May20, 1050
JUTE
FIBERHEMICELLULOSE : URONICACID
amount of undialyzed material being determined b y evaporating and drying t h e contents of t h e dialysis sac. Originally SO% of t h e stage I1 lignosulfonic acid failed t o dialyze under these conditions; after t h e first oxidation this value was 40%, and only 15% after t h e fourth. A Crude Calcium Oxylignosulfonate.-Nitrogen gas was bubbled through 30 cc. of a clear, centrifuged aqueous solution of t h e oxidized lignosulfonic acid until a n y chlorine dioxide had been expelled. T h e solution was shaken with small increments of calcium hydroxide until PH 4.5 was attained, and at this acidity t h e precipitated calcium sulfate was removed. After t h e clear mother liquor had been concentrated, the addition of 10 volumes of ethanol caused t h e precipitation of the crude product. Anal. Found: S , 3.1, 3.2; OCH3, 3.9, 3.8; C1, 6.6, 7.0; Ca, 13.3. (When corrected for 470 of calcium sulfate, t h e analysis became: S, 2.1; OCHa, 3.7; C1, 6.7; Ca, 12.1
%.) One gram of t h e crude calcium oxylignosulfonate was kept dispersed for one week in 150 cc. of water containing 7.5 g. of sodium hydroxide and 1.5 g . of sodium carbonate, and t h e suspension was then adjusted from pH 11.8 t o PH 7 with glacial acetic acid. T h e residue obtained b y evaporating the suspension was extracted with ethanol, was dissolved in 15 cc. of water and was reprecipitated with ethanol. After a second reprecipitation, t h e powder was dried by treatment with alcohol, then benzene, and finally in zlacuo over phosphorus pentoxide; yield 0.52 g. or 82%. Anal. Found: S , 2.7; C1, 0.6; Ca, 22.5, 22.4. Of the sulfur, 0.8% was present as free sulfate ion and 1.970 was combined; the apparent calcium content included a little sodium. T h e replacement of the sodium hydroxide-sodium carbonate buffer with saturated calcium hydroxide solution yielded a product with 1.87, of combined sulfur and only 1.4% of chlorine. Fractionation of a Zinc Oxylignosulfonate.-Zinc oxide, 5 g., was stirred with 200 cc of a solution containing 3.36 g. of oxylignosulfonic acid, and free of chlorine dioxide, until the pH was 4.9. T h e oxylignosulfonic acid had 1.2y0 of combined sulfur and 1.4y0 was present as sulfate ion; t h a t is, 40.3 a n d 47.0 mg., respectively. Next d a y the excess of zinc oxide, 0.32 g., was removed on the centrifuge; t h e clear liquor was concentrated t o about 50 cc. and was poured into 200 cc. of ethanol. T h e precipitate of zinc “oxylignosulfonate” was washed twice with 200 cc. of acetone before being dried in vacuo; yield 2.4 g. or 70% b y weight. Anal. Found: S , 0.9, 0.9; C1, 4.7, 4.5; Zn, 21.8, 20.2. T h e free chloride and sulfate content corresponded t o the presence of 0.13 g . and 0.07 g. of t h e respective zinc salts. Correction for these impurities gave t h e composition of t h e zinc oxylignosulfonate as S, 0.0; C1, 3.6; and Zn, 19.3.
[CONTRIBUTIOS FROM
THE
DIVISION
OF
2409
T h e ethanolic mother liquors and the acetone washings, when mixed, deposited a second fraction which was recovered and dried; yield 0.71 g. or 22%. Anal. Found: S, 0.5; C1, 6.2. Zn, 23 (cor. for 1% of zinc sulfate and 10% of zinc chloride). T h e analyses of aliquots showed t h a t t h e mother liquors contained 45 mg. of sulfur as sulfate ion, or 96Y0 of the original sulfate. T h e residue from these liquors was not analyzed because i t was extremely deliquescent, and also exploded when attempts were made t o dry it by heat. Since only 3.6 mg. of combined sulfur had been recovered in t h e second fraction of zinc oxylignosulfonate, the residue contained (40.3-3.6) m g . , or about 37 mg. T h e organic material in the residue weighed about l g., and therefore contained about 3.770 of combined sulfur. Isolation of a Barium Oxylignosulfonate.--A solution, 30 cc., containing 0.62 g . of oxylignosulfonic acid (S, 2.9% or 18 mg.) b u t no chlorine dioxide, was mixed with 10 cc. of cold, redistilled 47y0 hydriodic acid t o reduce the chloric acid present. After extracting the liberated iodine with ether, the aqueous solution was shaken with moist silver carbonate, freshly prepared from 11 g . of the nitrate and 5.5 g . of potassium carbonate. T h e insoluble silver salts were removed on the centrifuge, and were extracted with dilute hydrochloric acid. Since the extract gave no precipitate with aqueous barium chloride, the salts included no sulfate. T h e supernatant solution, now containing silver oxylignosulfonate and silver sulfate, was mixed with a n exact equivalent of dilute hydrochloric acid, and the precipitated silver chloride removed. Concentrated aqueous barium hydroxide was added t o the resulting solution of free oxylignosulfonic and sulfuric acids until the acidity was reduced to pH 4.8. T h e precipitated barium sulfate was removed and after ignition weighed 46.5 mg., corresponding t o 6 . 4 mg. of sulfur or t o 35% of the original amount. After concentrating the mother liquor t o 5 cc., t h e barium lignosulfonate was precipitated b y t h e addition of 50 ml. of ethanol, and was dried in vacuo; yield 0.45 g. Anal. Found: S , 1.7; Ba, 36.6. T h e sulfur content, 7.6 mg., was 42% of the original amount. Evaporation of the alcoholic filtrate t o dryness left 0.67 g. of salts. Anal. Found: Ba, 43.4; S, 0.2. T h e sulfur content corresponded t o 1.3 mg. (7 %).
Acknowledgment.--D. hl. S. wishes t o thank the National Research Council of Canada for assistance in the form of a Studentship, a Fellowship and two Summer Supplements. MONTREAL, CANADA
APPLIEDBIOLOGY, NATIONAL RESEARCH COUNCIL O F CANADA]
Uronic Acid Components of Jute Fiber Hemicellulose1s2 B Y €1.
c. SRIV.4STAVA3
AND
G. A. ADAMS
RECEIVEDSEPTEMBER 5 . 1958 Jute fiber hciniccllulosc produces upon partial hydrolysis a number of neutral and acidic sugars and oligosaccharides. T h e acidic components isolated by ion exchange and cellulose-column chromatography have been identified and shown t o be 4-U-methyl-~-glucuronicacid, ~ - 0 - ( ~ - ~ - m e t h y ~ - a - ~ - g ~ u c u r o n o p y r a n o s y ~ and, ) - ~ -tentatively, x ~ ~ o s e O-a-4-O-methyl-~glucuronopyranosyl-( 1 2)-O-p-~-xylopyranosyl-(1 + 4)-~-xylose. -f
The jute plant (Corchorus olitorius and C. capsularis) gives, after a proper treatment of retting, long glossy fibers which are used for making burlap and sacking materials. Associated with cellulose, the jute fiber has a considerable quantity (1) Issued as N.R.C. No. 5123. ( 2 ) A preliminary report of this paper has appeared in Chemistry 6’ Industry, 920 (1958). (3) Postdoctorate Fellow of the Sational Research Couucil.
of hemicellulose (about 30%).4 The structure of this hemicellulose has been studied by Sarkar, et aL5-’ On the basis of periodate oxidation data (4) P. B. Sarkar, A. K. Mazumddr and K. B. Pal, J . T e x t i l e I n s l . , 39, T i 4 (1948). ( 5 ) P. B. Sarkar, A. K. lhlazumdar and K. B. Pal, T e x t . Res. J . , 22, 529 (1952). (6) P. C. Das Gupta and P. B. Sarkar, ibid., 24, 705 (1954). (7) P. C. Das Gupta and P. B. Sarkar, ibrd , 24, 1071 (1954).
and chroi1iatograI)hic evidence these authors con- Syl)-D-XylOSe, was established in the following iiiancluded that the jute fiber hemicellulose was com- ner. Component 11, which had [ a I z 5 ~97", was posed of a chain of 1,4'-linked D-xylopyranose resi- converted to the methyl glycoside of the corrcsponddues arid that to Cs of every sixth D-XyloSe unit ing neutral disaccharide as described above. The there was attached a residue of X-O-methyl-~- latter gave upon hydrolysis D-XyloSe and 4-0glucuronic acid. They also claimed to have iso- methyl-D-glucose. The mode of linkage between lated an aldobiouronic acid to which was assigned the two sugar residues was established by niethyla3-0-(3-0-methyl-D-glucuronopy- tion studies. The methyl glycoside of the neutral the structure ranosy1)-D-xylose. T h e aldobiouronic acid com- disaccharide was methylated first with dimethyl ponent of many wood and cereal hemicelluloses sulfate and alkali and then with silver oxide and has been identified as 8-0-(4-0-methyl-a-~-glu- methyl iodide. The fully methylated disaccharide curoiiopyranosyl)-D-xylose.~-?o The identifica- gave upon hydrolysis approximately equirnolecular tion of 3-O-iriethyl-D-glucuroiiic acid and 3 - 0 - proportions of 2,3,4,6-tetra-O-methyl-D-glucose and (:3-O-niethyl-D-glucuroiiopyranosyl)-D-xylose as 3,4-di-O-methyl-~-xylose;the former was obtained components of jute fiber hemicellulose would thus in a crystalline form and the latter was characterindicate unique biosynthetic processes in the jute ized as 3,4-di-~-niethyl-~-xylonolactone.""These plant. A reinvestigation of the problem seemed data prove t h a t in the aldobiouronic acid (11) the tlesirable, an added reason being that the structural 4-O-rnethyl-D-glucuronic acid residue is joined by proofs o8ered by Sarkar and c ~ - w o r k e r s ~for - ~ the its reducing end to C z of the xylose moiety. The presence of 3-O-methyl-D-glucuroiiic acid were not a-configuration of the biose linkage is indicated based upon the identification of crystalline com- by the high positive rotation. X direct chemical ~",unds. proof for the configuration of the biose link in I1 This paper is concerned with the isolation and has been provided recently by Gorin and Perlin.zl identification of 4-O-inethyl-D-glucuronic acid (I) T h e aldotriouronic acid (component 111), which 2-0- (4-0-methyl-a-D-glucuronopyranosyl)- D-xylose was obtained in a crystalline form, had 11i.p. (11) and 0a-4-O-methyl-D-glucuronopyranosyl180-183" and showed [ a I z 5 ~ 58". Partial hy(1-2) -O-@-D-xylopyranosyl-(1-4) -D-xylose (111) drolysis of the aldotriouronic acid with lVsulfuric produced upon partial depolymerization of the jute acid and examination of the hydrolyzate by paper fiber hemicellulose. chromatography and paper electrophoresis showed Jute fibers were hydrolyzed with 0.3 N sulfuric the presence of D-XyloSe and another component acid for 7 hr. when a mixture of neutral and acidic which had the mobility of 2-0-(4-0-inethyl-a-~sugars and oligosaccharides was produced. Four glucuronopyranosyl)-D-XylOSe. acid components (I-IV) were isolated from the mixT h e aldotriouronic acid (111) was methylated in ture by ion-eschangc and cellulose column chroma- the following manner: I11 was converted to its tograp h y. corresponding methyl ester methyl glycoside derivThe identification of component I as -2-0ative (V) by treatment with iiiethanolic hydrogen inethyl-D-glucuronic acid is based upon the fol- chloride a t room temperature. Reduction of V lowing facts. Component T , which had [aI25) with lithium aluminum hydride",'? produced the 48" and equivalent weight 197, was converted neutral trisaccharide methyl glycoside (VI) which to the methyl ester methyl glycoside derivative by was methylated with methyl iodide and silver oxide boiling with methanolic hydrogen chloride and then to give a fully methylated trisaccharide which gave was reduced with lithium aluminum hydride21,2? upon hydrolysis equimolecular proportions of 3,3to produce the glycoside of the neutral sugar. The -k,O-tetra-O-methyl-D-glucose,2,:3-di-O-inethyl-D-syglycoside gave upon acid hydrolysis 4-0-methyl-D- lose and :3,1-di-O-iiicthyl-D-xq.lose. "3,4,(i-Tetraglucose which was identified by specific optical ro- 0-methyl-D-glucose was characterized as AV-phcnyltation, paper chromatography, paper electrophore- ~-glucosylainine",X,i,fi-tetramethyl ether,252,:I-tlisis and by transfoririing i t into the corresponding 0-methyl-D-xylose as ,li-phenyl-D-xylo])yr~~ii[)sylI)henylosasone. No 3-O-inethyl-D-glucose was amine '3,3-diinethyl ctherZ6 a n d ;S,-~-di-O-iiicttiylfountl. 011 D-xylose as ~.4-di-~-inethyl-D-~yloii~lactoiie.'" The structure of the aldobiouronic acid (coni- the basis of evidence outlined above, the altloponciit I I ) , '3-0-(3-0-inethyl-a-D-glucuronopyranotriouronic acid (component 111) is tentatively assigned the structure O-oc-i-O-inethyl-D-glucurono( 8 ) h. I-l iodide (20 wits separated on a n alumina coluinu (20 X 45 nim.).;6 addition of silver oxide (3 g . ) (1 g . T h e tetra-0-methylglucose component (411 mg.) was eluted was heated under reflux overliigllt. with chloroform after which the di-0-irietliylxylosc ( 2 0 5 s recovered by filtration aud cvapomg.) was displaced with methanol. F U C U O . After three such ~ n e t l ~ y l ~ t Identification of 2,3,4,6-Tetra-0-methyl-~-glucose.On removal of t h e solvent the sugar crystallized spontaneously. tioils, tlie partially methylated trisaccharide was co~npletely Recrystallization from diethyl ether-light pctroleum ether soluble in nictli>-liodide. TWOfurther mcthylatioris with iiiethyl iodide i i i i t l silver oxide g a v ~a clear sirup (114 I I I ~ . ) , (1: 1) yielded fine white needles, [ m ] * ' D +83 i 2' iii water [ n ] % +31" i n riit.th:t~iol(c 2 ) . (c I), m.p. and mixed m.p. 96'. Tlie 2,3,4,0-tetra-0A U U Z . ~ ; ~ i ~ for ( i , c ~ . I ~ ~ ocrI,{, ~ o ~.ix.!). ~ : FULIIKI : methy~-n-glucose(100 mg.) ill Irieth:iiiol (5 nil.) containing OCHS, 49.1. aniline (60 mg.) was refluxed fur 2 hr. 0x1p:irtial reniovd of t h e solvent the anilide cr tllizcd . ~iecr)-stallizatio~~ Hydrolysis of Methyl [0-~~2,3,4,6-Tetra-U-nlethyl-ufrom ethanol yielded 2 , 3 , 4 , 6 - t e t r a - 0 - n i c t i ~ ~ l - ~ ~ 7 - ~ , h c nglucosyl-( ?l-~1 + 2)-0-P-3,4-di-O-methyl-~-xylosyl-( 1 + 4 ) 1glucosylamine, m.p. 134-136", [ a I z j+233 ~ i- 1' in cliloro- 2,3-di-O-methyl-u-xylosidea n d Identification of Compoform (c l).*j nents.-The fully methylated trisaccharide (93 mg.) was Identification of 3,4-Di-O-methyl-~-xylose.-011 cliro- heated in a sealed tube a t 80' with 8% methanolic hydrogen niatographic examination in solvent C this sugar had a n chloride for 12 lir. Alethanol was removed and hydrolysis R, value of 0.38, identical with t h a t of 3,4-di-O-methyl-n- effected by heating with O.5N hydrochloric acid for 8 hr. on xylose and was distinguished from 2,3-di-0-nieth~1-~-xylose a boiling x i t e r - b a t h . The acid was removed with Aniberlitc ( R , 0.31) and 2,4-di-0-methyl-~-xylose (R,0.27). IR-45 and the methylated sugars were recovered as a sirup Anal. Calcd. for C7H1405: OCHI, 34.8. Fourid: OCH3, (88 nig. ). Chromatographic examination (solvents C and D ) showed the presence of three components which corre34.6. ondeti in color reaction and rate of movement t o authentic T h e sugar (80 mg.) dissolved in water ( 2 rnl.) containing les of 2,3-di-0-meth)-l-~-xylose, 3,4-di-G-methyl-obarium carbonate (70 mg.) was oxidized with bromine (10 e and 2,3,4,6-tetra-0-meth~.l-~-glucose. T h e IniXtUre drops) in t h e dark for 60 hr. T h e bromine was removed of t h e methylated sugars was resolved on filter paper using by aeration and t h e solution extracted exhaustively with C and the components were isolated in pure forni by chloroform. T h e chloroform extract was evaporated in solvcnt elution of appropriate portions of the paper. I n this manC'UCUO t o give a sirup (66 mg.) which was distilled, b . p . (bath (16 mg.), 2,3-di-0temp.) 120-160" (0.02-0.015 m m . ) . T h e distillate crj-stal- lier 2,3,4,6-tetra-~-methy~-~-glucose methyl-D-xylose (14 mg.) arid 3,4-di-0-methyl-D-xylosc lized on standing and after recrystallization frorn ether (12 mg.) were obtained. yielded 3,4-di-~-methy~-~-xy1ono~actone, m . p . and mixed Identification of 2,3,4,6-Tetra-O-methgl-n-g~u~ose.-~~~C iii.p. 66-67', [01]2'n -22 i 1' in water ( c 1 ) . 2 3 Characterization of 0-01-4-0-Methyl-~-glucuronopyranosyl- sirupy tctr~i-O-methyl-n-glucosewas refluxed with ethanolic ( 1 -+ 2)-O-P-n-xylopyranosyl-(1 ---* 4)-~-xylose.-The aldo- aniline t o afford ;\-phen)-l-o-glucos)-l~mi~le2,3,4,6-tetr;tD triouronic acid (fraction 3 ) was dissolved in the mininium methyl ether, m.p. and mixed m.p. 134-135', [ Q ] ~ ~$-?433" amount of water and t o the solution a few drops of 1- in acetone ( c O . i j (after recrystallization from ether).'j Identification of 2,3-Di-O-methyl-~-xylose.-The 2,3-dibutanol wiis added. Upon slow evaporation, tlic solution deposited needles which were filtered and rccrj,stallized O-methyl-D-x)-lose component, which was obtained as a birup, showed [ ~ Y ] +21" ~ ~ D in methanol ( c 1). from aqueous methanol. T h e crystals had n1.p. 180-183° Anal. Calctl. for Ci7HlrOa:OC& 34.5. I:ou~itl: O C h anti showed [ m ] 2 5 +58" ~ in water (c l ) . I 7 31 2 . Anal. Calcd. for C,6H23011(OCHa)~1/~II~O: C , 42.4; sirup (10 m g . j i w s refluxed for 2 hr. with aiiiline €1, 6.03; OCHa, 0.43. Fourid: C , 42.4; H , 5.90; OCEIa, (12The m g . ) and inethanol ( 2 ml.). On removal of t h e solvent, 6.34. the ntiilitle crystdlized and was recrystallized from ethyl An X-ray diffraction pattern of the crystals was identical acetate. The ~~~-phenyl-~-xylopyranosylarnine-2,3-diwith t h a t obtained from a sample of 0-01-4-O-mcthyl-n-glu- methyl ether had m.p. and mixed m.p. 123-124' and curonopyranospl-( 1 -+ 2)-0-~-~-xylopyranosyl-( 1 -+ 4 ) - ~ - showed [ a I z 6 D +I€@" i n ethyl acetate (c 0.4).26 xylose.'7 Identification of 3,4-Di-O-meth~l-~-xylose.-Thc chroPartial Hydrolysis of the Aldotriouronic Acid.--The altlo- matographic:illy pure 3,4-di-@rnethyl-D-xylose component triouronic acid (50 mg.) was dissolved in N sulfuric acid ( 5 had [ ~ U ] ~ ' D 25' in methanol (c 1). r n l . ) and t h e solution heated (100') in a sealed tube for 8 A n a l . C:tlctl. for Cli1IllO3: OCI&, 3-1.8. Fouiitl: OCHs, lir. T h e hydrolyzate WRS neutralized with barium carbon31.4. a t e and after filtration the solution was deionized with AmIt was traiisforined into :3,4-di-O-nicthyl-~-xylotlolactone, berlite IR-130 and then concentrated in vacuo t o a thin sirup. Paper chromatographic analysis of the hydrolyzatc :is described previously, which had m.p. and mixed m.p. 6667" and showed [ O ~ ] ' ~ D-23" (initial) in water (c 1) (after rcusing solvents A and E showed the presence of ~ - x y l o s cand crj.stallization from ethcr).*3 another component which had thc same R , as 2-0-(4-0~ncth~-l-rr-~-glucuronop!.ranos!~l~-o-u~lose. Examination o f Acknowledgment.---'The authors wish to thank the sirup h p paper clectrophorcsis using formate huffer, Ilr. J. IC. IIamilton for a specinicn of 0-a-4-07 (600 volts for 2.5 l i t - , ) : l i d sodium bicarbonate 1.025 i l f j 3 8 (700 volts for 2.5hr.), also iIldicated t h a t methy~-~-g~ucuronopyranosy~-( 1 + 2)-0-P-D-xy10tlic hydrolyzate was composcd of n-xylose and 2-0-(4-0pyranosyl-(1+4)-~-xylose and Dr. Mi'. H. Barnes
+
-
Inethyl-a-D-g~ucuronopyran~)s~~l)-n-xy~ose. Methyl [O-~-4-0-Methyl-~-glucopyranosyl-( 1 2 )-0-Pn-xylopyranosyl-( 1 -+ 4 ) ]-n-xyloside .-The aldotriouronic acid (133 m g . ) was converted t o tlie methyl ester methyl
for X-ray diffraction analysis. T h e technical assistance of Mr. A. E. Castagne and Mr. W. R. Rowsome is gratefully acknowledged. OTTAWA 2, CANADA
