STRUCTURE OF CORNHULLHEMICELLULOSE
Feb. 5 , 1957
695
acid (0.2005 g.)'g in water (15 ml.) was added concentrated usual way with methyl sulfate (162 ml.) and sodium hysulfuric acid (0.5 ml.) and the precipitated strontium sulfate droxide (405 ml. of 30%). The reagents were added in tenths during 2 hr. The methylation was completed by was filtered off. The filtrate was warmed on the waterbath at 70-75' for 1.5 hr. until i t was no longer optically heating the reaction mixture for 30 min., after which it was cooled, mixed with chloroform (200 ml.) and filtered active and treated with a solution of 2,4-dinitrophenylto remove sodium sulfate, the latter being washed with hydrazine (0.2509 g., or two equivalent proportions) in small amounts of chloroform. The chloroform layer was water (10 ml.), containing concentrated sulfuric acid (1.5 separated and the aqueous phase re-extracted twice with ml.), for 10 min. at 70-75' as described above. -4fter 48 hr. at room temperature a large crop of long, silky, yellow chloroform (50 ml.). The combined chloroform extracts were dried (sodium sulfate), freed from the solvent and the needles had separated along with a small amount of a microresidual liquid distilled giving: 1,2;3,4-di-O-isopropyIidene- crystalline orange-colored precipitate. These two crystalline products were separated by dissolving the yellow needles 6-O-methyl-~-galactopyranose, a colorless liquid, b .p. 125' in water at 60-65"; the insoluble orange crystals were fil(bath temp.), 0.001 mm., n z 5 ~1.4525, [ a I z 5 -64" ~ in tered off. On cooling the aqueous extract it afforded glychloroform (c 5 ) (yield almost quantitative); literature" oxylic acid-2,4-dinitrophenylhydrazone Jyield 0.1 186 g., [ a ] -63.2' ~ in tetrachloroethane. or 54.5% of the theoretical) m.p. 192 . Anal. Calcd. Hydrolysis of the 1,2,3,4-di-O-isopropylidene-6-O-meth~-l38.1; D-galactopyranose (30 g. ) was described previouslyls wlth for CsHeOsNd: c,37.8; H , 2.4; K, 22.0. Found: H , 2.4; N,22.0. aqueous acetone containing hydrochloric acid gave 6-0: It would seem that the above procedure is more advanmethyl-D-galactose (10.3 g.), m.p. and mixed m.p. 127.5 (after recrystallization from aqueous ethanol); literature'* tageous for the identification of small amounts of D'-(or L')methoxy-D-( or L)-hpdroxymethyldiglycolic acid than the m.p. 128' and CY]^:^ +114O + +77" in water. A solution of 6-O-methyl-~-galactose ( 5 g.) in 2% meth- method used by Jackson and Hudson4 who identified the glyoxylic acid moiety of the dicarboxylic acid, after hyanolic hydrogen chloride ( 2 5 ml.) was refluxed for 13 hr. drolysis of the latter, by oxidation with bromine to give when the rotation had become constant. Removal of oxalic acid. Thus for example, starting with 15 g. of the acid (Xg,COa) and solvent (in uacuo) gave a sirupy product which crystallized spontaneously. Recrystallization from hydrated strontium salt of the dicarboxylic acid, these methanol gave methyl 6-O-methyl-a-~-galactopyranoside,authors report a yield of 5.7 g. of barium oxalate. Experim.p. 138', [aIz3D +165O in water (c 1). Anal. Calcd. ments with 0.2-0.4 g. of the strontium salt of the dicarboxylic acid furnished negligible amounts of barium oxalate. for c,H1@6: OCHB,29.8. Found: OCHI, 29.8. Oxidation of Methyl 6-0-Methyl-~~-~-galactopyranosideOn the other hand, according to the procedure described with Periodate .-Methyl 6-~-methyl-a-~-galactopyranoside here, 0.4 g. of the hydrated strontium salt of D'-methoxy-D(1 8.) was oxidized with periodic acid in the usual way to hydroxymethyldiglycolic acid yielded approximately 0.24 give the sirupy dialdehyde, D'-methoxy-D-methoxymethyl- g. of glyoxylic acid 2,4-dinitrophenylhydrazoneand about diglycolic aldehyde, [ C L ] ~ ~ D 145' in water (c 1.5) changing 0.01 g. of the 2,4-dinitrophenylhydrazoneof pyruvic acidin 40 hr. to +119". The dialdehyde was treated with 2,4-dinitrophenylhydrazide,amounts that were adequate for hydrogen in the presence of palladium-charcoal as de- identification purposes. ( b ) The Glyceric Acid Moiety.-\Then the orange, scribed in the previous experiments. The sirupy product water-insoluble crystals, separated from the glyoxylic acid (0.74 8.) thus obtained appeared to consist only of unchanged dialdehyde recognized by its characteristic mutarotation, 2,4-dinitrophenylhydrazone in the preceding preparation, were recrystallized from hot ethyl acetate the crystalline [ a I z 5+153" ~ in water (c 1.5) changingin 50 hr. to +116'. 2,4-dinitrophenylhydrazone of pyruvic acid 2,l-dinitroBromine oxidation of the product from the hydrogenation phenylhl-drazide was obtained m.p. 316' dec. From two reaction in the presence of barium carbonate yielded the barium salt of the corresponding dicarboxylic acid. Hy- experiments, each starting with 0.2 g. of the strontium salt drolysis of this dicarboxylic acid and chromatography of the of D'-methoxy-D-hydroxymethyldiglycolic acid, a total of hydrolyzate using 1-butanol'acetic acid:water ( 2 : 1 : 1 ) re- 0.01 g. was obtained. The pyruvic acid is formed from vealed glyoxylic acid and 3-0-methyl-~-glycericacid. T h e glyceric acid bl a rearrangement of the pinacol type.2" Anal. Calcd. for CljH1?O9Sb: C, 40.2; H, 2.7; S , 25.0. former was characterized as glyoxylic acid-2,4-dinitroDhenylhydrazone, m.p. and mixed m.p. 192-193", while the Found: C , 40.6; H, 2.8; S,25.3. latter was recognized by comparison of its Rf value with Acknowledgment.-The authors thank the Office that of an authentic specimen. No 3-O-methyl-~-glycero1, which would arise by reduction of the C4 aldehydic group of Ordnance Research, U. S . Army, for financial aid under contract No. DA-11-022-ORD-999 which as in the case of methyl a-D-glucopyranoside, mas found. Identification of D'-Methoxy-D-hydroxymethyldiglycolic helped to defray the expenses of this work. Acid. ( a ) The Glyoxylic Acid Moiety.-To a solution of the (19) Prepared from methyl a-D-giucopqranoside by the method of strontium salt of D'-methoxy-D-hydroxymethyldiglycolic
c,
+
(17) K. Freudenberg and R . h l Hixon, Be?'.,66, 2119 (1923). (18) K. Freudenberg and I;.Smeykal, zbid., 59, 104 (1926).
[CONTRIBUTIOX FROM
THE
DEPARTMENT OF
Jackson and Hudson 4 (20) E Erlenmeyer, Be?' 14, 321 (1881)
ST PAUL. MISNESOTA
-4GRICULTURAL
BIOCHEMISTRY, UXIVERSITY O F MINNESOTA]
Structure of Corn Hull Hemicellulose. Part 111. Identification of the Methylated Aldobiouronic Acid Obtained from Methyl Corn Hull Hemicellulose1~2 BY R. MONTGOMERY A N D F. SMITH RECEIVED A
429, 1956 ~
~
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Corn hull hemicellulose has been methylated and the methyl derivative hydrolyzed. The cleavage fragments were separated by means of ion-exchange resins into a neutral and an acidic component. The latter was esterified and the resulting ester reduced to give methyl 2-~-(2,3,4-tr~-~-methy~-~-g~ucopyranosyl)-3-O-methyl-~-xylopyranos~de, m.p. 166-168', [or]% + 8 5 O in water. This proved that the aldobiouronic acid cleavage fragment of the methylated hemicellulose was methyl 2-~-(2,3,4-tri-~-xnethy~-~-g~ucopyranosy~uronic acid)-3-0-methyl-~-xylopy~dnoside and that the D-glucuronic acid end groups are linked directly to the main structural xylan framework of the polysaccharide (1) Paper N o . 3556, Scientific Journal Series, Minnesota Agricultural Experiment Station, University of Minnesota. (2) This research was done under contract with the U . S.Department of Agriculture and authorized by t h e Research and Marketing
Act of 1940. T h e contract was supervised by the h-orthern Utilization Research Branch of the Agricultural Research Service. Presented a t the 129th National Meeting of the A.C.S., Dallas, Texas, April, 1956.
K. MONTGOAIEKY A N D 17. Ssimr
696
Vol. 79
It has been shown p r e v i o ~ s l y t~h.a~t the graded 435;; potassium hydroxide and methyl sulfate. hydrolysis of corn hull hemicellulose gives rise t o The methylated hemicellulose was converted to %o-(-a-D-ghcopyranosyluronic acid)-D-xylopyra- the free acid by precipitation from solution in 1,4nose. This aldobiouronic acid has also been re- dioxane with :V hydrochloric acid and then freed ported t o be present in corn cob hemicellulose B,j from the latter by reprecipitation from 1,4-dioxane chagual gum6 and oat hulls.' I t s presence in with water. The resulting material, showing these and other acidic polysaccharides is easily [a]"D - 129' in I , k I i o x a n e arid -OCH:: &S.(i, demonstrated b y hydrolysis and formation of the tenaciously retained water which was rapidly rereadily crystallizable acetate of the niethyl ester absorbed if exposed t o the air after vigorous drytriethyl glycoside.3 ing. The present paper deals with the iiianner in Methanolysis of the methylated lieriiicellulosc which the aldobiouronic acid residues, ~ - O - ( C U - Dwith 3yoiiiethanolic hydrogeii chloride p 7 - e :i mixglucopyranosyluroiiic acid) - D - xylopy-anose, are ture of glycosides, the uronic acid ester groups in linked to the polysaccharide. The c c r i l hull heini- which were then saponified with 0.15 AT barium cellulose appeared to be essentially lioiiiogencous.% hydroxide. Since losses of fully methylated sugar Thus, about ( i 5 5 of the polysaccharide was pre- glycosides easily occur upon prolonged evaporation cipitated when a O.Gyoaqueous solution n-as gradu- of their aqueous solutions, these 1-olatile cleavage ally saturated with ammonium sulfate and this fragments were extracted with petroleum ether fraction had almost the same srJecific rotation as from the alkaline saponification solution bciore t h a t of the fraction which remained in solution. separatioii into the acidic and neutral sugar coiiiAlso. the acetyl derivative was fractionally pre- pocents on ion exchange resins w a s applied. T h e cipitated from chloroform-ether or acetoneeether neutral methylated cleavage fragments in the solutions with petroleum ether and the series of petrolcL11ii etlicr extract and in the ion-exchange fractions so obtained appeared to be similar. T h e resins cluate were combined and in\-estigated s e p 1.eniicellulose does not form an insoluble copper aratt1)-; these findings mill be reported in another complex with Fehling solution, a property which is communication. The acidic cleavage irngneiit in direct contrast t o the hemicellulose froni corn T V ~ Sesterified a n d then reduced nit11 lithium aluc ~ b and s by which means the latter was purified.q minuin hydridelO~ll t o gil-e crystalline niethyl 2I n order to determine the mode of union of the 0(2,:j,i-tri-O-metli!-l-cu-~-giucopyranosylj -3-O-rnecomponent sugars and of the aldobiouronic acid, tliyl-3-D-x!-lopyranoside (111).I ? The characterizathe heniicellulose acetate was methylated with tioi: of the latter, together ~ v i t hthe iirevious iclentification of tlie residue, 2-O-(cr-D-glUCOpyra~l~)SylH H ~iro!iic acicl)-D-xylose3,4 in tlie poij-saccharide pm-ecl t h a t the acidic cleavage fragment, resulting r iiiethaiiolysis of the n~etliylntetl corir hull niethpl 2 - 0 - [niethyl-(:!, Y O H O ;;,A-tri- 0 -methyl - cy - ~ ~ - ~ l u c o p ~ - r a ~ i o s ~ l ) - u r o n n t e ] :I-!~-iiiethyl-n-xylo~~~rari~)side (11). I t also pro\-et1 COOH that the units of D-glucuronic acid occupied terIliinal positions and were linked directly t o the iiiain structural xylan framework of the polysaccharide as in I.13 H OMe E H OH I Experimental ,
I
U
H
O
Me;w/ /
CH,OH
k-0 H
OMe
IJI
(3) I
