STRUCTURAL STUDIES ON AN ARABINOXYLAN FROM PEA SKIN (PISUM SATIVUM)

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1 STRUCTURAL STUDIES ON AN ARABINOXYLAN FROM PEA SKIN (PISUM SATIVUM) PART I. METHYLATION STUDIES N. BANERJI AND C. V. N. RAO Department of Macromolecules, Indian Association for the Cultivation of Science, Calcutfa-39, India Received April 4, 1963 ABSTRACT The polysaccharide extracted by 4% sodium hydroxide solution from pea skin (Pisum sati.dunz) was shown to be composed of D-xylose and L-arabinose in a ratio of 5:l. Hydrolysis of the fully methylated arabinoxylan yieided 2,3,5-tri-0-methyl-L-arabinose, 2,s-di-0-methyl- D-xylose, and 2-0-methyl-D-xylose in the molar ratio of 1:4.2:1.1 and traces of 2,3,4-tri-0- methyl-d-xylose and D-xylose. From these results and those of periodate oxidation studies, a structural formula for the arabinoxylan has been proposed. Substantial work has been done in the identification of carbohydrate constituents of pea (I), but no polysaccharide has been isolated from pea skin. The present paper describes the isolation and structural chemistry of an arabinoxylan that is present in pea skins. Powdered pea skins were extracted successively with a boiling mixture of benzene and alcohol, hot water, and 0.4% and 4% alkali solutions. The polysaccharide extracted by hot water gave a blue color with iodine and yielded only glucose on hydrolysis, indicating it to be a starch. The polysaccharides, extracted by 0.4% and 4Yo alkali solutions, gave on hydrolysis arabinose and xylose in the same ratio together with trace quantities of galactose. As the former one was obtained in very poor yield the later was selected for structural studies. The polysaccharide extracted from pea skin by 4y0 sodium hydroxide solution, having specific rotation --86.g0, gave on hydrolysis D-xylose and L-arabinose in the ratio of 5:l respectively. The polysaccharide was purified (2) by repeated complex formation with Fehling's solution. After three treatments the materia1 obtained had specific rotation -87.6' and gave arabinose and xylose in the ratio of 1:5. Galactose and uronic acid originally present in traces were removed during purifications. As there was no change in specific rotation or the ratio of arabinose to xylose after repeated purifications, the material was considered to be homogeneous. The arabinoxylan was methylated to yield a fully methylated derivative, having a weight-average molecular weight 5.12 X lo5. Hydrolysis of the fully methylated arabinoxylan yielded three major components, viz., 2,3,5-tri-0-methyl-L-arabinose, 2,3-di-0-methyl-D-xylose, and 2-0-methyl-D-xylose, which were characterized by their crystalline derivatives. Trace amounts of 2,3,4-tri-0- methyl-~-x~lose and D-xylose were also obtained. Column chromatographic analysis showed that tri-0-methyl-l-arabinose, di-0-methyl-d-xylose, and mono-0-methyl-d-xylose were present in the ratio of 1:3.81: 1. A small quantity of the mixture was resolved by quantitative paper chromatography and the sugars were estimated by alkaline hypoiodite method (3). The ratio of trimethylarabinose to dimethylxylose to monomethylxylose was 1:4.2:1.1, which is in close agreement with column chromatographic results. The above results indicate that the main chain of the arabinoxylan from pea skin is Canadian Journal of Chemistry. Volume 41 (1963)

2 BANERJI AND RAO: ARABINOXYLAN FROM PEA SKIN 2845 composed of D-xylopyranose molecules joined through linkages, with branching mainly through position 3 of certain xylose units. The majority of non-reducing end positions are occupied by L-arabinofuranosyl residues. Trace amounts of 2,3,4-tri-0- methyl-d-xylose probably originated from a few end points of the macromolecules. The trace quantities of D-xylose present in the mixture are of uncertain structural significance. The simplest structure of the repeating unit of the arabinoxylan which can accommodate the above facts is given below: Xp-D-xylopyranose residues; Ar--L-arabinofuranose residues From the negative rotation of the polysaccharide and its methylated derivative, it is concluded that the majority of the linkages are of P-type. The above structure is supported by the fact that during periodate oxidation, 0.84 mole of periodate was consumed per anhydro pentose unit, the theoretical value being 0.83 assuming the above structure. As the non-reducing end groups were arabinofuranose residues, no formic acid was liberated but an equal number of xylose residues (16.2%) at the branch points should remain unoxidized. The periodate-oxidized polysaccharide on hydrolysis yielded 14.2% of xylose, a figure in agreement with that expected theoretically. The fact that the arabinofuranose residues were present as single-unit side chains was further confirmed by subjecting the arabinoxylan and its methylated derivative to mild hydrolysis, taking advantage of the labile linkage of non-reducing arabinofuranose residues. Hydrolysis of the polysaccharide with dilute oxalic acid solution for 1.5 hours yielded a degraded polysaccharide and the supernatant liquid contained arabinose only. The degraded polysaccharide on hydrolysis afforded xylose (80.2%) and arabinose (5.4%) (these and subsequent values are expressed as percentages of the undegraded polysaccharide). The degraded polysaccharide was oxidized-with sodium metaperiodate and the xylose residues unattacked by periodate were estimated to be 6.1%. The fully methylated polysaccharide was heated with dilute aqueous methanolic hydrochloric acid under controlled conditions (4) and the degraded methylated polysaccharide was precipitated, remethylated, and subjected to methanolysis and hydrolysis. The hydrolyzate was found to contain 2,3,5-tri-0-methyl-L-arabinose, 2,3-di-0-methyl-Dxylose, 2-0-methyl-D-xylose, and traces of 2,3,4-tri-0-methyl-~-xylose and D-xylose. Quantitative estimation of tri-0-methylarabinose, di-0-methylxylose, and mono-0- methylxylose showed that the length of the repeating unit is about 16 pentose units and the ratio of 2,3,5-tri-0-methylarabinose to 2,3,4-tri-0-methylxylose is 12:l. Removal of arabinose residues from the methylated polysaccharide did not result in any substantial increase in the amount of trimethylxylose in the hydrolyzate of the degraded and remethylated product. These results together with those of periodate oxidation studies on the original and degraded polysaccharides show that arabinose is present as single-unit side chain in the arabinoxylan. The arabinoxylan obtained from pea skin is structurally similar to the arabinoxylan obtained from other sources, viz., from wheat (5, 6), rye flour (7), barley flour (4), but the length of the repeating unit varies from source to source. EXPERIMENTAL Methods The following solvent systems (v/v) were used for partition chromatography: (A) ethyl acetate - pyridine - water (8:2: 1) ; (B) n-butanol - acetic acid - water (4: 1:5), upper layer; (C) n-butanol-benzene-pyridine-

3 2846 CANADIAN JOURNAL OF CHEMISTRY. VOL. 41, 1963 water (5: 1:3:3), upper layer; (D) benzene-ethanol-water (169:47: 15), upper layer; (E) methyl ethq 1 ketone - water azeotrope; and (F) n-butanol-ethanol-water (4:1:5), upper layer. The spray reagents used were: (a) saturated solution of aniline oxalate in \\ ater (8); (b) aniline hydrogen phthalate; and (c) p-anisidine hydrochloride (37, solution in butanol). All specific rotations are equilibrium values. Unless otherwise stated all evaporations tvere carried out in vacuo at 30-40'. LVhatman Yo. 1 filter papers were used for paper partition chromatography. Large quantities of sugar mixture were separated by IVhatman No. 3MM papers. Isolation of the Polysaccharide Peas were kept under water overnight and the skins were separated by hand. They were dried in an oven at 35-40" and powdered. The powder (200 g) in batches mas extracted for 24 hours with a boiling mixture of benzene and ethanol (2:1, v/v), to remove fats and coloring matter. The material (200 g), which was free from fats and coloring matter, was extracted with hot water (4 1.) and the polysaccharide, precipitated by alcohol, was triturated and dried. Yield 0.4$,, moisture 9.2%, ash 0.05%, nitrogen , [CI']D~~ +98" (c, 0.5 in 4% sodium hydroxide solution). The polysaccharide developed a blue color with iodine solution and yielded only glucose when hydrolyzed. The remaining material Tvas subjected to extraction twice with 0.47, sodium hydroxide solution (750 ml each time) at room temperature in an atmosphere of nitrogen for 16 hours. The polysaccharide was precipitated by addition of the acidified extract to twice its x-olume of acetone. Yield 0.6%. The polysaccharide on hydrolysis gave xylose and arabinose in the ratio of 5:1, with traces of galactose. The material left after the above treatment was extracted four times with 47, sodium hydroxide solution (1500 ml) and the polysaccharide was isolated as described above. The product xvas dialyzed for 7 days, reprecipitated, and dried to yield an an~orphous powder. Yield 2%, moisture 11.2%, ash 1.2%, [a]$ -86.9" (c, 0.5 in 47, sodium hydroxide solution), pentosan (9) 96.77,, methoxyl 0.347,, uronic acid (9) 0.84Tc. Intrinsic viscosity 0.89 dl/g (in 4% sodium hydroxide solution). Hydrolysis, Identi$cation, and Esti?nation of Xylose and Arabinose The polysaccharide (1 g) on complete hydrolysis yielded xylose and arabinose together with a trace quantity of galactose. D-Xylose and L-arabinose were separated by paper chromatograph>-, The dry sirup containing D-xylose w-as crystallized from dry ethanol; m.p. and mixed n1.p "; [cy]d~~ +lgo (6, I in water), lit. +18' (11). The dry sirup corresponding to L-arabinose was crystallized from dry ethanol; m.p. and mixed m.p "; [a]n " (c, 1 in water), lit. +103" (12). The polysaccharide ( g) mas hydrolyzed with 2 N sulphuric acid and the component sugars were estimated quantitatively by the periodate method (lo), using D-ribose as reference sugar. Xylose and arabinose were found to be present in the ratio of 5:l. Puri$cation of the Polysaccharide.. The crude polysaccharide was purified by cornplexing mi-th Fehling's- solution (2). The arabinoxylan (20 g) was dissolved in 47, sodium hydroxide solution(l:5 1.) in an atmosphere of nitrogen, and freshly prepared Fehling's solution (1.5 1.) was added whereupon the copper complex precipitated. The precipitate was washed with ice-cold water (1 I.), and cold hydrochloric acid (0.5 N) was added to a suspension of the precipitate in water with stirring, until the copper complex decomposed giving, a slightly opalescent solution. The polysaccharide was precipitated by the addition of an equal volume of acetone and was isolated in the usual way. Yield 15 g, [cy]d~~ -87.6' (c, 1 in 47, sodium hydroxide solution). Intrinsic 7-iscosity 1.02 dl/g in 47, sodium hydroxide solution. Xylose and arabinose, estimated in the above manner, were found to be present in the ratio of 5: 1; galactose and uronic acid were absent. Methylation of the Arabinoxylan The arabinoxylan (5 g) was methylated by treatment first with di~nethyl sulphate and alkali and then with methyl iodide and silver oxide according to the method of Falconer and Adams (15, 16) to yield a fully methylated derivative (3.2 g) showing no hydroxyl absorption band in the infrared spectrum. OMe 38.05%; intrinsic viscosity 0.35 dl/g in chloroform; [CY]DZ9-58' (c, 1 in chloroform). iwolecular Weight Determination of the Methylated Arabinoxylan -411 measurements of the intensity of scattered light for molecular weight determination of the methylated arabinoxylan were carried out with a Brice-Phoenix light-scattering photometer (Model Oh ) using a semioctagonal cell. The concentration used was over a range of (1-4) X g/ml. The molecular weight was found to be 5.12X106, calculated by the dissymmetry method (17). lmethanolysis and Hydrolysis of the Methylated Arabinoxylan and Separation of Methyl Sugavs The methylated arabinoxylan (1 g) was refluxed with 37, methanolic hydrogen chloride (80 ml) till the rotation reached a constant value (18 hours). The resulting mixture of methyl glycosides was hydrolyzed with N hydrochloric acid (80 ml) till the rotation reached a constant value (16 hours). After neutralization

5 2848 CANADIAN JOURNAL OF CHEMISTRY. VOL. 41, 1963 filtered, and deionized. Chromatographic examination of the resulting solution showed the presence of 2,3,5-tri-0-methyl-L-arabinose and a trace of 2,3-di-0-methyl-D-xylose. The degraded methylated product, obtained in the above treatment, was remethylated twice by Purdie's method (21) to yield a degraded and fully methylated arabinoxylan (52 mg). OMe 38.77,, [cx]d~~-52.4" (c, 0.5 in chloroform). After methanolysis and hydrolysis of the methylated degraded polysaccharide followed by the usual treatments, the hydrolyzate afforded a sirup containing 2,3,5-tri-0-methyl-L-arabinose, 2,3-di-0-methyl- D-xylose, and 2-0-methyl-D-xylose with trace amounts of 2,3,4-tri-0-methyl-~-xylose and D-xylose as revealed by paper chromatography. The mixture was separated by quantitative paper chromatography and the amount of each sugar was estimated by alkaline hypoiodite. The ratio of trimethylarabinose, dimethylxylose, and monomethylxylose was 1.1:14.5:1.05. The tri-0-methyl pentose fraction was resolved chromatographically by solvent D into 2,3,5-tri-0-methyl-L-arabinose and 2,3,4-tri-0-methyl-D-xylose and quantitative estimation, by the alkaline hypoiodite method, showed them to be present in the ratio of 12:l. ACKNOWLEDGMENT The authors are thankful to Dr. P. Bagchi, Director of Research, East India Pharmaceutical Works Ltd., for suggesting the problem and his valuable advice and interest in the work reported in this paper. REFERENCES 1. A. POTTER, V. SILVEIRA, R. M. MCCREADY, and H. S. OWENS. J. Am. Chem. Soc. 75, 1335 (1953). 2. C. P. J. GLAUDEMANS and T. E. TIMELL. J. Am. Chem. Soc. 80, 1209 (1958). 3. E. L. HIRST, L. HOUGH, and J. K. N. JONES. J. Chem. Soc. 928 (1949). 4. G. 0. ASPINALL and R. J. FERRIER. J. Chem. Soc. 638 (1958). 5. R. MONTOGOMERY and F. SMITH. J. Am. Chem. Soc. 77, 3325 (1955). 6. A. S. PERLIN. Cereal Chein. 28, 370, 382 (1951). 7. G. 0. ASPINALL and R. J. STURGEON. J. Chem. Soc (1954). 8. R. H. HORROCKS and G. B. MANNING. Lancet, 256, 1042 (1949). 9. CHARLES DOREE. The methods of cellulose chemistry. 2nd ed. Chapman and Hall Ltd., London E. L. HIRST and J. K. N. JONES. J. Chem. Soc (1949). 11. J. K. GILLHAM and T. E. TIMELL. Can. J. Chem. 36, 410 (1958). 12. G. 0. ASPINALL, E. L. HIRST, and A. WICKSTROM. J. Chem. Soc (1955). 13. L. HOUGH, J. K. N. JONES, and W. H. WADMAN. J. Chem. Soc (1949). 14. L. BOGS, L. S. CUENDET, I. EHREXTHAL, R. KOCH, and F. SRIITH. Nature, 166, 520 (1950). 15. E. L. FALCONER and G. A. ADAX. Can. J. Chem. 34, 338 (1956). 16. G. A. ADAYS. Can. J. Chem. 36, 755 (1958). 17. P. DEBYE. J. Phys. Chem. 51, 18 (1947). 18. L. HOUGH, J. K. N. JOKES, and TV. H. TVADIVIAN. J. Chem. Soc (1950). 19. G. 0. ASP IN ALL^^^ R. J. STURGEON. J..Chem. Soc (1957). 20. P. FLEURY and J. LANGE. J. Pharm. Chim. 17, 107 (1933). 21. T. PURDIE and J. C. IRVINE. J. Chem. Soc. 83, 1021 (1903).

ACETONE DERIVATIVES OF d-ribose. II.

ACETONE DERIVATIVES OF d-ribose. II. BY P. A. LEVENE AND ERIC T. STILLER* (From the Laboratories of The Rockefeller Institute for Medical Research, New York) (Received for publication, June 14, 1934) The