Mannose-Containing Polysaccharides. I. The Galactomannans of

The galactomannans of lucerne and clover seed are found to be similarly constituted. Both polysaccharides are highly branched and contain n-galactopyr...
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Aug. 20, 1952

GALACTOMANNANS OF LUCERNE AND CLOVER SEEDS

carried out on each 10-ml. aliquot, after quenching by addition t o a large volume of water plus sodium acetate, by dilution to the proper concentration range and application of the colorimetric method of Lowry and Lopez.lD A Beckman spectrophotometer was used throughout the colorimetry, with the scheme calibrated a t a wave length of 700 mp.

[CONTRIBUTION FROM THE

DEPARTMENT OF

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Acknowledgment.-The suggestion of this overall project by Dr. Tenell L. Hill, and much useful discussion with D ~ ill ~ .and Avanual F, ~~~~l~~ are Fatefully BETHESDA14, MD.

ORGANIC CHEMISTRY. UNIVERSITY O F BRISTOL]

Mannose-Containing Polysaccharides. I. The Galactomannans of Lucerne and Clover Seeds1 BY P. ANDREWS,L. HOUGH AND J. K. N.

JONES~

RECEIVED JANUARY 10, 1952 The galactomannans of lucerne and clover seed are found to be similarly constituted. Both polysaccharides are highly branched and contain n-galactopyranose end-groups combined with chains of 1,4- or 1,g-linked D-madose residues, which are probably in the pyranose form.

Galactomannans are common constituents of the ungerminated seeds of leguminous plants, amounting in some cases to more than 40% of the total seed. They occur as mucilages in the endosperms of the seeds, from which they may be isolated by extraction with water. It is known that when the seeds germinate both the mucilage and the endosperm disappear, hence i t is argued that the galactomannan is a food reserve poly~accharide.~An extensive investigation of the seeds of more than 160 species of legumes by Wise and Appling4 and Anderson5has revealed that many of them contain galactomannans. The galactomannan of gum gatto (from Ceratonia siliqua seeds), commonly known as locust bean gum, is widely used in the textile, paper, food and other industries, and the purpose of examining so many other seeds was to find substitutes. The amounts of mucilaginous polysaccharide in the various seeds were found to vary greatly from one species to another; the highest yields of mucilage were obtained from carob seed (38% of the weight of the seed) and guar seed (35%). The composition of the galactomannans is also variable, although all those examined by these workers contained less galactose than mannose. For example, the molecular ratio of these sugars was 16:81 in Sophora japonica, whilst in guar seed galactomannan (“guaran’I6) the figures were 38 :58.5. Apparently, galactomannans derived from one species only are of variable composition, since the proportion of D-galactose to D-mannose in gum gatto has been variously reported as 16:84 by Hirst and Jones,7 20:80 hy Smiths and Wise and Appling,427 : 73 by Spadag a i i c l 1s : 82 by Lew and Gortner.lo Two other galactomannans are known in which ( 1 ) A summary of this paper w a s read in New York a t the XI1 International Congress of Chemistry on September 12, 1951. (2) Department of Chemistry, The University, Bristol, England. (3) Schulze, Landw. Juhrb., 23, 1 (1894); Bcr. d e u l . bot. Grs., 14, I36 (1 896). (4) L. E. Wise and J . W. Appling, I n d . Eng. Chem., Anal. Ed.. 16, 28 (1944). ( 5 ) E. Anderson, I n d . Eng. Chem., 41, 2887 (1949). (6) E. Heyne and R . L. Whistler, THIS JOURNAL, 70, 2249 (1948). (7) E. L. Hirst and J . K. N. Jones, J . Chem. SOL.,1278 (1848). ( 8 ) F. Smith, THISJ O U R K A L , 7 0 , 3240 (1948). ( ! I ) A. Spadn, All; S O C . ~ i o l e .nrnl. .\fodcnn, T O , 20 (193Q). ( I O ) B. Lcw ;rill1 I~

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GALACTOMANNANS OF LUCERNE AND CLOVER SEEDS

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solid (A) collected. Fehling solution (200 ml.) was added three contiguous hydroxyl groups. Excess ethylene glycol to the supernatant liquor, and the resultant insoluble copper was added after 342 hours, the solution dialyzed, and evapcomplex was separated by filtration under gravity on a coarse orated to dryness under reduced pressure. Hydrolysis of sintered-glass filter. The complex was washed with water, a portion of the oxidized material with Nsulfuric acid yielded and the copper removed by the addition of N hydrochloric some mannose, but no galactose. A quantitative deteracid (ca. 100 ml.) with vigorous stirring to its suspension in mination showed that the oxidized polysaccharide contained ice-cold water (1 1.) until solution was complete. The re- 9% of mannose (calcd. as CeH1006). A further sample of the galactomannan (0.3 9.) was subsultant viscous solution was filtered and the liltrate poured pended in water (20 ml.) and sodium metaperiodate soluinto alcohol (2 1.) with the formation of a white precipitate, which was collected on the iilter and washed with alcohol tion (0.5 N , 30 ml.) added. The solution was maintained until free from copper; it was further purified by solution a t pH 7 with dilute sodium hydroxide, and the reaction in water and reprecipitation with alcohol. The product allowed to proceed in the dark a t room temperature for 1U was washed first with absolute alcohol, then ether, and dried days. The oxidized polysaccharide was recovered as above, in vacuo, to give a white fibrous polysaccharide; yield 5.5 g. and a portion (ca. 10 mg.), when hydrolyzed with N sulfuric acid (2 ml.) and examined on the paper Chromatogram, was Found: S , < 0.3; sulfated ash, 1.3%; OMe, nil; titratio: found to contain no galactose and only a trace of mannose. ~ f 11 with 0.1 Nsodium hydroxide, negligible; [a]1 9 4-118 Methylation.-The galactomannan (6 g. ) was dissolved in water (c 0.4; observations made on a centrifuged solution, and concentration determined by evaporation to dryness in sodium hydroxide solution (300 ml., 40%) and methyl and weighing of residue). Only 62% of this desiccated sulfate (300 ml.) added dropwise with vigorous stirring. material was soluble in water and it was also incompletely After being stirred overnight, the mixture was neutralized soluble in 2 N sodium hydroxide. The aqueous solution with glacial acetic acid and dialyzed against tap water for gave no coloration with iodine. A qualitative examination 24 hours. The solution was then concentrated under reof the hydrolysis products on the paper ~hromatograrn2a.a~ duced pressure to cu. 100 ml. and the methylation procedure repeated by the addition of sodium hydroxide (50 g.) anti indicated the presence of galactose and mannose only. methyl sulfate (100 ml.), followed by more sodium hydroxide The residual seed material from (A) was further extracted with 6 % sodium hydroxide solution (1.5 1.) a t 70-80'. The (100 g.) and methyl sulfate (200 ml.). The mixture was insoluble material was removed on the centrifuge, and then neutralized, dialyzed, concentrated and rernethylated Fehling solution (100 ml.) added to the supernatant alkaline as described above. The final reaction mixture, after diliquor. Since the copper complex so produced was not com- alysis, was extracted with chloroform (3 portions of 700 pletely soluble in dilute hydrochloric acid, the copper was ml.); removal of the chloroform under reduced pressure removed by grinding it with acidified alcohol, the product gave the methylated galactomannan (5.4 g., OMe, 44.6%) washed with alcohol and ether, and dried in vacuo (yield 1 g.). as a crisp white solid. The methylated material was fractionated by boiling with Hydrolysis was found to give galactose, mannose and xylose, mixtures of petroleum ether (b.p. 40-60") and chloroform the latter sugar forming about half the total. This material (200 ml. for 2 hr. in each case), giving mainly one fraction was not further examined. Analysis of the Hydrolysis Products of the Purified Galac- (4.7 g.), soluble in 80% petroleum ether, but insoluble in tomman.-A dispersion of the galactomannan (0.45 9.) in 90%. This fraction had OMe, 45.0% and [ a ] 1 9 D +66 f .Ir sulfuric acid (20 ml.) was heated at 95' for 15 hours. 3' in chloroform (c 1.2). Fission of the Methylated Polysaccharide.-The methylThe reaction mixture was neutralized with barium carbonate, filtered and evaporated under reduced pressure to a ated material (1.45 g.) was dissolved in methanolic hydrosirup. The sugar mixture was separated by partition chro- gen chloride (2% by weight of hydrogen chloride, 30 ml.) matography on a column of hydrocelluloselg (22 X 2 cm.), and the solution boiled under reflux for 16 hours. The soluusing n-butanol half saturated with water as the mobile tion was then neutralized by the addition of a slurry of silver phase, to give D-galactose [m.p. and mixed m.p. 165'; carbonate in methanol, filtered and concentrated under re[ a ] I 8+79", ~ equil. value in water ( c 0.8)] and D-mannose duced pressure to a sirup. This sirupy mixture of glycosides 1m.p. and mixed m.p. 132"; [ ( Y ] ~ * D+14.5", equil. value in was hydrolyzed in N hydrochloric acid (50 ml.) a t 95'. water (c 1.2)]; both sugars were crystallized from methyl The optical rotation of the solution was constant after alcohol. about 12 hours heating. After 14 hours, the solution was The galactomannan (61.6 mg.) was hydrolyzed in a sealed neutralized with silver carbonate, filtered, treated with hytube with N sulfuric acid (2 ml.) for 15 hours at 100'. The drogen sulfide, again filtered and concentrated under retube was opened and the contents washed into a small duced pressure to give a nearly colorless sirup (1.42 g.) of beaker containing ribose (27.4 mg.); the solution was then reducing methyl sugars. neutralized with barium carbonate, filtered and concentrated Analysis of the Mixture of Methyl Sugars.-Examination to a thin sirup. The sugars were separated on a paper chro- of the mixture of methyl sugars o n t h e paper chromatograni matogram using a mixture of butanol, ethanol and water using as the mobile phase a mixture of benzene, ethanol and ( 4 0 : l l : l g ) as the mobile phase. After separation, the water (167:47: 15; top layer, clarified by the addition of a chroniatogram paper was dried in a current of air, and the little ethanol), indicated the presence of tetramethyl (R(; sugars determined by the method of Hirst and Jones.% 0.95),26trimethyl (Ro 0.63) and dimethyl (RG0.26) hex(Found: galactose, 1.79, 2.17; mannose, 2.27, 2.67; ri- oses. bose, 1.66, 2.00 mg.) Assuming complete recovery of the (This benzene-containing solvent has proved very efficaribose, these figures correspond to the production, from the cious for the separation of methyl sugars on the paper polysaccharide, of galactose, 26.6, 26.8, and mannose, 33.8, chromatogram; it gives a wide spatial separation of tetra-, 32.9 mg. (both calcd. as CsHloOb). The corresponding gal- tri- and dimethyl hexoses in 5 to 6 hours on Whatman Xo. 1 actose:mannose ratios are 1.00:1.27 and 1.00:1.23. filter paper a t 20'. It was also found of utility for the Periodate Oxidationzl of the Purified Galactomaxman.separation of methyl sugars on cellulose columns.) The galactomannan (280.0 mg.) was dispersed in water (25 A portion of the above mixture of methyl sugars was anaml.) in a steamed-out stoppered bottle, and potassium chlo- lyzed by separation on the paper chromatogram, followed by ride (3 g.) and sodium metaperiodate solution (0.36 N , 25 a quantitative determination of the components by oxidation i d . ) added. The reaction mixture was shaken, and as the for 15 hours with alkaline hypoiodite.*O (Found: in duplioxidation proceeded, the polysaccharide passed into solu- cate experiments, expressed as ml. of 0.01 Niodine consumed tion. At intervals, portions ( 5 ml.) of the solution were --"tetra," 3.36, 4.34; "tri," 0.86, 1.18; "di," 3.34, withdrawn, ethylene glycol (2 ml.) was added and, the for- 4.38). These results correspond t o an average tetra:tri:di mic acid content of the solution determined by titration with ratio of 4.00: 1.05:4.02. 0.01 N sodium hydroxide. [Found (quoted as polysacIdentification of the Methyl Sugars.-A portion of the charide yielding 1 g.-mole formic acid): 382 g. (96 hr.), 373 methyl sugar mixture (0.73 g.) was separated on a column g. (147 hr.), 371 g. (240 and 342 hr.).] The latter value of hydrocellulose (22 X 2 cm.) using n-butano1:petroleum corresponds to the presence of 43.7% of hexose, containing ether (b.p. 80-100") (40:60 v./v.) as the mobile phase.Ig As a result of a qualitative examination of small portions of (23) S. M. Partridge, Nature, 168, 270 (1946): Biochcm. J . . 42, 238 the effluent on the chromatogram, it was divided into three (1948). parts, which, on evaporation, gave: (24) L. Hough, J. K. N. Jones and W. H. Wadman, J. C h r n i . Soc.. 1702 (1950). (25) E. L. Iiirst and J. K. N. Jones, % b i d . ,1659 (1949).

(26) Rate of movement relative to tetramethylglucopyranose, see E. L. Hirst, L. Hough and J. K.N.Jones, i b i d . . 928 (1949).

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P. ANDREWS, 1,. HOUGHAND J. K. N .

JONES

Vol. 74

Periodate Oxidation.21-The galactornannan (261.0 mg.) was oxidized with potassium metaperiodate as described above; the yield of formic acid was determined by titration with 0.01 N sodium hydroxide solution. [Found (quoted as g. of polysaccharide yielding 1 g.-mole of formic acid): 379 (96 hr.), 370 (147 hr.), 375 (240 hr.), 374 (342 h r . ) ] . The latter value corresponds to 43.2% of the hexose residues possessing three contiguous hydroxyl groups. After hydrolysis of the oxidized material (recovered :I< described above), it was found to contain 10.5% of rn:cnnow (calcd. as C6H1006/. The polysaccharide, after oxidation for 10 days in the dark :it PH 7 with an excess of sodium metaperiodate, yielded neither galactow nor m:iriiiosc on hydrolysis with S sulfuric acid. Methylation and Hydrolysis.----The galactomaiiiian (4 g.) when methylated in the manner described above, gave the methyl derivative (.3.3 g.; OIMe, 43.7YOj. Extraction of this material (2.5 g . ) with mixtures of chloroform and petroleum ether (b.p. 40-60") gave mainly a fraction (2.3g.) with OMe, 43.8%, arid [el1%+76 i 2' in chloroform (c 1.3). hlethanolysis of the methylated galactomannan (1.50 9.) in the usual way, followed by hydrolysis of the glycosides with LV hydrochloric acid, gave a mixture of rcducing methyl sugars (1.46 g. j. Analysis of the Mixture of Methyl Sugars.---\Vhen ex:crnined on the paper chromatogram, using the benzene! ethanol/water solvent, the mixture appeared very similar to that obtained from the .methylated lucerne galactotriniannan. The relative amounts of tetra- ( R G( I . % ) , ( KG 0.03) and dimethyl ( R G0.26) sugars were determined by the alkaline hypoiodite method.*~l [Found (rF,sults quoted Fraction S, (0.25 g.) contairicd a dimethyl hexose only. iodine consumed): "tetra, 3.25, 3.94; .InaZ. Calcd. for CSH~BOG.: OMe, 29.8. Found: OMc, :ts ml. of 0.01 "tri," 1.02, 1.12; "di," 3.30, 4.051. These values corre28.7. It gave, after oxidation with bromine water, crysm.p. and mixed spond t o a n average tetra:tri:di molecular ratio of 3.36: talline 2,3-dimethyl-~-~-mannonolactorie, 1.00:3.43. A portion (1.07 g . j of the mixture of methyl i n . p . 108" after recrystallization from acetoneether. sugars was separated into its components by partition Anal. Calcd. for C~H~~OF,:Oble, 30.1. Found: Ohle, chromatography on a hydrocellulose columnly (22 X 2 cm. I , L'X.9. When heated with phenylhydrazine in ethyl alco- using the benzene/ethanol {water solvent. Three fractions holic solution, the lactone gave 2,3-dimethyl-~-mannonic were obtained: acid phenylhydrazideG which, when crystallized from ethyl Fraction 1, (0.45 g . ) , ICY]% +112" in water ( c 5.G) con:tlcohol, had m.p. 168' :cnd [ a ] I 9 ~-24" in water (c 17.8). tained 2,3,4,li-tetramethyl-D-galactose. Anal. Calcd. for C1Ji?20&2: S , 8.9. 1:ound: S , 8.7. .Ixul. Calcd. for tetramethyl hexose: ObIe, 52.5. Extraction of the Galactomannan from Clover Seed (oar. Found: Ohlc, 50.8. The sirup (0.30 9.) when boiled with Broad Red).-The polysaccharide was extracted from the aniline (0.11 g.) in absolute ethanol, gave 2,3,4,6-tetramilled seeds (100 g.) with water, and purified as the copper methyl-~-galactoseanilide (0.34 g.), m.p. and mixed in.]). complex, as described above, to give a white fibrous mate- 389-190°, [ u ] ~ % J +39', const. value in acetone (c 0.9). rial, yield 3.5 g. [Found: S , 0.9; sulfated ash, 2.270; liractiou 2 (0.12 g.j had [ ~ ] ' Y D-4' in water ( c 1.2). OMe, nil; titration with 0.1 N sodium hydroxide. negli.Inal. Calcd. for trimethyl hexose: OMe, 41.8. Found: gible; [e]~ R D +78 f 11' in water ( c 0.4; determined :is with the lucerne galactoniannanj] . This material was only 6370 OMe, 40.7. After oxidation with bromine water, it gave m.p. and .;oluble in water, and incompletely soluble in 2 S soditirn crystalline 2,3,6-trimethyl--p~-mannonolactone, inixed 1n.p. 81 after crystallization from acetoneether-hydroxide. petroleum ether. Hydrolysis of the Galactomannan:-Hydrolysis of :L sinall portion (ca. 10 mg.) of the material with N sulfuric .l?tal. Calcd. for &H1606: OMe, 42.2. Found: OMe, .rcid, followed by neutralization with barium carbonate and -11.5. The lactone, when heated with phenylhydrazine in examination of a concentrate 011 the chromatogram showed alcohol, gave 2,3,6-trirnethyl-~-mannoriic acid phenylhydrat h e presence of galactose and mannose only. A larger por- zide,? 1n.p. and mixed m.p. 131-132'. tion (0.5 9.) was similarly hydrolyzed, and the products Anal. Calcd. for C~IH.~O,&?:S , 8.5. Found: S , 8.2. separated on a cellulose column, giving D-galactose [m.p. Fraction 3 (0.33 g.) had [ a I i y-16' ~ in water (c 5.0). ;ind mixed m.p. 165", [a]1 5 +79.6", ~ equil. value in water ( r 1.2)] and D-mannose [m.p. and mixed m.p. 132", [aI1*D Anal. Calcd. for dimethyl hexose: OMe, 29.8. Found: -114.1O. equil. value in water (c 1.4)1: both sugars were OMe, 29.8. After oxidation with bromine water, the sugar crystallized from methyl alcohol. gave ~,3~ddirncthyl-r-D-mannonolactone,m.p. and mixed 11 (46.8 mg.) was hydrolyzed a5 above, 111.p. io!) . s added to the hydrolysate, which was rl?zal. Calcd. for CsH1606: OMe, 30.1. Fouud: OMe, tietitrdlizcd with barium carbonate, and the mixture of sug- 28..5. The lactone, when boiled with alcoholic phenylhydra; ;Lrs, after separation, estimated by the periodate oxidation ziiie, gave 2,3-dimethyl-o-mannonic acid phenylhydrazide, ' mcthod.3j (Fouiid: galactosc, 2.03, 2.18; inannose, 2.61, m.p. 167" antl [ a ] 1 8 ~-23" in water (c 0.7). 2.73; ribose, I .89, 1.99 rng.). Assuming complete recovery Acknowledgment.-One of us (P. A,) wishes to of the ribose, these figures correspond to the production from the polysaccharide of galactose, 19.0, 19.4 and man- thank the Department of Scientific and Industrial . (both calcd. as C&lo06). The corre- Research for a Maintenance Grant. mannose ratios are 'I .OO : 1.29 and 1.On :BRISTOL, ESGLASD I .27.

Fraction 1, (U.29 g . ) , which sirup contained 2,3,4,6tetramethyl-D-galactose (Ra 0.95), had [a]I b g 4-110" in XI ater (c 2.5). I d . Calcd. for C I O H ~ Q O Ohle, ~ : 52.5. Found: OMe, -50.8.The sirup ( 0 . Z g.) when heated under reflux with ,miline (0.10 9,) in ethyl alcohol (5 ml.) gave crystalline 2,3,4,6-tetramethyl-~-galactose anilide (0.26 g.), which had m.p. and mixed m.p. 190' and [aI1$D +40', const. value in icetone (c 1.2), after recrystallization from ethyl alcohol. Fraction 2, (0.09 g.) contained a trimethyl sugar (Rc, 1 1 tit? I antl L: small amount of a diniethyl sugar ( R G 0.26). .lmL. Calcd. tor trimethyl hexose: Obfe, 41.8. Fouiid: OXIe, 39.3. The two sugars were separated on a large sheet-paper chromatogram, using the benzeiie/etlianol/ \$,iter solvent, giving a sirupy trimethyl sugar (0.045 g. lzound: OMe, 41.2) with [ a ] I s-2' ~ in water ( c 1.1), and a miall amount of dimethyl sugar iThich was combined with Frtcction 3. The trimethyl sugar (ca. 5 mg.), after de; inethylation2i (with 48% hydrobromic acid (1 ml.) a t 100 tor 12 min.) and examination on the paper chromatogram g,cve a small amount of mannose, but no galactose. The w+ir was oxidized with bromine water in the usual way, m d the product, isolated as the sirupy lactone, did not crysI allize; when boiled with alcoholic phenylhydrazine it gave, i n 60% yield, 2,3,6-trimethyl-~-mannonicacid phenylhytlidzide,' m.p. 130" (raised to 131' when mixed with a n .cuthentic specimen, m.p. 131 " ) after recrystallization from t thy1 alcohol, and [ C Y ] % -2(Jc in water (c i).gJ.

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