325

(J. Appl. Glycosci., Vol. 48, No. 4, p. 325-330 (2001))

A New fJ-D-GalactanHaving 3-O-Methyl-D-galactosefrom Chlorella vulgaris

Kazutoshi Ogawa, * Masaki Arai, Hiroshi Naganawa,1 Yoko Ikeda' and Shinichi Kondo1

Department of Fundamental Science, College of Science and Engineering, Iwaki Meisei University (5-5-1, Chuodai-iino, Iwaki 970-8551, Japan) 'Institute of Microbial Chemistry (3-14-23 , Kamiosaki, Shinagawa-ku, Tokyo 141-O021, Japan)

The neutral from Chlorella vulgaris K-22 cells has been shown by the fragmen-

tation analysis, methylation analysis, and periodate oxidation studies to be a new type of ƒÀ-D-

galactan having 1,3-linkages in the backbone and 1,6-linkages in the side chain. This galactan also contains 6-O-glycosylated 3-O-methyl-ƒÀ-D- in the branching regions. The side chain has

fourteen residues of D-galactose including 3-O-metylated one(s). Mild acid hydrolysis of the galac

tan yielded three new , 6-O -(3-O -methyl-ƒÀ-D-galactopyranosyl)-D-galactopyranose, 6-

0 -ƒÀ-D-galactopyranosyl-3-O -methyl-D-galactopyranose, and 6-O -(3-O -methyl-ƒÀ-D-galactopyrano-

syl)-3-O-methyl-D-galactopyranose, together with 3-O- ƒÀ -D-galactopyranosyl-D-galactose, 6-O- ƒÀ -D-

galactopyranosyl-D-galactose, and 0 -ƒÀ-D-galactopyranosyl-(1•¨6)-O -ƒÀ-D-galactopyranosyl-(1•¨6)- D-galactose.

A glycoprotein-rich extract of Chlorella vulgaris ated sugar of the latter polysaccharide seemed to strain K-22 has exhibited biological response be characteristic of certain species or strains of the modifier (MRM) activity,11)and a extracellular gly genus Chlorella. We were interested in a mode of coprotein isolated from C. vulgaris K-22 culture linkages of the mono-O -methylated saccharide in medium has shown antitumor activity and antimet the polysaccharide, so we set out to study the astatic immunopotentiation, and its chemical structure of the polymer. moiety had a /9-1,6-D-galactopyraose backbone.2,3) Three new disaccharides containing 3-O -methyl- Therefore, from the cell may be D-galactose, were obtained by the mild acid hy suitable as taxonomic markers of the strain having drolysis of the neutral polysaccharide. Three physiological activities and their structural analyses known di- and containing only D- are significant. galactose were also isolated together with mono Blumreisinger et al.4) showed that the distribu saccharides, D-galactose, and 3-O -methyl-D-galac tion of neutral in the cell walls of tose. Therefore, the polysaccharide seems to be a new chlorococcal algae provides useful knowledge for type of polysaccharide, ƒÀ-D-galactan. In this paper, strain identification (according to this theory, the isolation and structural determination of the new Chlorella vulgaris strain K-22 is classified into a disaccharides obtained from the galactan are described. /galactose type). In our investigation of The structure of the galactan is also discussed on from extracts of commercial the basis of fragmentation analysis, methylation preparation of Chlorella vulgaris K-22 cells, we analysis, and periodate oxidation studies. found an acidic polysaccharide, glucuronorham- nans5)and a neutral polysaccharide having 3-O - MATERIALS AND METHODS methyl-D-galactose as a constituent.6) The methyl General methods. Optical rotaions were taken * Corresponding author . on a Jasco DIP-370 or Perkin-Elmer 241 polarime 326 J. Appl. Glycosci., Vol. 48, No. 4 (2001)

ter. Mass spectra were determined with a Jeol JMS Rf 0.27; [a ] D + 32° (c 0.39, water) (lit.8) [a ]D+

SX102 mass spectrometer (FAB mode). 'H-NMR 31°); FABMS (pos.): m/z 343 [M+H]t ,ƒÀ-D- spectra were measured with a Jeol JNM-EX400 or Galactopyranosyl- (1•¨ 6) -ƒÀ-D-Galactopyranosyl- (1•¨

JNM-A500 spectrometer using sodium 3-trimethyl 6)-D-galactose (6): 9.4 mg from silylpropionate (c = 0) as an internal standard, and fraction, Rf 0.10; [a ] D + 17° (c 0.26, water) (lit.8) 13C -NMR spectra were recorded with 1 ,4-dioxane [a ]D+20°); FABMS (pos.): m/z 505 [M+

(ƒÂ = 67.4), in D20 at 40•Ž. Ascending paper chro H] +. 3-0 -Methyl-D-galactose6): 26.9 mg from di matography was carried out using Toyo No. 51 A saccharide fraction, Rf 0.77. D-Galactose from filter paper (Toyo Roshi Kaisha, Tokyo), eluted fraction: [a ] p + 76° (c 0.64, with 6 : 4 : 3, 1-butanol-pyridine-water and de water) (lit. 9) [a ]D+80.2°). tected with silver nitrate reagent. Preparation of the oxidized galactan.10) Galac

ƒÀ-v-Galactan . The purified neutral polysac tan (284 mg) was oxidized with 0.02 M sodium charide (ƒÀ-D-galactan, 1.4 g) was prepared from metaperiodate for 12 days at 5•Ž in the dark, then the dry cells of C. vulgaris K-22 (700 g, Chlorella reduced with sodium borohydride. The neutralized

Industry Co., Tokyo) according to the method de solution was dialyzed against water for 2 days, and scribed in a previous paper.6) freeze-dried to give a solid. The solid was re- Mild acid hydrolysis (Fragmentation analysis). oxidized for 6 days and treated in the same way.

Galactan (50 mg X 18) was hydrolyzed with 0.2 M The polyalcohol was hydrolyzed with 0.05 M sul trifluoroacetic acid (10 mL) in a screw-capped furic acid for 18 h at 20•Ž, then neutralized, dia tube for 2 h at 100•Ž. The hydrolyzate from 18 lyzed, and freeze-dried (75 mg, 27% yield). tubes was collected and then evaporated to dry- Complete acid hydrolysis of galactan and oxi ness. The residue was fractionated by gel filtration dized galactan. The polymer (5 mg) was hydro

(15 X 900 mm column) on Bio-Gel P-2 (fine, 45- lyzed with 0.75 M sulfuric acid for 4 h at 100•Ž. 90 pm, Bio-Rad Laboratories, California), eluting Sugar analysis of the hydrolyzate was done as with water at 40•Ž. Fractions of 40 drops (1.3 mL) alditol acetates by GLC using a Shimadzu CBP5 were collected. The eluate was divided into four capillary column (50 m X 0.2 mm, 220•Ž, carrier fractions, tube Nos.75-80 ( fraction), gas N2). Nos. 81-86 (In-), Nos. 8795 (di-), and Nos. 98-103 Methylation analysis. Galactan or the oxidized

(mono-). Concentration of each fraction yielded galactan (15 mg X 4) was methylated twice by 49, 74, 196, and 229 mg of solids, respectively. Hakomori's method"' (in the case of oligosaccha

Isolation of . Further purifi rides 2 mg each were used), and then treated using cation of these solids by preparative paper chroma the method of Lindberg.12) The partially methylated tography (eluted with 6 : 4 : 3, 1-butanol-pyridine alditol acetates were analyzed by GC-MS on a Jeol water) gave six oligosaccharides. A mixture of JMS-AX505H mass spectrometer, using a Shima new disaccharides 1 and 2: 11.9 mg from trisac dzu CBPS or CBP1 capillary column (50 m X 0.2 chride fraction, Rf 0.48; FABMS (pos.): m/z 357 mm, 200•Ž, carrier gas N2).13)

[M + H] +; FABMS (neg.): m/z 355 [M - H] -. 'H- and 13C-NMR data, see Table 1. New RESULTS AND DISCUSSION 3: 7.0 mg from tetrasacchride fraction, Rf 0.64;

[a ]+38°(cD 0.37, water); FABMS (pos.): m/z The neutral polysaccharide, galactan was iso 371 [M+H] +, FABMS (neg.): m/z 369 [M- lated from a defatted preparation of C. vulgaris K- H] -. 'H-and 13C-NMR data, see Table 2.3-0 -ƒÀ-D- 22 cells using the method described in our previ Galactopyranosyl-D-galactose (4): 17.0 mg from ous paper.6) Complete acid hydrolysis of the galac disaccharide fraction, Rf 0.37; [a D+51° (c tan gave 3-0 -methyl-D-galactose and D-galactose 0.15, water) (lit.'' [a ] D+63° ); FABMS (pos.): in the molar ratio of 1.0 : 7.1. The configuration m /z 343 [M + H] +. 6-0 -ƒÀ-D-Galactopyranosyl-D- of D-galactose was determined by its optical rota galactose (5): 31.3 mg from disaccharide fraction, tion value. /9-OD-OGalactanfromChlorella vulgaris 327

Table 1. 1H-O and 13C-ONMR data for a mixture of 6-O0 -O(3-O0 -Omethyl-Ofl-OD-Ogalactopyroanosyl)-OD-Ogalactopyranose (1) and 6-O

0-OƒÀ-OD-Ogalactopyranosyl-O3-O0-Omethyl-OD-Ogalactopyranose (2).a

A Spectra (ƒÂ, ppm) were measured in D2O at 40•Ž with a Jeol JNM-OA500 spectrometer. 1H-O and 13C-Ochemical-Oshift as-O signments were made by DEPT, 1H-O1H COSY, 13C-O1H COSY and HMBC experiments. 6Assignments are exchangeable .

Table 2. 1H-O and 13C-ONMR data for 6-O0 -O(3-O0 -Omethyl-OƒÀ-OD-Ogalactopyranosyl)-O3-O0 -Omethyl-OD-Ogalactopyranose (3) .a

a Spectra (~ , ppm) were measured in D2O at 40•Ž with a Jeol JNM-OA500 spectrometer. 1H-O and 13C-Ochemical-Oshift as-O signments were made by DEPT, 1H-O1H COSY, HMQC and HMBC experiments. 328 J. Appl. Glycosci., Vol. 48, No. 4 (2001)

Mild acid hydrolysis of the galactan with 0.2 M Acid hydrolysis of disaccharides 4 and 5 (Rf trifluoroacetic acid, followed by gel filtration on a 0.37 and 0.27, respectively), and tisaccharide 6

Bio-Gel P-2 column and preparative paper chro (Rf 0.10) gave only D-galactose. These homoge matography, yielded one monosaccharide, five di neous oligosaccharides were identified as 3-O -ƒÀ- saccharides (1, 2, 3, 4, and 5), and one trisaccha D-galactopyranosyl-D-galactose (4),7) 6-O -ƒÀ-D- ride (6). The monosaccharide (Rf 0.77) was identi galactopyranosyl-D-galactose (5),8) and 0 -ƒÀ-D- cal with 3-O -methyl-D-galactose6)according to the galactopyranosyl- (1•¨6) -O - ƒÀ-D-galactopyranosyl- spectra data including 1H-NMR. (1-;6)-D-galactose (6)8) by their MS and NMR A mixture of two new disaccharides 1 and 2 (Rf spectra, and optical rotation values. The results of 0.48) was obtained and a new disaccharide 3 (Rf methylation analysis of 4, 5, and 6 also supported 0.64) was isolated as colorless solids. On gel filtra their structures, as shown in Table 3. tion, disaccharides 1 and 2 were eluted in the tri The oxidized galactan was prepared from the saccharide fraction. This could be ascribed to an parental galactan by exhaustive periodate oxida apparent increase of volume caused by methyl tion, followed by reduction and hydrolysis,]°' and groups introduced on the saccharide residue, as re- its yield was 27% of the weight of the original ga ported by Grellert and Ballou.14) lactan. Complete acid hydrolysis of the oxidized The positive and negative-ion FABMS spectra galactan yielded 3-O -methyl-D-galactose and D- of the mixture of 1 and 2 showed m/z 357 [M+ galactose in the molar ratio of 1.0: 2.4. H] + and m/z 355 [M-HI -, indicating a molecu As shown in Table 4, several galactitol deriva- lar formula C,3H24O11. The extensive NMR studies tives were detected by methylation analysis show- showed that it is a mixture (approximately 3 : 2) of ing the presence of 3-O-, 6-O-, and 3,6-di-O - disaccharides, 6-O -(3-O -methyl-ƒÀ-D-galactopyra glycosylated galactose moieties both in the galac nosyl)-D-galactopyranose (1) and 6-O -ƒÀ-D-galacto tan and the oxidized galactan. Molar ratio of 3-O - pyranosyl-3-O -methyl-D-galactopyranose (2). But, methylgalactose (29%) in the complete acid hy the mixture was difficult to isolate as separate drolysis of the oxidized galactan coincided with compounds. As shown in Table 1, 1H- and 13C that of 2,3,4,6-tetra- and 2,3,4-tii-O-methylgalactoses chemical-shift assignments of 1 and 2 were made (total 30%) derived from 3-O -methylgalactose in by DEPT, 1H-1H COSY, 13C-1H COSY, and the methylation analysis. These results showed that HMBC experiments. The anomeric configuration the backbone of the galactan has 3-O -glycosylated of 3-O -methylgalactose in 1 or galactose in 2 were galactose residues and 3,6-di-O -glycosylated resi determined to be ƒÀ by J,,2. Moreover, J,,2 of the due in the branching regions. Furthermore, the reducing-end sugars showed that 1 and 2 are ano methylation analysis also suggested that the side meric mixtures (approximately 1 : 2, respectively). chain consists of fourteen sugar chains including The positive and negative-ion FABMS spectra of 3 ten 6-O -glycosylated galactose residues, three 6- showed m/z 371 EM +H] + and m/z 369 EM- 0 -glycosylated 3-O -methygalactose residues, and H] -, indicating C,4H26011. 1H- and 13C- NMR spec one nonreducing terminal galactose (Fig. 1). tra of 3 (Table 2) showed it to be a disaccharide Thus, by fragmentation analysis, methylation consisting of two molecules of 3-O -methyl-D- analysis, and periodate oxidation studies, the neu

galactose and having a ƒÀ-(1•¨6) glycosidic link- tral polysaccharide from Chlorella vulgaris K-22 age. There were a - and ƒÀ-anomeric proton signals cells was shown to be a new type of ƒÀ-D-galactan

(J,,2=4 and 8 Hz, respectively) for the reducing- having 1,3-linkages in the backbone, 1,6-linkages end sugar as a mixture of approximately 1 : 2. The in the side chains, and 3-O -methylgalactose in the

configuration of two molecules of 3-O -methyl- branching regions. galactose in 3 was assigned by its optical rotation Branched ƒÀ-D-galactan containing both 1,3- and value. Thus, the structure of new disaccharide 3 1,6-linkages (branched 3,6-D-galactan) is exempli was elucidated to be 6-O -(3-O -methyl-ƒÀ-D- fied by the arabinogalactan (Type II) from larch- galactopyranosyl)-3-O -methyl-D-galactopyranose. wood.15-"' The main molecular framework of the ƒÀ-D-Galactan from Chlorella vulgaris 329

Table 3. Methylation analysis of oligosaccharides 4, 5, and 6.a

Fig. 1. A possible partial structure of ƒÀ-D-galactan.

Data were analyzed by GC-MS using a Shimadzu CBP1 capillary column. bperacetylated (1-deuterio)-tetra- or tri-O - methylgalactitol. `Relative to that of 1,5-di-O-acetyl-(1- deuterio)-2,3,4,6-tetra-O -methylgalactitol. d+, + +, + + + : approximate molar ratio, based in the area of each peak.

Table 4. Substitution patterns of Galp residue by methylation analysis of the galactan and oxidized galactan.a

Data were analyzed by GC-MS using a Shimadzu CPB5 capillary column. The galactan and oxidized galactan con tained 12% and 29% of 3-O -methyl-D-galactose, respectively. The oxidized galactan was obtained in 27% yield from the galactan. bperacetylated (1-deuterio)-tetra-, tri-, or di-O -methylgalactitol. `Relative to that of 1,5-di-O -acetyl-(1-deuterio)- 2,3,4,6-tetra-O -methylgalactitol. arabinogalactans found in plants is basically L- tein of C. vulgaris K-22 cell and that from extracts arabinosyl-substituted branched 3,6-D-galactan, but of the same cell may result from the difference of has no mono-O -methylated galactose as a constitu their roles in the cell. ent.15)On the other hand, some algae have sulfated galactans containing D-galactose, 2-O -methyl-L- We thank Mr. Shuichi Kasama and Ms. Sanae Fukuda galactose, 4-O -methyl-D-galactose, and/or 6-O - for assisting with the experimental work, and Mr. Isao methyl-D-galactose as constituents. Some galacto Maruyama of Chlorella Co., Ltd. for the Chlorella cell oligosaccharides having the above mono-O - preparations. methylated were obtained from their REFERENCES partial acid hydrolyzates and partial acetolyzates (Aeodes ulvoidea 18,19)and Pachymenia car 1) T. Hasegawa, M. Okuda, M. Makino, K. Hiromatsu, nosa20,21).To the best of our knowledge, disaccha K. Nomoto and Y. Yoshikai: Hot water extracts of rides having 3-O -methyl-D-galactose, 1, 2, and 3 Chlorella vulgaris reduce opportunistic infection with are new ones, so the neutral polysaccharide of Listeria monocytogenes in C57BL/6 mice infected Chlorella vulgaris seems to be a new type of with LP-BM5 murine leukemia viruses. Int. J. Im munopharmacol, 17, 505-512 (1995). polysaccharide, a new galactan. Examination of 2) K. Noda, N. Ohno, K. Tanaka, N. Kamiya, M. Okuda, physiological activities of this galactan is the sub T. Yadomae, K. Nomoto and Y. Shoyama: A water ject for a future study. The difference between the soluble antitumor glycoprotein from Chlorella vul structure of galactan from extracellular glycopro garis. Planta Med., 62, 423-426 (1996). 330 J. Appl. Glycosci., Vol. 48, No. 4 (2001)

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