DECEMBER, 1962
METHANOLYSIS OF FUCOIDAK. I
Methaiiolysis of Fricoidan.
4267
I. Preparation of Methyl a-L-Fucoside and L-Fucose1
RICHARD G. SCHWEIGER Research Laboratory of Kelco Company, San Diego, California Received M a y 25, 1962 Fucoidan from Macrocystis pyrifera is subjected to methanolysis. The polysaccharide is depolymerized to its monomer, and the sulfate groups (which previously had been found to be very stable) are removed quantitatively. Methyl a-Lfucoside can be obtained in crystalline form in high yield, so the procedure may be applied as a preparative method for methyl a-L-fncoside and, after subsequent hydrolysis, L-fucose.
Fucoidan is a water-soluble polysaccharide which mannitol in crystalline form from the hydrolyzate of occurs in brown alga in various proportions an extract of F U C ~ Lserratus S L. depending on the species of alga and, to a lesser The present article gives information on another extent, on locat,ion and season.2a It was first method of depolymerization, namely, methanolysis isolated and described by Rylinzbas a polymer with of fucoidan. This produces methyl a-L-fucoside in fucose as the unit. It is now generally accepted high yields, so it can be used as a preparative that fucoidan is composed mainly of L-fucose units method. Crystalline L-fucose may be obtained which are sulfated3in the 4-position and connected also if, before the isolation of the methyl L-fucoside, through a-(1+2) linkage^.^ However, there are the solution is refluxed with mineral acid. still doubts about the details of the structure, Fucoidan, isolated from the exudate of Macrobranching, and the presence of other sugar unit~.~86cystis pyrijera, was treated with methanolic Fucoidan and methylat'ed fucoidan have been hydrogen chloride under various conditions. In depolymerized by hydrolysis and by acetolysis in all experiments, methyl a-L-fucoside was isolated order to solve structxre problems. In other in- as the principal crystalline compound, the yield vestigations, the main object mas the prep- of which depended greatly on the concentration of aration of L-fucose, which is difficult to syn- hydrogen chloride, the amount of fucoidan per thesize.e Giinther and Tollens' were the first liter of methanol, and the reaction temperature, who obtained L-fucose from seaweed extracts in but very little on the reaction time. The yield was preparative quantities. Since the direct crystal- determined by the weight of methyl a-L-fucoside lization after hydrolysis was difficult, t'hey formed which actually could be crystallized rather than on and separated the phenylhydrazone and t'hen the reducing power of the hydrolyzed reaction mixdecomposed it, wit'h benzaldehyde t o obtain ture, the optimum of which need not be identical crystalline L-fucose. Essentially the same method with optimum yields of crystalline product. Only hut using different seaweeds was applied later by the fractions of crystals with sufficient purity (cor. ~ et aZ.,lO suc- rect melting point) were considered in these deterClark8 and by Hudson, et ~ 1 Black, ceeded in crystallizing L-fucose directly from the hy- minations. drolyzate of a sample of fucoidan from Pelvetia canTable I indicates optimum yields at about 60'. aliculata. l1 Trabertl? separated L-fucose from D- A rapid decrease of the yield is noticed a t lower temperatures probably because of insufficient (1) Presented before the Division of Carbohydrate Chemistry, 142nd National Meeting of the American Chemical Society, Atlantic City, depolymerization. The slight decrease a t higher N. J., September, 1962. temperatures probably is due to degradative ( 2 ) (a) W. A. P. Black, J . Sei. Food Agr., 5, 445 (1954); F. N. Woodward, ibid., 2, 477 (1951); D. J. Wart, Can. J . Botany, 33, 323 (1955); impurities (deeper color of the reaction mixture), M. M. V. Holdt, S. P. Ligthelm, a n d J. R. Nunn, J . S e i . Food Agr., which make crystallization more difficult. 6, 193 (1955); F. Karawara, Japanese Patent 4334, (Oct. 22, 1952); Chem. Ahstr., 47, 8846c (1953); C . Sannie, C o m p t . Rend., 231, 874 (1950). (b) H. Kylin, Z. Physiol. Chem., 33, 171 (1913); 94, 337 (1915). (3) E. G. V. Percival and A. G. Ross, J . Chem. Soc., 717 (1950). (4) J. Conchie and E. G. V. Peroival, i b i d . , 827 (1950); 4. N. O'Neill, J . Am. Chem. Soc., 76, 5074 (1954). ( 5 ) T. Dillon, K. Kristensen, and C. O'hEochdha, Proc. Roy. Irish Acad. Sect. B . 55, 189 (1953): E. T. Dewar, Chem. Ind. (London), 785 (1954). (6) 0. T. Schmidt and E. Wernioke, Ann., 558, 70 (1947); R, Iselin and T. Reiohstein, Helu. C h i m . Acta, 29, 508 (1946). (7) A. Giinther and B. Tollens, Ber., 23, 2586 (1890). (8) E. P. Clark, J . B i d . Chem., 6 4 , 6 5 (1922). (9) R. C. Hockett, F. P. Phelps, and C. S. Hudson, J . Am. Chem. Soc., 61, 1658 (1939). P. Black, W. J. Cornhill, E. T. Dewar, and F. N. Woodward, J . Sci. Food AoT.,4. 85 11953). (11) W. A. P. Black, E. T. Dewar, and F. N. Woodward, ibid., 3.122 (1952). (12) C. H. Trahert, .lrch. P h a r m . , 293, 278 (19fi0).
TABLEI METHANOLYSIS OF FUCOIDAX AT DIFFERENT TEMPERATGRES Fncoidan, 150 g . ; Methanol, 1000 InL; Hydrogen Chloride, 50-60 g. Temperature
20-25 60 80
L-Fucoside, g .
3 1 14.0' 2 5b 10.4a 2 . 85b 2.75c
110
10.5= 1.55b a First fraction of crystals. Third fraction of crystals
M.p.
Sirup,
153-156 157-158 140-152 155-157 147-155 105-120 156-150 152-1 56 Second frnvtion of
g.
21 17 4 21
21
rrystals.
SCHWEIQER
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VOL.27
TABLE I1 Final evidence was obtained by isolation and charMETHANOLYSIS OF FUCOIDAN USINQDIFFERENT AMOUNTS acterization of methyl p-r,-fncoside as the potassium O F IfYDROOEN CHLORIDE Temp., GO'; Methanol, 1000 ml.
acetate adduct. The quantity of methyl p-Lfucoside may be roughly estimated by comparing HC1,g. L-Fuooside, g. M.p. Sirup, g. the rotation of the mixture with the rotation of 10 0 ... 1.4 the pure a- and P-forms, -197' and + 1 4 O , re20 0.v 155-158 9 spectively.g 5.Gb 106-119 If the fucoidan is purified by reprecipitation with 40 14.4a 148-155 8.6 5.30 122-143 alcohol, the L-fucose content and, correspondingly, 84 14.2a 147-156 19.4 the yields after methanolysis increase. This in1.P 119-144 crease, however, does not compensate for the high First fraction of crystals. Second fraction of crystals. losses of fucoidan during its purification. Instead of methyl a-L-fucoside, L-fucose may be The results of varying the hydrogen chloride obtained if the solution of the fucoside is heated concentration (Table 11) show that crystalline with dilute sulfuric acid. The free sugar then can L-fucoside is obtained only if more than 10 g. of be readily crystallized in a yield of 50-60%. hydrochloric acid/l. of methanol is present. OptiIf hydrochloric acid is used, the crystallization of mum yields are produced with 45-70 g./l. L-fucose is difficult. Similarly, I,-fucoside can be crystallized only if Summarizing all results, methanolysis under the the amount of fucoidan/l. of methanol (Table 111) conditions described above produces methyl a-G is below 600 g. fucoside and, to a smaller extent, methyl P-Lfucoside as the only compounds which crystallize TABLE I11 directly. The high yields of these two derivatives METHANOLYSIS OF VARYING AMOUNTS OF FUCOIDAN and the lack of chromatographic evidence for Mcthanol, 1000 ml. ; Hydrogen Chloride, 40-50 g. oligosaccharides establish that fucoidan is comL-Fucoside, g./150 g. pletely depolymerized by cleavage of the glycosidic Fucoidan, g. fucoidan M.p. Sirup, g. linkages and that the sulfate groups (which are 600 0 ... fucose, the mixture waa refrigerated for 10 days. Crystals occasionally were removed from the sides of the flask with a spatula. The crystallizate was filtered off on a fritted-glass funnel, washed with a cold mixture of methanol and ether, and dried; -75.0' f 1.5' (c 1.3, yield, 7.5 g., m.p. 136-138', [cy]'% water). When admixed with an authentic sample of Lfucose, the m.p. was undepressed. Preparation of L-Fucose.-Fucoidan (75 g.) was subjected to methanolysis a t 60' for 24 hr. using 500 ml. of methanol and 25 g. of hydrogen chloride. The procedure from here until after passage through the cation- and anion-exchange resin columns was the same as described above. The neutral, colorless solution, however, then waa concentrated to a smaller volume, acidified with sufficient sulfuric acid to make it N with respect t o the acid, and refluxed for 5 hr. After filtration, precipitation of the sulfuric acid with barium hydroxide, and treatment with activated carbon and some Amberlite IR-1204 H+) cationexchange resin, the colorless solution was concentrated in vacuo to a sirup. The sirup was diluted with hot methanol to which ether was added. This mixture waa left in the refrigerator for crystallization, the crystals being occasionally removed from the sides of the flask. After about 2 weeks, L-fucose waa filtered off on a fritted-glass funnel, washed with a cold mixture of methanol and ether, and dried. The yield waa 7.6 g., m.p. 136-139O, [a]% -75.3 f 1.4' ( c 1.3, water). When a sample was mixed with authentic L-fucose, the m.p. waa undepressed. The noncrystallizing sirup weighed 29.5 g . (18) S. M. Partridge. ibid.. 164,443 (1949) (19) A. J. Watters, R. C. Hockett, and C. 9.Hudson. J. Am. Chem. Soc., 66, 2199 (1934).
