ROY L. WHISTLERAND H. E. CONRAD
3544
and distillation of ethanol t o remove a n y further borate impurityl6 the sirupy product was heated at a temperature of 55-60' for 4-5 hours t o effect lactonization (yield 1.35 g.). Separation of the Phenylhydrazides of D-Gluconic and L-Idonic A~id.~-"~'-The sirupy misture of the lactones (1.35 g.) mas dissolved in ethanol (15 ml.) and phenylhydrazine (0.75 ml.) mas added, T h e reaction mixture was refluxed on a boiling water-bath for 40 minutes. The solution was allowed to cool slowly at room temperature whereupon D-glUCOniC acid phenylhydrazide separated in the form of fine white crystals. T h e crystals wcre filtered, washed with ethanol and dried (yield 280 mg.). The filtrate and washings were combined and concentrated to a small volume in oacuo and left overnight t o crystallize. A further amount (0.20 9.) of gluconic acid phenylhydrazide was obtained in this way. This procedure was carried out five times until no more crystals separated. The total yield of the phenylhydrazide of D-gluconic acid was 0.504 g., and after a further crystallization from water it showed 4-11.5' in water (c 0.7), m.p. and miscd m.p. 198-201". The;e values are in good agreement with those (m.p. 198-201 , [alZoD4-12" in mater) recorded for D-glUCOlliC acid phenylhydrazide.I4 Anal. Calcd. for C12HlsOcXz: C, 50.4; H, 6.39; N, 9.8. Found: C, 50.46: H, 6.21; ?;, 9.67. After the isolation of the D-gluconic acid phenylhydrazide the concentrated mothcr liquors wcre kept a t 2" for 2 1 hours, whereupon a mass of crystallinc-like material v a$ deposited. The mother liquor v a s rcmoved and the solid residue triturated with ice-cold ethanol. Recrystallization of this material from ethanol yiclfled L-idonic acid phenylhydrazide (0 73 g.), m.p. 115-117" with previous sintering at 100-105", [a1Iz2~4-12.5' in water (c 1.6). One sample showed m.p. 117-120' with previous siutering a t 107'. Crystallization from other solvents, such as methanol, I ,4-dioxane, ethanol-ethyl ether or benzene, produced no change in the m.p. of the product. When obscrved under the polarizing microscopc the product did not appear to bc (17) R Fischcr a n d I' I'nssmnre, Bci , 2 2 , 2728 (1881)).
[CoNrRrnurrov
FROX TIIE
DFPARTMIXr
Vol. 76
crystalline. Micheel recorded m.p. 1lS" with previous sintering a t 102", and [ a l z of10.5 ~ (water) for L-idonic acid phenylhydra~ide,~.s while Nef reported m.p. 100-1 10" for the enantiomorphic compound which showed [e] 2 0 -12.4' in water.8 A n d . Calcd. for C I ~ H I S O ~ NC, Z : 50.4; 11, 6.39; X, 9.8. Found: C, 50.03; H,G.6; N,9.3. Epimerization of D-Gulono-r-lactone.-A solution of Dgulono-y-lactone (2 g.) in water (12.5 ml.) containing pyridine (0.9 g.) was heated in a sealed glass tube for 97 hours in a boiling water-bath. The solution was clecolorizetl (charcoal), diluted with water and evaporated in v u c z ~ oto give a sirupy product. The latter was heated in vacuo for 2 hours at 50-55' in order to lactonize the mixture of Dgulonic and n-idonic acids. The sirup was dissolved in hot methanol (10 ml.) and the cooled solution seeded with a crystal of D-gulono-y-lactone. When the crystallization a t room temperature was complete the supernatant liquid was removed, concentrated and a further crop of the crystnllinc D-gUlOno--,-hCtOllC removed. Crystallization was carried out until no more crystals appeared upon cooling to 0'. The sirupy product obtained upon removal of the solvcnt was chiefly D-idono-y-lactone (yield 1.18g.).cfG,*o Formation of the Phenylhydrazide of D-Idonic Acid.When a small portion of thc above sirup was treated with phenylhydrazine in boiling ethanol as described ahovc, the phenylhydrazide of D-idonic acid was obtained. After "crystallization" from ethanol the product hntl in .I>. 1 1.311-2' with previous sintering a t 97". Attempts to "recrystallize" the phcnylhytlrazitlc from methanol, 1,l-dioxane, ethyl ether and water failed to raisc the m . p . and i n a11 cases the rnaterinl appeared to I x ninorQhOUS.
Acknowledgment.-'e wish to acknowledge the generosity of the Xorthern Regional Research Laboratories (Peoria, Illinois) for the calcium 5keto-D-gluconate trihydrate used in this work. Sr. PAUL~ I I N N E S O T A
OF
BIOCHEMISTRY, PURDUE UNIVERSITY]
2-0-(D-GalactopyranosyluronicA c i d ) - L - r h a m n o s e from Okra Mucilage1 B Y ROYL. I~IIISTLER AND H. E. CONRAD RECEIVED JASUARY 14, 1!1,54 Okra mucilage on partial acid hydrolysis yields a mixture of oligosaccharides which can bc scparatcd by chromntogtapliy on carbon and cellulose columns. Among thc acidic oligosaccharides, there are a n aldobiouronic acid and two aldotriouronic acicl)-L-rliniiitioic. The acids. By the methylation method the first of these iq shown t o be 2-O-(~-galaetopyranosyluronic aldotriouronic acids arc shown to be galactosyl -+ (galactosyluronic acid) -+ rhamnose and a (galactosyluronic ncitl) -+ rhamnosyl + galactosc. The occurrence of these oligosaccharides as hydrolytic fragments of okra mucilage suggest< t h a t thc linkages involved are present in the polysaccharide.
Okra obtained by water extraction of individual acidic components is achicvcd by redefatted okra pods is a polysaccharide cornposed of chromatography on a column of cellulose. D-galactose, L-rhamnose and D-galacturonic acid Determination of physical constants and monounits. A . previous investigation of the structure of saccharide components for each of these oligosacthis mucilage2 has shown that partial hydrolysis charides indicates that one is an alclobinuronic ncitl gives rise to three galactobioses, one of which h3s composed of D-galacturonic acid arid r,-rhniiinosc. and t h a t the other two are aldotriouronic ncttls, been proved to be 4-O-~-galactopyranosyl-~-galactosc. Here is reported the isolation and characteri- each of which is composed of gal~cturonicncitl, zation of an aldobiouronic acid obtained upon in- galactose and rhamnose. Upon complete methylation of the aldo1,iouronic complete acid hydrolysis of okra mucilage. Two aldotriouronic acids are also isolated in low yields acid, followed by reduction with lithilliii ,iluiiiinuin hydride and hydrolysis of the reclitcctl iii,itcrinl, from the hydrolyzate. A mixture of acidic oligosaccharides is obtained there are obtained 3,4-di-0-methyl-r,-rhnmnosr ,itit1 from the mucilage hydrolysate by use of carbon 2,3,4-tri-~-methyl-~-galactose. The lattcr c o n chromatography.? Separation of the mixture into pound arises from the reduced galacturonic .icitl (1) Journal Paper Xo 774 of t h e Purdiie Aqricultiiral 1'~perimcnt part of the molecule. From the isolation of tlicsc Station, Lafzyette, Indizna methylated sugars i t can be directly conclutlc tl (2) R I, Whistler and I1 E Conrad, T F I IJor ~ R U A I 76, IC:? (19.54) ( 3 ) R I, Whistler and Il T' niir-o rbzd , 72, 677 (1950)
(4)
T IIoiigh, J R K Tonrc ? n d IV M W i d n i 7 n \
( 10 $ 8 )
i r f i i II
162 I
~
2-O-(D-GALACTOPYRANOSYLURONICACID)-L-RHAMNOSE FROM O K R A
July 5 , 1954
3545
TABLE I ACIDICOLIGOSACCHARIDES FROM OKRAMUCILAGE HYDROLYSATE
a
Yield,
%
R, ga1.a
I, aldobiouronic acid
1.7
0.97
11, aldotriouronic acid
1.0
.42
111, aldotriouronic acid
0.5
.19
Component
Monosaccharide components
D-Galacturonic acid L-Rhamnose D-Galacturonic acid t-Rhamnose D-Galactose D-Galacturonic acid rhamnose D-Galactose
[aIz5D
Equiv. wt. of B a s a l t Calcd. Found
$15.1°
407
446
.....
570
489
+52,2
570
576
Ratio of distance traveled by unknown on a paper chromatogram t o distance traveled by D-galactose in solvent C.
neutrality and the mixture was chromatographed on a 44 X 265 mm. column of cellulose. Solvent A was used to elute the neutral oligosaccharides. Barium salts of the acidic oligosaccharides were washed from the column with water, concentrated t o a sirup (2 8.) and rechromatographed on another cellulose column of the same size using solvent B. Since the irrigatin lution was acidic, the acidic oligosacd from the column in the free acid charides were rem form. Fractions were collected through use of an automatic fraction collectorg and analyzed qualitatively by paper chromatography. Chromatographically similar fractions were combined. By this means there were obtained three acidic oligosaccharides whose characteristics are given in Table I . Methylation of Aldobiouronic Acid (Component I) .Chromatographically pure sirup (I .05 g.) was dissolved in water (8 ml.) and dimethyl sulfate (8 ml.) was added. While stirring this mixture a t Oo, aqueous sodium hydroxide (12 ml., 40%) was added dropwise over an 8-hour period. The reaction mixture was stirred an additional 15 hours during which it warmed t o room temperature. It was then cooled again t o O0, solid sodium hydroxide (7.2 g.) added and dimethyl sulfate introduced dropwise with stirring as before. After a final reaction period of 15 hours, the solution was acidified with dilute hydrochloric acid and extracted continuously for 16 hours with chloroform. The chloroform extract was concentrated in vacuo t o a sirup (0.57 g.) which was twice methylated with methyl iodide (5 ml.) and silver oxide (2 g.) t o give a fully methylated sirup (0.52 g.) with [cY]*~D+54.4" ( c 1.74 in water). Reduction and Hydrolysis of Methylated Sirup.-The sirup was dissolved in anhydrous ether (20 ml.) and the solution mas added dropwise t o a vigorously stirred slurry of lithium aluminum hydride (1.2 g. in 40 ml. of anhydrous Experimental Paper Chromatography.-Chromatographic separations ether) a t room temperature. After a reaction time of 1 hour, excess hydride was destroyed by cautious addition of were made on filter papcr strips and cellulose columns at room temperature using one of the following solvents in the water. On evaporation of the ether the aqueous mixture was acidified with dilute hydrochloric acid and extracted volume ratios indicated: (A). butanol-1, pyridine, water (6:4:3); (B) butanol-I, acetic acid, water ( 2 : l : l ) ; (C) continuously for 15 hours Kith hot chloroform. Removal ethyl acetate, acetic acid, formic acid, water (18:3: 1: 4 ) ; of the chloroform left a sirup (0.50 g.) which was refluxed (D) butanol-1, ethanol, water (5:1:4, top layer); and (E) with methanolic hydrogen chloride (20 ml., 4%) for 4 hours. benzene, ethanol (170:50), saturated with mater. @-Anisi- Water (20 ml.) was added, methanol evaporated and the resulting aqueous solution heated a t 100" for 2 hours. Neudiue hydrochloride8 was used to detect the sugars and their tralization with Ambcrlite IR-4B resin and concentration of derivatives on paper chromatograms. Hydrolysis of Okra.-Fifty grams of okra mucilage was the hydrolysate gave a sirup (0.50 g.) which on paper homogeneously dispersed in 2 1. of water a t 80" and aqueous chromatography was found to contain two components with R, (g = 2,3,4,6-tetra-0-methyl-~-glucose) values of 0.90 sulfuric acid added t o a 5y0 concentration. The mixture was hydroly7ed a t 80" with stirring for 4 hours. On cooling, and 0.75 in solvent D. These values suggested the presence the mixture was neutralized with 750 g. of barium hydroxide of 3,4-di-O-methyl-~-rhamnose and 2,3,4-tri-o-methyl-~and the barium sulfate removed by filtration. Barium ions galactose. Identification of Hydrolysis Products.-The above hyin salt formation with the acidic oligosaccharides were removed by passing the solution through a column of Amberlite drolysate was separated on 4 large sheets (18.5 X 22.5 in.) of Whatman KO. 1 paper with solvent D. Upon paper IRJ120 resin Effluent was then added t o the top of a 44 X ionophoresis in borate buffer a t @H10, the rhamnose com21% mm. column3 of Darco; Celite (1: 1) and monosacchaponent moved toward the anode, which suggested that it was rides mere removed by washing the column with 8 1. of water 3,4 - di - 0 -methyl - L - rhamnose.10 Oxidation with bromine and were discarded. Acidic oligosaccharides were regave crystals moved with 5 1. of 10y0 ethanol. After concentrating the water t o 3,4-di-O-methyl-~-rhamnonolactone eluate t o a sirup ( 3 . 5 g.) barium hydroxide was added t o which on recrystallization from ether-heptane had m.p. 78-79' and [cx]*~D -154' (c 0.21, water) -116" (48 hours). (6) R. E. Gill, E. 1,. Hirst and J. K. N. Jones, J . Ckem. SOL.,1469 (1939). Anal. Calcd. for C~Hi405: C, 50.5; H , 7.4. Found: ('2) R.S. Tipson, C. C. Christman and P. A. Levene, J . B i d . Chem., C, 50.5; H, 7.3.
that the original acidic disaccharide is 2-O-(D-galaCtopyranosyluronic acid) -L-rhamnose. This disaccharide has been isolated previously from hydrolyzates of slippery elm m ~ c i l a g e flaxseed ,~ mucilage6 and Plantago ovato Forsk m ~ c i l a g e . ~ The two aldotriouronic acids recovered from the cellulose column have been partially characterized. Monosaccharide units on the reducing ends of these trisaccharides are identified by oxidizing the aldotriouronic acids with bromine water to convert the terminal reducing sugar units to glyconic acids. On hydrolysis of the oxidized oligosaccharides followed by chromatographic separation of the components and spraying with p-anisidine reagent, the sugars giving rise to the glyconic acids are found to be uncolored. This evidence suggests t h a t a galactose unit is a t the reducing end of one aldotriouronic acid and that a rhamnose unit is a t the reducing end of the other. Further structural evidence is obtained by chromatographic identification of the sugar derivatives obtained by hydrolysis of the methylated and hydrogenated trisaccharides. Together these data suggest t h a t one of the acids is a galactosyl 4 (galactosyluronic acid) --c rhamnose and that the other is a (galactosyluronic acid) + rhamnosyl + galactose.
-
128, GO9 (1939). (7) R.A. Laidlaw and E. G. V. Percival, J. Chenz. SOL.,528 (1950). (8) 1,. Hough, J. K. N. Jones and W. 13.Wadman, ibid., 1702 (1950).
(9) J. L. Nickson and R. I,. Whistler, A n d . Chem., 26, 1425 (1953). (10) A. B. Foster, Chem. a w l Ind., 828 (1952).
2,3,4-Tri-~-methyl-~-galnctose was identified as its characteristic anilide derivative. After recrystallizatioii from absolute ethanol, the 11i.p. was 167-168". aluuZ. Calcd. for CI6HP3OIX:X, 4 . 7 . Found: S , 4.7. An X-ray diflraction patterii of the crystals \vas identical with that of :til authentic specimen. Test for Reversion.-On paper chromatography of a solution of D-g:dactose xiid L-rhamnose in equimolar concentrations ivhich had been stibjectvd to the sanic conditions rolysis of okra tnucilage no oligosaccharide rvetl, sho.5 i.1g f liat the altlobioitroiiic acid IrolJ..;i.; of okra inucilagc i i not :i rcvcrsioii
for the altlobiouroriic acid. Paper chromatography in solvent D of a hydrolysate of a small amount of the reduced product showed the presence of two trimethylgalactoses antl a dimethylrhamnose. The inaiti quantity of the reduced sirup v a s remethylated aiid then hydrolyzed t o yield tetrnmethylgalactose, a trimethylgalactose and a dimethylrhantnose as showu by paper chromatography in solvents I1 antl E. Attempts to isolate these derivatives were UIISL:C~CSSful bccause of the small amounts presetit. By using the bromine osidatioii procedure described above, rhamnose was showti to be a t the reducing eiid of component 111. Paper chromatography of a hydrolysate of completely methylated conipoiient 111 indicated the resence of a tlimcth!-lrhatiinose, :i tlitiieth~lga1:ict~iroiiicacirl :tntl t e t r a i n e t ~ i ~ - l ~ r l a c t o s e .
Characterization of Aldotriouronic Acids.-Bromitte ositlatioti of L: sninil anioutit of coniputiciit I I folio\\.(i l 11)- aciil Acknowledgment.--The authors wish to express l i ~ - ~ l r o l ~a i-i i~l i 11:~pcr s cliroiii:ttogr:tljli~~(Jf tlie liyilrul~~\:itv iiit1ic:itctI tli:tt gcii:tcloyL-is ;it tlic rrtlticitii: cittl t r f tlii> tritheir thxiks to Dr. I,. IJough for supplying the auliaride siitce g:tl:ictow, l l ~iiig > i ) c i . i t o\iclizi.tl t o gxl:tt,thentic speciiiieri of ~ , ~ ~ , l - t r i - O - m e t l i y l - i ~ - g ~ ~ l ~ t ~ ~ t ~ ~ s e t o i i i c w i J , did I t ( J t a1ipc:tr 011 t l i v e ~ t r ~ ~ t i t : t ~ o of g r thi. ~ i i It!.i ~ ~ a~iilitleusetl for X-rny cc)iri~xirisu~i.
[ C O K T R I n L v l I f ~ SFRC)X I ~ E F E S C ERESE:ctivccompound. The latter coinpoutid W:LS itletil ti>- tlcgr:itlation with phosphotus pctitachloride :is 1 - 1 n c t l i y l - 2 - l i ~ i ~ r o s ~ ~1-methyl-1 ~ r o p ~ l -propcti~~l ~nc~th:iricplio~~~hoii;ttc.
lowered the yield of cyclic product to Xiyo. hloreover :L n e w optically active compound was forrnetl yield. This new compound had a rotation and it gave analytical values in good agreement with the empirical forinula CeIll,04P. Its infrared spectrum has a band a t e'M)O CIII. - - I due t o 0-13 stretching vibrations. X medium band at 1710 c~ii:-~ is assigned to C-C stretching vibrations or :til :mociatctl ester group lic.cuuse C 0 stretching vibrations would be espected to give a stronger absorption bnnd. The strong :tbsorption band a t 1 2 2 c i ~ i . - ~with a n :hsorption h n c t on the shoulder a t 1Yi.i ciii: * is :issigned to stretchiiig T,ibr:iticms o f the 1' 0 group:j- a n t i :.I ' I iciii: is :tssigned'2" to the P-0-C linkage. Since the infrared spectrum of the optically active IZ = I ) and 2-iiiethyl-S-oxo-l,3-dioxa-2-phospliacycloheptane (I, I i and I-l d i c l ~ l o r ig~ilv~i i~q a inirturc. o f 8-chloro- structure of t l i c l i l ~ ~ r n ~ ~ h ~ ~ s ~ , h o r - l n ! c . t l ? : l n ~ ~ l i i ~ s ~ ~ l ~ ~ ctlil-1 hytlri)geli i:ieth:iiie])lic~s~)hc,llalc :tiit1 di-3CH C I 1 0 1 I ci rc I I cliloroetliyl ~ i i e t l i a n e ~ ~ l i o s ~ ~ l i t ~ ~ ~ : ~ t e . 0 CII; lieceutly' it was s h o ~ ~ that - i i D - ( - j-i',::-butanetliol :ind niet~innel,liosplionyl dichloricle in methylene chloride anti in the prrsence of pyridine gave a 7,jC'; yield of thc cyclic product -3,1,;,-triiiiethq.l--clxo1 ,:3-dios~t--3-pliosph:t~~yclope~it:~iie (I, I< a n c l I
