Sulphated polysaccharides of the grateloupiaceae family

Carbohydrate Research

Elssner PrInted

Publuhrng m Bclgnun

Company

SULPHATED FAMILY PART

IV’.

POLYSACCHARIDES

MIXHYLATION

J R NUNNANDH November

ANALYSIS

OF THE GRATELOUPIACEAE

OF PHYLLYhENAN

AND

DESULPHATEXI

PHYLLYMENAN

PAROB

Chemrstry Department, (Received

145

Amstc_dam

Rhodes Umversay, 25th,

Grahamstozvn (South Africa)

1969)

Methylatlon studies of phyllymenan and desulphated phjllymenan mdlcate that the Dgalactosidlc linkages are essentially a-(1-+3) and /I-(144) Th.s information, together with previous data, has led to a partial structure for the polysacchande. INiODUCTION

Previous work on phyllymenan’, a sulphated polysacchande from the red seaweed Phyll>menla cornea, revealed that the polymer contains D-galactose, 6-02-0-methyl-D-galactose, xylose, and methyl-D-galactose, 4-0-methyl+galactose, ester sulphate, that all of the sulphate ester groups are attached to units linked through posltlon 3, and that the polymer yields a bgh proportion of PO+D-galactopyranosyl2-0-methyl-~galactose and a lesser amount of 4-0-/3-D-galactopyranosyl-D-galactose residues on hydrolysis In thrs paper, we report the results of methylation studies of desulphated phyllymenan and phyllymenan Itself. RESULTS

AND

DISCUSSION

The positions of the glycosldlc linkages were deduced by methyIatlon analysis of desulphated phyllymenan Phyllymenan was desulphatedl with methanohc hydrogen chloride The desulphated polymer was partially methylated ln methyl sulphoxlde with methyl sulphate and sodmm hydroxide, and methylatlon was completed m N,N-dlmethylformamlde with methyl iodide and sliver oxide After hydrolysis, the products were fractionated by gradient eluhon from a charcoal-Cehte column and charactensed (molar ratxos 111brackets) as 2,3,4,6-tetra-0-methylgalactose (1 6), 2,4,6-tn-0-methyl-D-galactose (10 l), and 2,3,6-tri-0-methyl-D-galactose (8 ‘7)_ The presence m the native and desulphated polymers of a fugh propomon of monomethyl ethers of galactose renders dticult an unambiguous mterpretation of the results of the methylaoon analysis. Nevertheless, some conclusions can be drawn if these results are considered together wth those of partial hydrolysis, penodate oxldatlon, and a&ah-treatment stu&es ofphyllymenan and desulphated phyllymenan’. Curbohyd

Res , 14 (1970) 145-150

146

J

R. NUNN,

H. PAROLI’

The 2,3,6-tri-0-methyl-D-galactose IS considered to have arisen from (144)~hnkec D-galactose and (l-+4)-linked 2-U-methyl-D-galactose resrdues m the polymer The Jsolatlon of 4-O-/?-D-galactopyranosyl-D-galactose and 4-0-/3-D-galactopyranosyl-2 O-methyl-D-galactose (1)from a partial hydrolysate of phyllymenan supports thr conclusion, and mdicates that at least the majority of the (1+4) linkages have the /I-D cor&guration The 2,4,6-trr-O-methyl-D-galactose IS considered to have ariser from (143)-lmked D-galactose and (143)~hnked 6-0-methyl-D-galactose residues rr the polymer The presence of galactose and 6-0-methylgalactose m the hydrolysatc of desulphated, penodate-oxrdised phyllymenan, coupled with the absence of branch mg m desulphated phyllymenan (as mdrcated by the absence of di- or mono-0 methylgalactoses m the hydrolysate of methylated, desulphated phyllymenan), supporn this conclusion The isolation, on partial hydrolysis of phyllymenan, of a small propor Don of a disaccharide’,, to which the structure 3-&x-(2-O-methyl-D-galactopyranosyl) D-galactose (2) has been assigned, suggests that at least some of the (1+3) lmkages have the CL-Dconfiguration In common with aeodan3 and other polysaccharides, phyllymenan was difficult to methylate Repeated treatment with methyl sulphate and aqueous sodmm hydroxide afforded a partially methylated product. Further methylation was achieved by dispersing the product in methyl sulphoxide and treatmg the dispersion with Purdie’s reagents4 The Infrared spectrum of the final product showed a small hydroxyl peak. The methylated polysaccharide was hydrolysed, and the components were separated by cellulose-column chromatography 2,3,4,6-Tetra-0-methylgalactose, 2,3,6-tri-Umethyl-D-galactose, 2,4,6-tri-U-methyIgalactose, 2,6- and 4,6-di-O-methyl-D-galactose, 2-O- and 4-0-methyl-D-galactose, and D-galactose were isolated The results of the methylation of phyllymenan must be treated with some caution because of the extreme difficulty encountered m lmethylatmg the polysaccharade, and hence the uncertamty of the extent of methylation The major methyl ether isolated, tzz , 4,6-d:-0-methyl-D-galactose could only have arisen from (1+3)linked D-galactose 2-sulphate (or the correspondmg 2-sulphated 6-O-methyl derivative) residues m the polymer The Jsolation of the 2,4,6-tri-O-methyl derivative suggests that not all of the (l-+3)-linked D-galactose and/or 6-O-methyl-D-galactose residues are sulphated The 2,3,6-trl-U-methyl-D-galactose IS consldered to have arisen from (1+4)-lmked D-galactose and (1+4)-lmked 2-O-methyl-D-galactose residues m phyllymenan The 2,6-di-0-methyl-D-galactose could have arisen from (1+3)-lmked D-galactose and/or 6-0-methyl-D-galactose residues carrymg sulphate at position 4 The alternatrve umts, VIZ, (1+4)-lurked D-galactose 3-sulphate and the corresponding 6-O-methyl derivative, are alkah labile and are therefore ruled out. On the other hand, it may be due to undermethylation The D-galactose and mono-Omethyl-D-galactoses are almost certamly due to undermethylation, since their presence 1s not consistent with the followmg observations (u) the ester sulphate content of phyllymenan, (b) the absence of branching m the macromolecule, and (c) the high yield of 2,3,6-tn-U-methyl-D-galactose m the hydrolysate of methylated, desulphated phylfymenan. carbohyd Res , 14 (1970) 145-150

MEITIYLATION

STUDIES

OF PHYLLYMENAN

147

At present, no unique structure can be proposed for phyllymenan However, the followmg partial structure IS conststent wtth the accumulated evtdence

[?+o*] I-J R = H and lessfrequentlyCH3, R’

= SO;

and lessfrequentlyH, R” = CH3andlessfrequentlyH

A lmear, repeating structure is shown, not because an alternating sequence of (143) and (l-4) hnkages has been conclustvely proved, but because the presence of approximately equal proportions of (1+3) and (l-4) linkages m the desulphated polymer is suggestive of an alternating structure The recent isolation m this laboratory of a trisaccharide’, which furmshes 1 and 2 on partial hydrolysis, mdlcates that at least some regions of alternatmg structure do exist

Concentratton of solutions was carried out at 40”/20 mm, and specmc rotations were measured m water Paper chromatography was carrted out on Whatman No 1 paper with (I) 8 2 1 ethyl acetate-pyndme-water and (2) 18 3 1 4 ethyl acetate-acetic actd-formtc acid-water p-Amsidme hydrochloride’ and amhne-diphenylamme-phosphoric acid6 are sprays a and b, respectively. Thin-layer chromatography (t 1 c ) was carrted out on glass plates coated with Sthca Gel G contammg calcmm sulphate as bmder, employing 85 7 butanone-water as solvent RGnland RTafGvalues refer to the rates of travel of sugars relattve to galactose and 2,3,4,6-tetra-0-methylgalactose, respecttvely. Infrared spectra were recorded on a Beckman IR-8 spectrophotometer Desttlplzatron of plzyllynzezzan - Phyllymenan (10 g, [c&,~ +63 3”, SO:-, 19 62%) was shaken with 0 15~ methanohc hydrogen chloride (750 ml) for 48 h at room temperature, and the desulphated polysaccharide was isolated m 72% yield as described previously’ (Found SO:-, 0 0%) Metlzylutzon of deszdphated plzylIymezzan The polysaccharide (2 0 g) m methyl sulphoxide (50 ml) was treated wtth methyl sulphate (7 5 ml) m the presence of powdered sodmm hydroxtde (15 g) under an atmosphere of mtrogen The methyl sulphate was added m two equal portrons at Intervals of 1 h Further addition of methyl sulphate and sodmm hydroxide was made after 2 h, and the mixture was vrgorousIy stirred overnight, dduted with water, neutrahsed, and extracted with chloroform The washed chloroform extract was dried (Na,SO,) and evaporated to a glass (1 61 g) (Found OMe, 23 5%) Paper chromatography (solvent I) revealed the presence of galactose, 2-O- and 6-0-methylgalactose, 2,6- and 4,6-dt-O-methylgalactose, 2,3,6- and 2,4,6-tn-0-methylgalactose, and 2,3,4,6-tetra-0-methylgalactose The partially methylated polymer (1 2 g) was dtssolved m NJV-dtmethylformamrde (15 ml) and vrgorously strrred wrth methyl Iodide (15 ml) and srlver oxtde (15 g) for CorbohydRes , 14 (1970) 145-150

148

J.

R

NUNN,

H

PAROLE

24 h at room temperature. The product (I 04 g) was rsolated after a further addmon of methyl rodrde and srlver oxrde (Found: OMe, 43.8%). A very small hydroxyl peak was observed in the I r spectrum (KBr drsc) Hy&olysrs of desnlphated, methyiated phyllymenan, and separatron of the products. - The methylated polysaccharrde (0 54 g) was hydrolysed with N sulphurrc acrd for 6 h, neutrahsed (BaCO,), and concentrated to a syrup (0 45 g) Paper chromatography (solvent I) revealed the presence of 2,3,6- and 2,4,6-trr-O-methyl- and 2,3,4,6-tetra-O-methylgalactose The syrup was apphed to a column (48 x 4 8 cm) of charcoal-Cehte and eluted wrth a lmear gradrent, from O-2%, of aqueous butanone (5 4 htres) followed by 2-3% aqueous butanone (4 0 lures) Fractions (45 ml) were collected and, on the basrs of paper chromatography (solvent _Z),were recombmed into the followmg three fractrons. Fractron I (206 mg) was eluted wrth 2 02-2 18% aqueous butanone (630 ml) and was chromatographrcally mdrstmgurshabIe (I?,,, 0 73, solvent I) from 2,4,6-trr0-methylgalactose The syrup, [LX&~+88 3”, crystalhsed as the hydrate, m p 83-85”, on standmg The amhne derrvatrve, after recrystahlsatron from ethyl acetate, had m p 168-169” and mixed m p (wrtb an authentrc sample of 2,4,6-trr-O-methyl-Nphenyl-D-gaIacrosy~amme3) 169-170” Fractron 2 (178 mg) was eluted wrth 2 25-2 4% aqueous butanone (580 ml) and was chromatographrcally mdrstmgmshable (R -,,,,,o 0 79, solvent I) from 2,3,6-trr-Omethylgalactose The syrup, [c&~ +97.3”, was converted mto the lactone, whrch had m p and nuxed m p (wrth authentrc 2,3,6-trr-O-methyl-D-galactono-1,4-lactone3) 99-100”. Fractron 3 (36 mg) was eluted wrth 2 65-2 80% aqueous butanone (600 ml) and was cbromatographrcally mdrstmgr.ushabIe (R,, 1; solvent I) from 2,3,4,6-tetra-Omethylgalactose The amhne derrvatrve, after recrystdlhsatron from ethanol, had m p. and mrxed m p (with authentrc 2,3,4,6-tetra-O-methyl-N-phenyl-~-galactosylam~ne’) 188-189”. Methylatlon of polysaccharlde - The polysaccharrde (26 2 g, [cL]~~+63 3”, so:-, 19 62%) m water (500 ml) was treated slowly and simultaneously (under an atmosphere of mtrogen) with methyl sulphate (150 ml) and 30% aqueous sodmm hydroxrde (450 ml) wrth vrgorous strrrmg during 7 h The mixture was then stirred for a further 17 h After repeatmg the methylatron procedure a further srx times, the mrxture was dralysed against running tap-water (10 days) and concentrated to ca 500 ml, and the add&on of the above reagents was repeated a further SIX times The polysacchande (24 g) was Isolated from the mrxture by freeze-drying, after dralysrs and concentratron T 1 c (spray b) of a neutrahsed acid-hydrolysate revealed galactose together with a large number of rts methyl ethers The methylatron procedure was therefore repeated a further five times on a portion (16 g) of the partrally methylated material The polysaccharrde (15 8 g) was Isolated as above (Found OMe, 17.1; N, 0 3; SO:-, 16 38%) T I c of a hydrolysate revealed the presence of galactose, CO-methyl-, 2-O-methyl-, 2,6-dr-O-methyl-, 4,6-dr-O-methyl-, 2,3,6-trr-O-methyl-, and 2,4,6-trr-O-methyl-galactose, together with mmute traces of 2,3,4-tn-U-methylCwbohyd

Res , 14 (1970) 145-150

METHYLATION

STUDIES

149

OF PHYLLYhENAN

and 2,3,4,6-tetra-U-methyl-galactose The partially methylated polymer (2 3 g) was mixed with dry methyl sulphoxlde and vigorously stlrred for 2 h at room temperature to disperse the polysaccharlde Sliver oxide (30 g) and methyl lodlde (60 ml) were added, and the mixture was stirred for 24 h at room temperature Two further addltrons of Purdle’s reagents4 were made, and each time the nuxture was &u-red for 24 h at 50” A large volume of water was added to preclpltate the salts, the mixture was centrtiuged and dlalysed, and the polysacchande (1 1 g) was isolated by freeze-drying (Found OMe, 24 7; N, 0 27; SO:-, 6 7%) A very small hydroxyl peak was observed m the 1 r spectrum (KBr disc) Hydrolysrs of mettzylated polysacclzunde

and separation of the products

-

The

methylated polysacchande (1 g) and N sulphunc acid (15 xn1)were heated on a bollm,o water-bath for 16 h The hydroljsate was neutrahsed with barium carbonate, filtered,

and concentrated to a brown syrup (0 9 g), which was applied to a celIulose column (45 x5.4 cm) and the methylated sugars were eluted with solvent 2. Each fraction (ca 60 ml) was analysed for sugars by t 1 c (spray b), and slmllar fractions were combmed, evaporated to dryness under reduced pressure, and extracted with ethyl acetate or methanol The extract, after removal of the solvent, was placed under high vacuum to remove the last traces of acetlc acid, the followmg products were subsequently Identfied Tubes 1-5 contamed the degradation products (86 mg) Tzrbes 6 and 7 contained 2,3,4,6-tetra-0-methylgalactose (28 mg) The syrup was decolounsed ulth charcoal and converted mto a crystalhne amlme denvatlve wluch, after recrystalhsatron from ethanol, had m p and mixed m p (with authentic 2,3,4,6-tetra-O-methyl-N-phenyl-D-galactosylamlne) 188-l 89”) ht * m p 189-190”. Tube 8 gave a syrup (30 mg), [a]; f89” (c 0 56), chromatograplucally (RTMG 0 86, grey spot, spray b) ldentlcal with 2,3,6-tn-O-methylgalactose The denved lactone had m p and mixed m p. (with authentic 2,3,6-tri-0-methyl-D-galactono-1,P lactone3) 99-100” Tubes 9 and 10 contamed a nuxture (102 mg) of 2,3,6-tn-O-methylgalactose (R,, 0 66, blue&-OfG 0 86, grey spot, spray b) and 2,4,6-tn-0-metbylgalactose green spot, spray b) The 2,3,6+somer constituted ccz 80% of the nurture Tubes 11-14 afforded a syrup (67 mg) wluch was shown by paper and thmlayer chromatography to be a mixture of 2,3,6-trl-, 2,4,6-tn-, and 2,6-&-U-methylgalactose, together wth a trace of 2,3,4-tn-U-methylgalactose and an unldenttied sugar (&MG 0 7, Whatman No 1, carmme spot, spray a). Tube 15 contained no carbohydrate Tzzbe 16 afforded a syrup (51 mg, &nlG 0 53 ; grey spot, spray b) which crystalhsed from ethyl acetate and, after recrystalhsatlon from the same solvent, had [a]b6 +47 1 (2 mm) --, f85 8” (c 0 7), m p. and nuxed m p (w1t.h authentic 2,6&-Omethyl-D-galactose) 128-129”, ht8 [c& +45+ +88”, m p 128 5-130’. Tubes 17 and I8 contamed a nuxture (103 mg) of 2,6- (&MG 0 53) and 4,6-dl-Omethylgalactose (RTMG 0 33, blue spot, spray b) The 4,6-dlmethyl ether, whch Curbohyd Res , 14 (1970) 145-150

150

J R

NUYN,

H

PAROLE

constituted ca. 70% of the nuxture, readily crystalhsed when the syrup was mlxed with ethyl acetate Tubes 29-24 afforded a syrup (103 mg) which crystalhsed (charcoal) from ethyl acetate The product (m p 140-142”), after two recrystalhsatlons from the same solvent, had m p 146-147”, [a]h6 + 131 2 (2 mm)-, +72” (c 0 64) The mlxed m p with a sample of 4,6-dl-O-methyl-D-galactose3 isolated from methylated aeodan was 142” The Infrared spectra of the two sugars were Identical The denved phenylozasone had m p and mixed m p 154-155” Tubes 25-35 contamed no carbohydrate Tubes 36-41 contained 2-O-methylgalactose (58 mg, RGal2 2, solvent 2) which crystalhsed from ethanol-ethyl acetate and, after recrystalhsatlon from the same solvent, had [oL]~~+85 2” (c 0 74), m p and mixed m p (with authentic 2-O-methylD-galactose ‘) 147-149” Tztbe 42 contamed no carbohydrate Tubes 43-54 afforded a chromatographlcally pure syrup (99 mg, RGIII1 80, solvent 2) which crystalhsed from ethanol and, after recrystalhsatlon from ethanolethyl acetate, had [c&,’ +49 (5 mm)+ +73 0” (c 0 63), m p and mlxed m p (with authentic 4-O-methyl-D-gaiactose3) 202-204” Tubes 55-56 contained no carbohydrate Tubes 57-63 contamed galactose (228mg), [a]g + 81” (c 0 66), m p and mixed m p 166-167” The derived galactanc acrd had m p and mixed m p 212-2 13” REFERENCES 1 Part III J R NUNN A?.?) H PAROLE, Carbohyd Res ,9 (1969) 265 2 J R NUNN AND H PAROLIS, unpubhshed results 3 J R NUNN AND H PAROLIS, Carbohyd Res, 6 (1968) 1 4 -I- PURDIE AND J c IRVINE, J Ckm SOC, (1903) 1021 5 L

HOUGH,

J K

N

JONES, AND W

6 S SCHWIMMERAND A

H

WADMAN,

J

Chem

Sot,

BEVENUE,Scrence, 123 (1956) 543 7 3 R NUNN AND H PAROLIS, Carbohyd Res, 8 (1968) 361 8 A L CLINGMAN AND J R NUNN. J Chem Sot, (1959) 493 Cerbohyd

Res , 14 (1970) 145-150

(1950)

1702