4420
G. G. S. DUTTONAND K.
HUNT
VOl.
so
ponent is a concentration anomaly. 3 7 - 3 0 This granules dispersed in a buffered, amyl alcoholanomaly can be explained if one assumes that the saturated solution for 20 hours was less hydrolyzed sedimentation coefficient of the slower component than an amylopectin obtained from starch granules is greatly dependent on the concentration as ob- dispersed for three hours in an unbuffered solution. served above for that of the faster moving com- 3. The above results illustrate that (a) similar ponent. light-scattering molecular weights are obtained For simplicity let us assuiiie that the equation using iV KOH, water arid 6 31 LiBr as solvents; obtained by Johnston antl Ogstori'" is valid3?: (b) sedimentation constants of immature corn CSobS/CSo = (Sf- .S,,,ixt)/(Sf - S,) where C>O')S and amylopectins :Ire extremely large ; (c) immature C'," are the observed antl initial coiicentratioris oi w t x y corn amylopectin consistently behaves as a t,he slow coiiiponeiit; .S,,nlixL and S, are the sedi- statistically branched polymer (acid hydrolysis mentation coeflicients of the slow cotnponent in the studies) ; arid id) variations in the method of dispresence o f the fast ct)uq)oiieiit anct alone; and persing starch granules with lithium bromide do Sf is the sedimeritatioii coeKicieiit of the fast coiii- iiot appear to alter the molecular weight of the ponent. .4gain for siniplicity, let us assume that in ainylopectixi. 'These results indicate that interthe 0.5$;; solution the value of S s , m i x t is equal to that inolecular hydrogen bonding is absent in the disfound in the l?(, solution, that is, 17 Svedbergs, persed ainylopectin samples and that amylopectins and that iii the 0.29;~solution thc value of 5&ixt have broad sizedistributionssuch as found inA-.R-Bs is equal to that found in the 0.5%)solution, that is, type polymers. 4. Immature waxy corn starch has 20 Svedbergs. Using the values for the fast and two components. The fast moving coniponent slow components of waxy I amylopectin listed in may be :i chemically aggregated anylopectin while 'l'able \', the value of CsobS/Csofor the 0.5%., solu- the slower moving component may be an aITly10tionis(1-11 - l i ) / ( l 4 1 - 20) = l . ~ S a i i d f o r t h e 0 , 2 ~ opectin or glycogen which does not exist as a chemisolution is (29(i - 20)/(2996 - 94) = 1.87. Thus the cally aggregated "tetramer" or which is an irnjbservecl relati\-e concentratioiis for the slower purity from the pericarp or ovary. The apparent cunponeiit in the 0.2:',, solutio11 may be as much as increase in the relative concentration of the slower 4/3 times as large as in the 0.5% solution. component when the starch solution is diluted is most likely due to ii concentration anomaly in thti Conclusions schlieren ultracentrifuge pattern. 1 . Starch granules can be completely dispersed Acknowledgment.-The authors wish to take by refluxing in neutral (ior 8 -11 LiBr solutions li. I,. Sinsheimer for this opportunity to thank Dr. without degrading the amylopectin to any cletectable extent. 2. In dispersing starch granules by the use of his light-scattering and differential rerefluxing in amyl alcohol solutions, a buffer must be fractometer instruments and Dr. [V. F. Harringused. A i l amylopectin obtained from starch toil and Dr. 11. Rougvie for their assistance a ~ i d suggestions on matters related to the ultracentri(37) iV. I-. I I ~ i r r ~ i l ~ taoi lfdl 11. K . Schachman T H I S] O I J K N . l I . , 76, fuge kind to other problems connected with this 3.-133 ( 1 953) research project. The ultracentrifuge facilities (38) R 'l'rnutmar: \., S Schumaker, W. I;. H a r r i n g t c u : i r i d H. I;. Schachman, J Chitii I ' h r ~ . 2. 2 , 555 (1954). Tverc made available through a grant from 'The (39) IS,
1ttn.A
The Constitution of the Hemicelluloses of Sitka Spruce (Picea sifchensis). I. Composition of the Hemicellulose and Identification of 2-0-(4-0-Methyl-nglucopyranosiduroni c Acid) -D-xylo s e BY G. c;. s. I ~ I J T T O NAND I(.HUNT Ri?C%IvEn h f A R C l I 1X, 195s
The hemicelluloses of Sitka spruce which were extracted from the chlorite tiolocellulose with 10% aqueous potassium hydroxide gave upon hydrolysis D - X ~ I C I S ~ ,D-mannose, D-galactose, D-glucose, ( ?)L-arabinose and a n aldobiouronic acid. XI1 of the neutral sugars except arabinose were identified a s crystalline derivatives. T h e aldohinuronic acid has been identified as 2-0-(4-~-methyl-oc-~-glucuronosyl) -~-xylose.
Sitka spruce sawdust was delignified by treatment with sodium chlorite and the hemicellulose (1) A preliminary account of this work was presented a t the 40th Annual Conference, Chemical Institute of Canada, in Vancouver, Canada, June, 1957, and a t the 133rd A. C. S. Meeting in San Francisco, Calif., April, 1958. The Sitka Spruce sawdust was obtained from the Powell River Co. Ltd. through the courtesy of Dr. R . F. Patterson t o whom w e express o u r thanks. This work is abstracted from a thesis submitted by K. H u n t for the M.Sc. degree, April, 1957, and was supported by the National Research Council of Canada to whom we evpress our thank,.
was obtained from the holocellulose by alkalilit, extraction. T J p o n hydrolysis the hemicellulose gave an aldobiouronic acid and a mixture of neutral sugars. The acidic component was separated by ineans of ion-exchange resins and the neutral sugars separated on a cellulose column using l-butanol saturated with water a t 5". All of the sugars with the exception of arabinose were characterized ds crystalline conipouiids. The aldobiouronic acid
HEMICELLULOSES OF SITKA SPRUCE
Aug. 20, 1958
442 1
was shown to be 2-0-(4-0-methyl-~-glucopyrano- Isolation of Sitka Spruce Hemicellulose.-The sawdust, as received, was delignified in 100-g. batches by treatment siduronic acid)-D-xyloseby the following evidence. sodium chlorite and acetic acid." The average yield Paper chromatographic investigation of the acidic with of holocellulose was 67 g. The original sawdust and the component showed it to contain mainly an aldobio- holocellulose when air-dried were found t o contain 6.6 and uronic acid, some free acid and small amounts of 4.4% of water, respectively. Holocellulose (ca. 70 9.) was higher oligosaccharides. A pure sample of the aldo- extracted at room temperature with 10% potassium hydroxide ( 1 1.) for 4 hr., and after filtration the residue was biouronic acid was obtained by separating the re-extracted with a further 500 ml. of alkali The two acidic mixture on a cellulose column using ethyl filtrates were combined and the residue washed with water acetate-acetic acid-formic acid-water. Cleavage until the total volume was 2 1. Acidification of the alkaline of the aldobiouronic acid with 8% methanolic hy- solution with glacial acetic acid gave no precipitation but hemicelluloses were obtained as an impure brown solid drogen chloride followed by treatment with ammo- the by the addition of 2 volumes of ethanol; yield 5.5 g., [ C Y ] , ~ ~ D nia yielded the crystalline amide of methyl 4-0- -17.4" ( c 0.52 in water). The preferred method of isolation methyl-a-D-glucopyranosiduronicacid.2 After re- was to add Fehling solution (200 ml.) to the alka1i:ie exmoval of this uronic acid derivative, hydrolysis of tract ( 2 1.) and centrifuge off the green gelatinous precipitate which formed on standing overnight. T h e precipitate the neutral sugar glycoside gave D-xylose charac- was washed with aqueous alkali and alcoholic alkali to reterized as the di-0-benzylidenedimethyl acetal. move excess Fehling solution, and finally with methanol. The point of attachment of the 4-O-methyl-~- (Although the precipitated copper complex does not appear glucuronic acid unit to the D-xylose was determined readily soluble in excess Fehling solution, an excess map inits precipitation and therefore the optimum ratio of from a study of the completely methylated disac- hibit Fehling solution was determined separately.15 X ratio of charide, obtained by lithium aluminum hydride re- 1:10 appeared suitable). The complex was decomposed by d ~ c t i o nand ~ , ~methylation of the aldobiouronic es- suspending i t in methanol and adding hydrochloric acid. ter. Hydrolysis of the methylated disaccharide The liberated polysaccharide was washed with methanol until free of chloride ion and then dried by solvent-exchange; and chromatographic separation of the components yield 6.0 g. of hemicellulose I. After electrodialysis :i on a cellulose-hydrocellulose column6 using buta- sample had [ C X ] ~ D-26.3' (c 1.5 in water) and equiv. wt. none-water as the irrigating solvent7yielded 2,3,4,6- 1232. T h e supernatant from t h e copper complex was ditetra-0-methyl-D-glucose and 3,4-di-o-methyI-~- alyzed against running water to remove salts and evapot o small bulk. Acidification and addition of ethanul xylose. The former was identified as the crystalline rated yielded material (0.3 g . ) having [ C Y ] ~ O D -31.9" (c 2 in water) sugar8 and the latter as the crystalline 3,4-di-0- which was not examined further. Attempted Direct Extraction of Hemicelluloses from methyl-~-xylono-6-~actone. This evidence shows that the aldobiouronic acid Whole Sawd~st.1~-Spruce sawdust (100 g.) was extracted 16 hr. with hot ethanol-benzene (1:2, 900 ml.). The is 2-0-(4-0-methyl-D-ghcopyranosiduronic acid)- for sawdust was washed with hot water, air-dried and then exD-xylose and the work of Bishop10suggests that the tracted at room temperature with sodium hydroxide (0.1 A:, linkage is of the a-type. This acid is identical with 900 ml.) for 48 hr., and the highly colored solution disthat obtained by hydrolysis of other hemicellulose carded. The residue was re-extracted with sodium hy(1 N , 700 ml.) and the polysaccharide isolated V I U materials and it is observed1' that &O-methyl-~- droxide its copper complex; yield of hemicellulose 11, 0.13 g. having glucuronic acid is commonly, but not always,12 [cu]*'D -13.9' (c 1.0 in water). T h e residue from this exlinked to position 2 of the D-xylose fragment. The traction was washed with water, delignified by the chlorite existence of other aldobiouronic or higher acids is treatment and the holocellulose extracted with potassium (loyo,1 1. and 0.5 1.). The polysaccharide i s ) possible since paper chromatographic examination hydroxide lated via its copper complex, yielded hemicellulose 111 (3.33 of the acidic fraction showed several components. g.), . [ a I Z 2 D -52.6' (c 1.0 in HzO). Samples (10 mg.) of The present paper reports the constitution of the hemicelluloses I, I1 and 111 were hydrolyzed and examined chromatographically. All showed the presence of xylose, main constituent of this mixture. mannose, arabinose, glucose and galactose. There were It is of interest to note that in the case of beech no obvious differences in the concentration of the sugars i.1 wood, the hemicelluloses may be extracted from the the three samples, but no quantitative analyses iverr whole wood by 1 N aqueous sodium hydroxide,13 carried out. Quantitative Analysis of Hemicellulose I.-Hemicellubut when this was tried on Sitka spruce only a negliI (10 9.) was hydrolyzed with sulfuric acid (1 dV, 300 gible amount of polysaccharide was isolated. I t lose ml.) on a steam-bath for 12 hr. The solution was neutralmay well be that this is another illustration of the ized (BaC03) and t h e filtrate passed through a column of differences between the hemicelluloses of deciduous Amberlite IR 12016 and then through a column of Duolitr A-4." Evaporation of the eluate yielded the neutral sugar.: and evergreen trees. as a sirup (6.8 g.).
Experimental ,211 evaporations mere carried out under reduced pressure and at a bath temperature not exceeding 40". ( 2 ) F. Smith, J . Chem. Soc., 2646 (1951). (3) L. J. Breddy and J. K. N. Jones, ibid., 738 (1945). i-1) h l . Abdel-Akher and F. Smith, N a f u r e , 1 6 6 , 1037 (1950). ( 3 ) B. Lythgoe and S. Trippett, J . Chem. Soc., 1983 (1950). ( 6 ) J. D. Geerdes, B. A . Lewis, R . Montgomery and F. Smith, Anal. Chum., 26, 264 (1954). (7) L. Boggs, L. S. Curndet, I. Ehrenthal, R . Koch and F. Smith, N a t u r e , 166, 520 ( 1 9 5 0 ) . (8) J. C. Irvine and J. Vi. H. Oldham, J . Chem. Soc., 1744 (1921). (9) S. I?. James and F. Smith, ibid., 739 (1945). (10) C. T . Bishop, Can. J . Chem., 3 1 , 134 (1953). ( 1 1 ) G. .4. Adams and C. T . Bishop, THISJOWRNAL, 7 8 , 2842
(1956). (12) D . J. Brasch and L. E. Wise. TafiPi, 39, 768 (1956). ( 1 3 ) I. R . C. McDonald, J . Chem. Soc., 3183 (1952).
The acid fragments were eluted from the Duolite resin with sodium hydroxide (1 N,50 ml.) and the eluate passed through a fresh column of Amberlite IR 120. Concentration yielded the acidic component (1.9 g.) as a hygroscopic glass, [CZ]~'D $82" (c 0.5 in water). Anal. Calcd. for C12H20011: OMe, 9.1; equiv. i v t . , 339. Calcd. for C7H120,: OMe, 14.7; equiv. w t . , 20%. Found: OMe, 10.9; equiv. wt., 274, 276. A portion of the neutral sugars (90 mg.) was streaked o ' i two sheets (8'' X 22") of Whatman No. 1 paper. One sheet was developed in 1-butanol-water for 168 hr. and the other in butanone-water for 100 hr. The former solvent enabled D-xylose, D-galactose and D-glucose to be determined while the use of the latter solvent led to the determination (14) L. E. Wise, M. Murphy and A. A. D'Addieco, Paaer T r a d e J . , 122, [21, 35 (1946). ( 1 5 ) G. G.S. Dutton and F. Smith, THISJOURNAL, 78, 2505 fl9,56). ( 1 6 ) Supplied by Rohm & Haas Co.. Philadelphia, Pa. (17) Supplied by Chemical Process Co., Redwood City, Calif.
4422
G . G.S . DUTTONAXD K. HUNT
Vol. 80
of D-XyIOSe, D-mannose and L-arabinose. Estimations were charcoal yielded a pale yellow sirup (126 mg.). This sku? done using the phenol-sulfuric acid method.'* The followwas dissolved in methanol saturated with ammonia at 0 ing results were obtained, using a Coleman 14 spectro(13 ml.) and the solution kept for 24 hr. at 3". When the photometer: xylose, 42.1; mannose, 38.8; glucose, 8.6; solution was evaporated a clear sirup was obtained which galactose, 6.9; and arabinose, 4.2y0. largely crystallized in 24 hr. The crystals were washed A second portion of the neutral sugar sirup ( 1 9 . ) was with cold ethanol and the filtrate saved. The solid was readded to the top of a cellulose column (40 cm. by 2.75 cm. crystallized from ethanol containing a trace of water and i.d.1 and the, column developed with 1-butanol saturated yielded methyl 4-0-methyl-cu-D-glucopyranosiduronamide, with xvater a t 5" a t a l i o m rate of 9 ml./hr. Fractions were m.p. 233-236' and mixed m.p. 233-237°.2 The ethanol collected at 30-min. intervals. washings were concentrated t o a sirup (43 mg.) which was Identification of Component Sugars.-1. Tubes 100-150 hydrolyzed with sulfuric acid (1 N,4 ml.) for 12 hr. on the gave a trace of material having a n Rr value identical with steam-bath. After neutralization (BaC08) and treatineilt rhamnose and giving the same yellow color with p-anisidine with charcoal a clear sirup (18 mg.) was obtained. Since trichloroacetate spray; i t has not been otherwise ideritified . the sirup did not crystallize the di-0-benzylidene dimethyl 2. D-XyloSe was obtained crystalline from tubes 160-200 acetal reagent (0.5 ml.) was added and after 24 hr. the solid and had m.p. and mixed m.p. 143-145', [ c Y ] ~ ~+19.4' D was recrystallized from chloroform-petroleum ether giving (c 0.38 in water, equil.); it was further confirmed as its diD-xylose-di-0-benzylidene dimethyl acetal, m .p. aiid mixed 0-benzylidene dimethyl acetal, m.p. and mixed m.p. 208in .p. 207-208'. 209'. 3. D-Mannose was not obtained crystalline owing to Preparation and Identification of Methyl 2-0-(2,3,4,6-Tetcontamination with D-xylose and L-arabinose, tubes 220ra-0-methyl-~-D-glucopyranosyl)-3,4- di - 0 -methyl - D - xylo340; it was identified as its phenylhydrazone, n1.p. and pyranoside.-A second portion of the aldobiouroliic acid mixed m . p . li7-177.5', [ a I z 3 D +27.8 ---t 34.4' (93 hr., (162 mg.) was converted to t h e ester glycoside by refluxing c 0.4 in pyridine).1° 4. The arabinose was not separated with 270 methanolic hydrogen chloride. ilfter iieutralizafrom the mannose, tubes 230-340. The mother liquor from tion (XgzCOB)the glycoside was reduced by refluxing in the mannose phenylhydrazone preparation was heated on tetrahydrofuran with lithium aluminum hydride (0.5 9.) for the steam-bath for 2 hr. with Amberlite IR 120 resin t o re1 hr. T h e reduced product was isolated via its acetate and move excess phenylhydrazine and the filtrate concentrated. deacetylated as previously described .I6 T h e neutral disacPaper chromatographic examination in phenol-water (80 :20 charide (153 mg.) was dissolved in water (6 ml.) to which \-./v.) showed a strong spot for arabinose, b u t treatment of was added sodium hydroxide (1.5 g.). Dimethyl sulfate the filtrate with diphenylhydrazine hydrochloridew failed to (1.5 ml.) was added over 3.5 hr. with vigorous stirring. yield a n y crystalline material. AII attempt was made t o During the final half-hour the temperature was raised tn 70' prepare the diphenylhydrazorie from the crude neutral sirup, to hydrolyze excess dimethyl sulfate. T h e cold methylation but this also failed. A% portion of the Iieutral sirup, [ c Y ] ~ ~ Dsvlution was brought to pH 8.9 with sulfuric acid and cou+32.2" (c 0.2 in water), xvas fermented for 4 days with tinuously extracted with chloroform overnight. Concentration of the chloroform yielded a sirup (88 ing.). This yeast and then examined chromatographically. The presence of D-xylose, arabinose and D-galactose was demonsirup together with 175 tng. from a previous experitneiit was strated, but D-mannose was absent. I t was still not posdissolved in tetrahydrofuran (12 ml.) to which was added sible t o obtain arabinose diphenylhydrazone, but since the pulverized sodium hydroxide (2.3 g.). Dimethyl sulfate optical rotation was [ c x ] ~ ~+41' " ~ after fermentation, it was (3.0 ml.) was added to the vigorously stirred solutioii over assumed the arabinose belonged t o the L-series. 5. T h e 3 hr. and t h e reaction stirred at room temperature for a ~-gluccisesirup did not crystallize, but r a s identified as the further 16 hr. Sufficient water was added tn dissolve the K-p-nitrophenylglucosylamine, m.p. and mixed m.p. 172salts, the solution heated to 60" for 1 hr., cooled arid ex1'T3°.2' 6. T h e D-galactose did not crystallize and an tracted with c h l o r o f o r n ~ . ~T~h e fully methylated disacattempt to prepare the N-~-nitrophenylgalactosylamine charide was obtained as a pale yellow sirup (111 nip.), b.p. yielded insufficient material to purify. i\ccordingly a por(bath temp.) 12S150" (0.08 mm.), and was hydrolyzed tion of the Iieutral sirup was oxidized with nitric acid and with sulfuric acid (1 Lr,\ 10 ml.) for 12 hr. on the steam-bath. the derived mucic acid had m . p . and mixed m.p. 203°.22 Tlie acid was neutralized by passage through Duolite A%-4 Separation of the Aldobiouronic Acid.-Chroinatograpliic and the cluate concentrated to a sirup (120 lug.). Cliroinaexamination of the acidic fraction obtained above when detographic examination iii butarione-water azeotrope showed veloped iii ethyl acctate-formic acid-acetic acid--water only two spots corresponding to tetra-0-n~etliylliexose and (18: 1 : 3 : 3 v./v.) showed five distiTict spots haviug R, di-0-inetliylpentose. T h e sirup was streaked oil Whatman So. 3 h l N paper (18" X 22") and developed in the same values 1.33, 0.64, 0.47, 0.38 and 0.11 ( R xylose = Rx = 1.00) and several fainter ones. The value 1.33 curresolvelit. T h e appropriate zones were eluted with a mixture sponded with 4-0-methyl-~-glucurunicacid arid that of 0.64 of acetone and methanol (50:50 v./v.). Concentraticiri of with 2-~-(4-~-methy~-~-g~ucuronosy~)-o-~y~ose from roestone solution gave 2,3,4,G-tetra-O-m et Iiyl-~-glucose nhich crystallized spontaneously and was recrystallized from ether, erii hem10ck.l~ The acidic mixture iyas accordingly fractionated on a cellulose column and the fraction having R, m.p. and mixed m.p. 92-93", T h e other solution gave 0.64 was investigated further. This fraction was slightly a sirup (22 mg.) having [ C X ] ~ ' D+lso (c 0.16 ill nietkmol); contamined with small amounts of fractions R, 1.38 a.iid lit. values for 3,4-di-@methyl-~-xylose + 1 3 O 8 and -k22.loZ4 R, 0.47 and it was found later that a purer fraction could be in niethanol. The sirup was dissolved in x t t e r (1 ml.) alid excess bromine added. T h e solution vas kept in the dark obtained by carrying out the separation on sheets of Whatman 3 M M paper. a t room temperature iri a tightly stoppered Bask fur tic0 Methanolysis of Z-O-(4-0-Methyl-~-glucopyranosiduronic days and the excess brorniue removed by aeratioii. The Acid)-n-xylose.--A portion of the aldobiouronic acid (198 solut~ionwas neutralized with silver carboiiatc, filtered a n d mg.) was dissolved in methanolic hydrogen chloride (87,. the residual silver removed with hydrogen sulfide. The 10 nil.) and sealed in a Carius tube. T h e tube was heated final sirup was distilled, b.p. (bath temp.) 150-170° (0.0; for 7 hr. at 105" and then the contents diluted with methanol mm.), and yielded a clear sirup (12 mg.) which crystallized arid neutralized (Ag2C08). Evaporation gave a dark on nucleation. The 3,4-di-O-methyl-~-xylono-8-lactone was brown sirup (157 mg.) which after two treatments with recrystallized from ether and had m.p. and mixed m.p. (We are grateful to Dr. J . IC. N. Jones for ail 61-64'. (18) M. Dubois, K. Gilles. J. K. Hamilton, P. A. Kebcrs and F. authentic sample.) Smith. Nature, 168. 167 (1951); A w l . Chem., 28, 350 (1956). (19) C. L. Butler and L. 11. Cretcher, Tars JOUXNAL, 53, 43.58 (1931). ( 2 0 ) C. Neuberg. Ber., 88, 2243 (1900). (21) F. Weygand, W. Perkoa and P. Ruhner, ibid., 84, 594 (1951). (22) T. Posternak, Hclu. Chim. A d a , 19, 1007 (1936).
VANCOUVER, B. C., CANADA (23) E. L. Falconer and G . A. Adams, Con. .I. Chcln., 84, 338 (1956). (24) J. D. Geerdes and F. Smith, THISJ O U R N A L , 77, 3569 (1955).
