groups in C-I may be mainly located at position 4 of the 3-linked fucosyl backbone. In a previous study, the IR spectrum of C-I also indicated that most sulphate groups were mainly located at C-4 of fucosyl residues [ 11.

Table

3. Methylation

Position of OMe groups

Fucosyl

2,394 3,4 24 23 2,3 3 2

4

n.d., not detected.

It was also shown that LMF contained small proportions of 2-, 3- or 4-mono-0-deuteriomethylated terminal galactose, 2,4,6-tri-O-methylgalactose and 6-O-deuteriomethyl-2,4-di-O-methylgalactose (data not shown). Although the result of deuteriomethylation of the methanolysates also suggested that some sulphate groups may be attached at various positions of fucosyl and galactosyl residues in addition to 3-linked fucosyl residues, it is not known whether minor deuteriomethylation occurred by desulphation or cleavages of glycosidic linkages during methanolysis. The methanolysates of methylated C-I also contained large amounts of undeuteriomethylated 2,3-di-substituted fucosyl residues, indicating that the fucosyl side-chain may be attached at a part of position 2 of the 3-linked fucosyl backbone. Because a molar ratio of sulphate and fucose in the present study did not agree with that calculated from the composition of C-I, some fucosyl residues with more than two sulphate groups may exist in C-I, or partial desulphation may have taken place in the course of the first methylation of fucoidan with Me1 by the method of Hakomori [6]. The present results suggested that C-I may have the partial structures 1 as a major part and 2 and 3 as minor parts. C-I is a new type of anticoagulant fucoidan because the structure of C-I differed from that of other known fucoidans, which consist mainly of a 2-linked fucosyl backbone with sulphate groups located at position 4 in addition to 2,3- and 2,4-branched fucosyl residues [7-lo]. C-I contained 2,3-disubstituted glucuronic acid residues and several linkages of galactosyl residues as minor components. The detailed structure of the minor portion in C-I remains to be clarified but the chain length of galactose portions is suggested to be short comparing with the fucose portion, because the fragments containing galactose were detected only in the low M, fraction obtained after methanolysis of methylated C-I. EXPERIMiNTAL

General.C-I was purified from the brown seaweed E. kurome as described previously [l]. Carbohydrates in column eluates

analysis of high and low M, products (HMF and LMF) from methylated C-I by methanolysis

Glycosyl residues

2,4 4 2 2

531

Position of OCD, groups

2 3 4

Deduced glycosidic linkages Terminal Terminal Terminal Terminal 4443333,43,4z3z3-

(pyranosyl) (pyranosyl) (pyranosyl) (pyranosyl)

HMF 0.2 0.4 3.0 2.5 n.d. n.d. n.d. 25.8 6.1 36.0 5.2 0.5 15.4 4.1

Mol. % LMF 4.5 2.0 8.9 6.3 0.5 1.1 2.5 17.3 7.4 18.7 trace trace 17.5 n.d.

538

T. NISHINO et al. SOi t

SO; i 4

+3)-Fucpa-(

I-3)-Fucpa-(

FucporFucfu-(

l--2)

>Fucpu-

( I--

2

c

l-3)

14 ,FucpaSO; -) 4 Fucpa - ( I--2

I-3)

1-o

so; -3).Fucpn-(

2 +3)-Fucpa-(

( l--

)

3

were monitored by the PhOH-H,SO, method [ll]. Acetyl content was determined by the method of ref. [2]. Neutral oligosaccharides were hydrolyscd with 2 M TFA at 121” for 1.5 hr [12]. Hydrolysates were analysed by TLC on cellulose (Merck) with EtOAc-pyridine-HOAc-H,O (5: 5: 1: 3) and detected with alkaline AgNO, [13]. Sugars were converted into alditol acetates [14] and analysed by GC [15]. FID/GC analysis was performed on a DB-1 capillary column (0.25 pm film thickness, 30 m x 0.25 mm) in the splitless mode. The flow rate of the carrier gas, N, , was 0.9 ml min _ ’ The oven temp. was prog. at 60” for 1 min, 30” mini to 180” and then 3” mini to 220”. Molar ratios of sugars were calculated from peak areas and M,s of the corresponding alditol acetates. Methanolysis of C-I. C-I (42.6 mg) was methanolysed in 91 mM MeOH-HCl (3.4 ml) for 24 hr at room temp. [4]. The reaction mixt. was dil. with H,O, neutralized and desalted on a column (2.6 x 96 cm) of Sephadex G-25. The carbohydrate was further fractionated on a column (1.5 x 29 cm) of AG 1 x 8 (Cl -) resin, and the neutral and acidic frs obtained by eluting with H,O and 1 M HCl, respectively. The neutral fr. was further fractionated by gel filtration on a column (2.5 x 50 cm) of Bio-gel P-2 at 55” with H,O to give a monosaccharide fr., four oligosaccharide frs (oligo I-IV) eluted in the regions for di-, tri-, tetra- and pentasaccharide, respectively, and the highly polymerized-oligosaccharide fr. (POF) in the near void volume. Metkylation analysis. (i) C-I was methylated once by the Hakomori method [6] in order to prevent /I-elimination [16]; the completeness of formation of alkoxide was checked usmg triphenylmethane [17]. The methylated polysaccharide was purified using a Sep-Pak C,, cartridge according to the procedure of ref. [lS] except that EtOH was used as eluent. The carboxyl groups of polysaccharidcs were reduced with NaBD, in 90% EtOH-THF (2773) [18, 191. The resulting carboxylreduced, partially 0-methylated polysaccharide was hydrolysed with 90% HCO,H at loo” for 6 hr, followed by hydrolysis with 1 M TFA at 100” for 2 hr, and the partially methylated sugars reduced with NaBH, to the corresponding alditols. These were then acetylated with Ac,O m the presence of NaOAc at 121” for 3 hr [18]. (ii) Neutral oligosaccharide frs from C-I by methanolysis, were reduced with NaBD, at room temp. for 18 hr in 1 M NH,OH to convert them into their corresponding oligosaccharide-alditols. These were recovered by desalting with a AG 50W x 8 (H’) resin and methylated as described above. The fully methylated oligosaccharide-alditols were hydrolysed with 2 M TFA at 121” for 1 hr, and then converted into their corresponding alditol acetates. (iii) For desulphation, fully methylated C-I prepared according to (i) was subjected to methanolysis (91 mM MeOH-HCl) at room temp. for 24 hr. After solvent was removed

in an air-stream, the methanolysates were dtssolved m 50% DMSO, purified using a Sep-Pak Cis cartridge in order to remove Me sulphates, remethylated with CD,1 and purified using the same cartridge. The products were fractionated into frs of high (HMF) and low M, (LMF) on a column (1.5 x 26 cm) of Sephadex LH-20 using CHCI,-MeOH (1:l) [20]. Each fr. was hydrolysed with 2 M TFA [15], and then converted into alditol acetates. Partially methylated alditol acetates were analysed by GC and GC-MS [20,21]. GC was performed on a DB-1 capillary column wtth splitless injection and the temp. prog. was at 60” for 1 min, 18” min-’ to 150” and then 2”min-’ to210”. Theflow rateofthe carrier gas, N,, was 0.9 ml min-‘. GC-MS was performed on a SPB-1 capillary column (0.25 pm film thickness, 25 m x 0.25 mm i.d.) at 12&210” (2” mini) with splitless inj. and operated at an ionization voltage of 70 eV with an ionization current of 300 PA. The flow rate of the carrier gas, He, was 0.9 ml min-‘. Peaks were identified on the basis of then R, and fragmentation patterns. The molar ratios for each sugars were calibrated using the peak areas and FID response factors [22] on GC. GC-MS of methylated oligosaccharide-alditols. Per-O-methylated oligosaccharide-alditols derived from oligo I and II according to (ii) were purified by HPLC. HPLC was carried out on a column (6 x 150 mm) of YMC-pack and developed with MeOH-H,O (7: 3). Purity of methylated oligosaccharidealditols was assessed by TLC on silica-gel PR-18 using MeOH-H,O (7:3) and detection with the 1-naphtol-H,SO, reagent [23]. GC-MS of the purified methylated oligosaccharide-alditols was carried out on a SPB-1 capillary column according to the method of ref. [24]. The temp. prog. was at 180-310” (4” mm’). EIMS was performed at 70 eV with an ionization current of 300 PA, and CI (iso-butane) at 250 eV and accelerating voltage 3 kV. CI [25] and EI [26] fragment ions [A, J andalditol (aid)] were used to determine the structure of per-0-methylated ohgosaccharidealditols. NMR. ‘H (400 MHz) and i3C (100 MHz) NMR spectra of C-I were obtained from soln. in D,O at 80” using and sodium 3(trimethylsilylhrropane-1-sulphonate-d,(TSP) as mt. standard. Acknowledgement-The authors thank MS A. Nakagawa MS C. Sakabe for their assistance with GC-MS.

and

REFERENCES 1. Nishino, T., Yokoyama, G., Dobashi, K., FuJihara, M. and Nagumo, T. (1989) Curbohydr. Res. 186, 119. 2. McComb, E. A. and McCready, R. M. (1957) Anal. Chem. 29, 819.

Fucoidan from Ecklonia kurome 3. Ray, T. C., Smith, A. R. W., Wait, R. and Hignett, R. C. (1987) Ear. J. Biochem. 170, 357. 4. Percival, E. (1968) Carbohydr. Res. 7, 272. 5. Hussein, M. M. D., Fouad, S. T. and Fattah, A. F. A. (1979) Carbohydr. Res. 72, 177. 6. Hakomori, S. (1964) J. Biochem. (Tokyo) 55, 205. 7. Conchie, J. and Percival, E. G. V. (1959) J. Cbem. Sot. 827. 8. O’Neil, A. N. (1954) J. Am. Chem Sot. 76, 5074. 9. Cote, P. H. (1959) J. Chem. Sot. 2248. 10. Mian, A. J. and Percival, E. (1973) Carbobydr. Res. 26, 147. 11. Dubois, M., Gilles, K. A., Hamilton, J. K., Rehem, P. A. and Smith, F. (1956) Anal. Chem 28, 350. 12. Yamada, H., Otsuka, Y. and Omura, S. (1986) Planta Med. 311. 13. Trevelan, W. E., Procter, D. P. and Harrison, J. S. (1950) Nature 166,444. 14. Alhersheim, P., Nevins, D. J., English and Karr, A. (1967) Carbohydr. Res. 5, 340. 15. Yamada, H, Kiyohara, H., Cyong, J.-C. and Otsuka, Y. (1987) Carbohydr. Res. 159,275.

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16. McNeil, M., Darvill, A. G. and Alhersheim, P. (1980) Plant Physiol. 66, 1128. 17. Rauvala, H. (1979) Carbohydr. Res. 72, 257. 18. Waeghe, T. J., Darvill, A. G., McCneil, M. and Alhersheim, P. (1983) Carbohydr. Res. 123, 281. 19. Dutton, G. G. S., Mackie, K. I., Savage, V. and Stephenson, M. D. (1978) Carbohydr. Res. 66, 125. 20. Kiyohara, H., Yamada, H. and Otsuka, Y. (1987) Carbohydr. Res. 167, 221. 21. Lindherg, B. (1972) Methods Enzymol. 28, 178.

22. Sweet, D. P., Shapiro, R. H. and Alhersheim, P. (1975) Carbohydr. Res. 40,217. 23. Dishe, Z. (1962) Methods Carbohydr. Chem. 1,478. 24. Yamada, H., Kiyohara, H. and Otsuka, Y. (1987) Carbohydr. Res. 170, 181. 25. Chizhov, 0. S., Kadentsre, V. I., Solovyov, A. A., Levonowich, P. E. and Dougherty, R. C. (1976) J. Org. Chem. 41, 3425. 26. Kochtkov, N. K. and Chizhov, 0. S. (1966) Adu. Carbohydr.

Chem. 21.39.

An anticoagulant fucoidan from the brown seaweed Ecklonia kurome.

The structure of a alpha-L-fucose-rich, sulphated polysaccharide (C-I) with a potent anticoagulant activity, which was isolated from the brown seaweed...
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