J. D. GEERDES.4ND F. SMITH
3572
Vol. 77
Anal. Calcd. for C17HaoO~l:OMe 45.3; equiv. wt., 410. spot while the spot produced by the neutral sugar from the Found: OMe 45.0; equiv. wt., 425. aldobiouronic acid was light tan in color. Heavy spotting on paper chromatograms indicated possible contamination with Methanolysis of Methyl 2-0-( 2,3,4-Tri-O-methyl-~-glucuronosyl)-3,4-di-0-methyl-~-xyloside.-Asolution of the mono-0-methyl-D-xylose so the material was purified by sheet fully methylated aldobiouronic acid in methanol containing paper chromatography. The 3,4-di-0-methyl-~-xylose so D (c 0.7, in methanol). hydrogen chloridz (8yo)was heated (sealed tube) for 8 produced was a sirup, [ c Y ] ~ ~+22.1" A solution of the 3,4-di-O-methyl-~-xylose (15 mg.) hours a t 110-115 The reaction mixture was neutralized with silver carbonate, filtered, and the filtrate evaporated in water (1 mi.) from the fully methylated aldobiouronic t o a sirup. The material wa5 extracted with ether, the acid was oxidized with bromine (0.1 ml.). The reaction ether extract concentrated and hydrolyzed by heating with mixture was allowed to stand in the dark for 50 hours after which time the di-0-methyl-n-xylose had disappeared from -V' sulfuric acid on a boiling water-bath until the rotation became constant. The hydrolysate was neutralized with the solution as determined by descending paper chromatogbarium carbonate, filtered, concentrated in BUCUO t o a volume raphy. The excess bromine was then removed by aeration, the hydrogen bromide neutralized with silver carbonate and of 10-15 ml. and passed successively through a column of ion-exchange resin Amberlite IR120 and of Duolite A4 t o the resulting solution filtered. Residual silver ions were remove the acid component. The eluate was evaporated removed by passing hydrogen sulfide through the solution t o give the neutral sugar component. Paper chromato- which was then filtered and evaporated in Vacuo to a sirup. graphic examination of this neutral sugar fraction using The latter was heated a t 95-100" for 3 hours a t 15 mm. methyl ethyl ketone-water azeotrope as the irrigating sol- pressure in order to bring about lactonization. Upon nuvent revealed a heavy spot having a n Rfvalue of 0.57. cleation of the resulting sirup with 3,4-di-O-methyl-~Comparison standards of 2,3-di-0-methyl-n-xylose and xylono-&lactone complete crystallization took place a t once. Recrystallization from ether pielde! long colorlesz :~,3-di-0-methyl-D-xylosegave spots having Rr values of 0.56 and 0.63, respectively. The wide variation in Ri needles having m.p. and mixed m.p. 67 , [ a ] " D -51 values between 3,5-di-O-rnethyl-~-xylose and the sugar in (25 minutes); -23" (69 hours, constant).10 question discounted the possibility that the former was the Acknowledgment.-The authors wish to thank derivative obtained from the fully methylated aldobiouronic Dr. E. V. White for a sample of 3,4-di-o-methyI-~acid. When sprayed with p-anisidine-trichloroacetic acid1* xylono-&lactone, and the Graduate School of the the 2,3-di-0-methyl-~-xylose gave a dark reddish-brown
.
(18) L. Hough, J. K. N. Jones a n d W. H Wadman, 1702 (1950).
J Chcm. Soc ,
University of Minnesota for financial support. ST. PAUL,MINX.
[CONTRIBUTION FROM THE DIVISIONO F AGRICULTURAL BIOCHEMISTRY, UNIVERSITY O F MINNESOTA]
The Constitution of the Hemicellulose of the Straw of Flax (Linum Usitatissimum Sp.). 11. Hydrolysis of the Methylated Hemicellulose BY J. D. GEERDES AND F. SMITH*^^ RECEIVED JANUARY 28, 1955 The hemicellulose isolated from delignified flax straw by extraction with alkali has been shown t o be a branched chain 4-~-methy~-~-glucurono-~-rhamnoxylan. The side chains, are composed of single units of 4-O-methyl-~-glucuronicacid which are attached t o position 2 of a n-xylopyranose unit of the xylan molecular framework of which L-rhamnopyranose units are also an integral part. The methylated flax straw hemicellulose gives upon hydrolysis: 2,3,4-tri-0-methyl-~xylose (1 mole), 2,3-di-0-methyl-~-xylose(105 moles), 2-O-methyl-~-xylose(2 moles), 3-O-methyl-~-xylose(15 moles), 2.4-di-0-methyl-~-rhamnose (2 moles) and 2,3,4-tri-0-methyl-~-glucuronic acid (15 moles). The general structural features of the polysaccharide are discussed.
J An aldobiouronic acid, 2-0-(4-0-methyl-~-glu- with the proposed linkage in other ~ y l a n s . ~ The curonosy1)-D-xylose, has been shown3 to be a com- high proportion of xylose in the hemicellulose was ponent of the hemicellulose of flax straw. This pa- shown by the fact that crystalline D-xylose could per is concerned with the main structural features be obtained readily from the neutral fraction of the hydrolysate of the flax hemicellulose. of the hemicellulose itself. Paper chromatographic examination of the sugars In order to ascertain the mode of union of the produced from the acid hydrolyzed hemicellulose building units of the flax hemicellulose the latter showed xylose to be the principal component with was acetylated6 and then methylated with methyl a small amount of a second neutral sugar component sulfate and alkali. Fractional precipitation with having an Rfvalue which corresponded to that of the usual solvents gave products which appeared to L-rhamnose. I n addition there was an acidic com- be homogeneous as determined by specific rotation, ponent consisting of 2-0-(4-O-methyl-~-glucuronomethoxyl values, and neutralization equivalents. syl)-D-XylOSe. Following methanolysis with 2y0 methanolic hyWhen the hemicellulose was hydrolyzed with acid drogen chloride under conditions which retain the there was a marked increase in optical rotation. sugar acid in the form of a disaccharide, the glycoThis is generally attributed to the cleavage of sides were separated into neutral and acidic corn,&glycosidic bonds which would be in agreement ponents using ion-exchange resins. The acidic com(1) Paper N o 3311, Scientific Journal Series, Minnesota Agricultural ponent was shown3 to be methyl 2-(2,3,4-tri-OEiperiment Station methyl-~-glucuronosyl)- 3 - 0 - methyl - D - xyloside. ( 2 ) Extracted from a thesis submitted by J D Geerdes t o t h e gradu. .
a t e faculty of t h e University of Minnesota in partial fulfillment of t h e requirements for t h e degree of P h . D . , 1953. This paper was presented at t h e 125th A C.S. merting i n Kansas C ~ t 1954. y ( 3 ) J. D . Gcerdes and F S m i t h , THISJ O I J R S A L , 77, 3563 (1955)
(4) W . N. Haworth and E. G. V. Percival, J . Chem. Soc., 2850 (1331). ( 5 ) R.J . M c l l r o y , i b i d , 121 (1943). ( 6 ) J . F Carson and W D. Rlaclay, THISJ O U R N A L , 70, 293 (1948)
July 5 , 195.5
HYDROLYSIS OF METHYLATED HEMICELLULOSE
3573
Furthermore, the introduction of a methyl group by ing point showed a depression of 16’. This left methylation into the 4-position of the xylose unit only the 2,4-di-O-methyl-~-rhamnoseto be tested ; of the isolated methyl aldobiouronic acid estab- a specimen of the anilide of 2,4-di-O-methyl-~lished that in the polysaccharide the xylose moiety rhamnose was found to give no depression when is joined to other units through position 4. Since mixed with the unknown di-0-methyl-L-rhamnose i t is also joined through its reducing group, this anilide obtained from the flax hemicellulose. The particular xylose residue clearly constitutes a X-ray powder diffraction patterns of the two anilides branch point in the molecular complex. It may be offered further confirmation that the di-0-methylfurther deduced that the 4-O-methyl-~-glucuronic L-rhamnose compound under examination was inacid is attached as a single side chain unit to the 2- deed the 2,4-di-O-methylderivative. position of xylose by a glycosidic bond in the origiFrom the above experimental evidence, certain nal polysaccharide molecule since only the 3-0- structural features of the hemicellulose can be demethyl-D-xylose is obtained from the methylated duced. I t is clear that the 2,3,4-tri-O-methyl-d-xyaldobiouronic acid of the methylated polysaccharide. lose is derived from terminal xylopyranose units in The mixture of neutral sugar glycosides obtained the polysaccharide and from the large amount of from the methanolysis of the methylated flax straw 2,3-di-O-methyl-~-xyloseobtained it is concluded hemicellulose was hydrolyzed and resolved into that the main body of the polysaccharide consists of its components on a cellulose-hydrocellulose col- D-xylose units of the pyranose type linked, as al~ m n .The ~ mixture was found to contain 2,3,4- ready pointed out, through positions l and 4. The 2- 2-O-methyl-~-xyloseis derived from units of xylose tri-O-methyl-D-xylose, 2,3-di-O-methyl-~-xylose, 0-methyl-D-xylose, 3-O-methyl-~-xylose,2,4-di-0- which form branch points in the molecule; clearly methyl-L-rhamnose, a small amount of D-xylose and these units are joined through positions 1 and 3. 4-0-(2,3-di- 0-methyl - D - xylopyranosyl) - 2,3- di - 0- The isolation of 2,4-di-O-methyl-~-rhamnoseindimethyl-D-xylose, cates that this sugar is mutually joined to other The 2,3,4-tri-0-methyl-~-xylosewas identified units of the polysaccharide through positions 1 and 3. by qualitative chromatography, by rotation and by Considering the mole ratios of the cleavage prodthe fact that its anilide had the same Rfvalue as an ucts of the methylated hemicellulose (see Table I) authentic specimen of 2,3,4-tri-0-methyl-~-xylose it will be apparent that the polysaccharide moleanilide. The identity of the 2,3-di-O-methyl-~- cule has one aldobiouronic acid residue associated xylose was established by its transformation into the with 7 D-XylOSe units. Discounting for the prescharacteristic crystalline anilide.* The identity of ent the minor components, the average “repeating the xylobiose, 4-0-(2,3,di-0-methyl-~-xylopyranounit” consists of one 4-O-methyl-~-ghcuronicacid syl)-2,3-di-O-methyl-~-xylose, was establishedby the unit attached as a side chain to a position 2 of one observation that hydrolysis of it gave 2,3-di-0- of eight D-XylOSe units. This would correspond to a methyl-D-xylose. When the monomethyl sugar “repeating unit’’ weight of 1457 for the methylated fraction obtained from the column separation was polysaccharide which is in accord with the value seeded with 2-O-methyl-~-xylose, crystallization (1485) of the neutralization equivalent. The numoccurred, yielding this same crystalline sugar. ber of terminal units (2,3,4-tri-0-methyl-~-xylose) From the mother liquor of the %O-rnethyl-~-xy- found indicates there are 15 of these repeating lose there was also isolated a small amount of crys- units in a single molecule having as the reducing talline 3-O-methyl-~-xylose. ends D-XylOSe or L-rhamnose. The L-rhamnose The component (no. 3, Table 111) leaving the units if not in a terminal reducing position are locolumn between the 2,3-di-0-methyl-~-xylose,and cated in the main chain of the molecule. The exact the 2,3,4-tri-0-methyl-~-xylose fractions had an Rf position of these rhamnose residues is not known value which corresponded to that of a di-0-methyl- but it is clear that they cannot arise either from L-rhamnose. Although it could not be induced to branching points or from non-reducing terminal crystallize it readily gave a crystalline anilide. Since positions. The amount of 2-0-methyl-~-xylose 3,4-di-O-methyl-~-rhamnose crystallizes very read- obtained from the methylated polysaccharide indiily i t was deduced that the unknown di-0-methyl cates that there is an average of 2 xylose units a t rhamnose was probably not the 3,4-di-O-methyl which branching occurs in the xylose chain in addiderivative, a view supported by the fact that 3,4- tion to the 15 xylose units of the chain which form di-0-methyl-L-rhamnose gave a sirupy anilide. That branch points for the attachment of 4-O-methyl-~the compound was a di-0-methyl derivative of L- glucuronic acid side chains. rhamnose was demonstrated by the fact that diTABLE I rect methylation of the crystalline anilide with THE HYDROLYSIS PRODUCTS OF METHYLATED FLAXSTRAW silver oxide and methyl iodideg afforded the known HEMICELLULOSE anilide. crystalline 2,3,4-tri-O-methyl-~-rhamnose Mole r a t i o The possibility that it was 2,3-di-O-methyl-~Sngar derivative (approx 1 2,3,4-Tri-0-methyl-~-xylose 1 rhamnose was next examined. The melting point of the anilide of 2,3-di-O-methyl-~-rhamnosewas 2,3-Di-O-methyl-~-xylose 105 almost the same as the anilide of the unknown di2-0-Methyl-D-xylose 2 0-methyl-L-rhamnose anilide but the mixed melt3-0-Methyl-D-xylose 15 (7) J. D. Geerdes, Bertha A. Lewis, R. Montgomery and F. Smith, A n a l . Chem., 16,264 (1954). (8) I. Ehrenthal, R . Montgomery and F. Smith, THISJ O U R N A L , 76, 5509 (1954). (9) I. Ehrentbal, M. C. Rafique and F. Smith, i b i d . , 74, 1341 (1952).
2,4-Di-O-rnethyl-~-rhamnose 2,3,4-Tri-0-methyl-~-glucuronic acid
2 15
Although the trace amounts of xylose formed by hydrolysis of the methylated polysaccharide could
J. n. GEERDESAND F. SMITH
3.774
1'01. 77
Acetylation of Flax Hemicellulose.-A solution of the hemicellulose (7.2 g.) in formamide (100 ml.) was treated with pyridine (65 ml.) followed by acetic anhydride ( 5 0 ml.), the latter being added gradually with stirring t o keel) the temperature below 45". Two hours after the addition of the acetic anhydride the reaction mixture was poured into a 1 % hydrochloric acid solution (1 1.) with stirring, whereupon the acetylated polysaccharide precipitated as a gel. This was allowed to remain in the acidic solution overnight to leach out pyridine and formamide from the gel. P-D-Xylp 1[@-D-Xylp 117-48-D-Xylp 1 ~--@-D-sylp 1The acetylated product was filtered, washed with water, resuspended twice in absolute alcohol, filtered and dried. Ignition showed that the product contained very little, if 4-0-3Ir-o-D-(:Ap ,, any, ash. The yield of crude acetylated hemicellulose W:LS I 95% of theory. of Flax Hemicellulose Acetate.-A solution The rhamnose units being joined by 1-3 bonds must of Methylation the hemicellulose acetate (13 9.) in 1,4-dioxane (100 ml.) form part of the main chain in which case this por- was treated simultaneously with 45y0 potassium hydroxide (250 ml.) and methyl sulfate (75 ml.). The reagents were tion of the molecule would be indicated by added during a 3-hr. period with vigorous stirring and the - 4 - ~ - x y l p 1-3-~-Rhap l--l-D-Sylp 1-temperature was kept at 45", acetone being added when For convenience the molecule is written with a free necessary to keep the polysaccharide in solution. The rewas completed by heating for 1 hr. in a boiling waterreducing group. This may or may not be the case action bath to decompose the excess methyl sulfate and to expel for it is conceivable that the structure depicted the excess of the acetone, whereupon the partially methylmay be joined to another one and thus may pro- ated product separated. Sufficient boiling water was then vide a branching xylose unit which would give rise added t o dissolve the salts and the product was filtered. methylated hemicellulose was dissolved in acetone and to the formation of the 2 - 0 methyl-~-xyloseupon The remethylated in the same manner as before. After 5 methylmethylation. ations had been applied in this way using acetone as the This additional information concerning the hemi- solvent the methylated product was dissolved in aqueous cellulose of flax straw together with that already acetone (the minimum amount of acetone was used) and the solution treated with dilute hydrochloric acid to pH 5 whrreknown about the hemicellulose of esparto grassl0,l3 upon the methylated polysaccharide was precipitated. wheat s t r a ~ , corn ~ . ~ cobs,* ~ pear cell-wall1° and Further addition of acid failed t o precipitate more product. Phormium tenaxb emphasizes the view that in all After removing the methylated flax straw hemicellulose these polysaccharides there is present a framework thus precipitated, by filtration, it was dried in vacuo and in acetone (70 ml.), When the acetone solution consisting of D-xylopyranose units. The properties dissolved was poured slowly with stirring into light petroleum ether of these hemicelluloses vary considerably which (10 vol.), the methylated flax straw hemicellulose was ohwould now seem to be due to the nature of the side tained as an amorphous almost white powder which after chains which are attached to the main structural filtration and drying in vacuo amounted to 9.9 g. and had framework of xylopyranose units and to the extent [ a ] " D -46.2' (c 1.0, in chloroform), equiv. wt. 1485. Anal. Calcd. for a methylated polysaccharide with the of regularity or irregularity of the branching. In repeating unit proposed (formula I ) : OMe, 37.6. Calcd. esparto grass hemicellulose the pure polysaccharide for a methylated pentosan: OMe, 38.7. Found: OMe, contains few if any other sugar unitslO but in the 36.6. case of the wheat straw h e m i c e l l u l ~ s e ~the ~ ' ~side The methylated hemicellulose (8.3 g.) was dissolved in chains consist of L-arabinose units. In the wheat acetone (160 ml.) and light petroleum ether (b.p. 3C-60") added slowly in increasing amounts with stirring to efstraw hemicellulose studied by :Idams13 the side was fect fractional precipitation. The properties of the five chains consist of L-arabinose and D-glucuronic acid fractions ohtained are given in Table 11.
conceivably arise from demethylation'O or incomplete m e t h y l a t i ~ nthe , ~ ~ possibility should not be overlooked that it represents a unit in the molecule a t which multiple branching occurs. From these facts a simplified structure I for flax straw hemicellulose may be tentatively proposed.
i ]
[
units, while corn cob hemicelluloses have been obtained which have L-arabinose units8 and glucuronic acid units14 attached to the basic anhydroxylose framework. In the case under discussion, the side chains consist almost entirely of units of 4-0methy~-~-g~ucuronic acid. Experimental l5
Hydrolysis of Flax Straw Hemicellulose .-After removing the acidic compound from the selectively acid-hydrolyzed hemicellulose with ion-exchange resins (see preceding paper) the neutral sugar fraction crystallized spontaneously upon removal of solvent and gave D-xylose m . p . and mixed m.p. 146', [ c Y ] * ~ D+18,6" (c 1.2, in water). Qualitative paper chromatography of the mother liquor using solvent A gave spots having Rivalues of 0.49 and 0.65 corresponding to Dxylose and L-rhamnose, respectivel!.. ( I O ) S. K . Chanda, R . L. Hirst, J K. N Jones and E . C: V.Percival, Soc., 1289 (1950). (11) S. K. Chanda, E . L Hirst and E . G . V. Percival, ibid., 1240 (1951). (12) W . h-.Harvorth, E . L. Hirst and Elsie Oliver, ibid , 1917 (1934). (13) G. A. Adams, Can. J. Chem., 3 0 , 698 (1952). (14) R . L. Whistler and L. Hough. THISJ O U R N A L , 75, 4918 (1963).
J. Chem.
(15) F o r the partition chromatographic analyses t h e following solvents were used: A, phenol saturated with water; B, methyl ethyl ketone: water azeotrope; C, 1-butanol-ethanol-water ( 4 : 1:5).
TABLE I1 FRACTIOXAL PRECIPITATION OF METHYLATED FLAXSTRAW HEMICEILULOSE Iiraction
Total petroleum rther added (ml.)
- constitutional significance. OMe, 24.5. acetate ( c 1.0). AnaZ. Calcd. for C13H~BOiS: Found: OMe, 24.5, I t failed t o give a dibenzylidene dimethyl acetal derivative. ( e ) The Isolation of 2-0-Methyl- and J-O-Methyl-~-xyAcknowledgement.-The authors wish to thank lose from Component S.-Component 5 , consisting of a D in water (c 1.2). Paper the graduate school of the University of Minnesota sirup (39.7 mg.), had [ C L ] ~ ~$30" for financial support during the initiation of this chromatographic examination using solvent B indicated (21) T h e authors are grateful t o D r . W. N. Lipscomb of t h e University of Minnesota for carrying o u t these determinations
[COTTRIBUTION FROM
THE
work. ST. PAUL,MINNESOTA
DEPARTMEUT OF CHEMISTRY OF THEUNIVERSITY OF CALIFORNIA AT Los ANGELBS]
Synthesis and Properties of Certain Spiro [4.4]nonenes1 BY DONALD J. CRAMAND B. L.
VAN DUUREN RECEIVED OCTOBER 27, 1954
The synthesis of l-spiro(4.4]nonene (\TII), 1,3-spiro[4.4)nonadiene (111) and 1,6-spiro[4.4]nonadiene (XVIII) are described, as well as the behavior of I11 as a dienophile in a Diels-Alder reaction.
The objective of this investigation was the exploration of the chemistry of the spiro [&.4]nonenes, the compound 1,3,6,8-spiro[4.4]nonatetraene (I) being the ultimate target for the work. Since 1,Gdiketospiro[4.4]nonane (TI); was an obvious but expensive starting material, the model synthesis of 1,3spiro[4.4]nonadiene (111) from the more available l-ketospiro[4.4]nonane (117) was developed first. 0 /I
I
0 I1 0
! _ < > - II I11
11'
The monoketone IV3 was converted to its oxime (1) This research mas conducted under contract AF 33(616)-146 with t h e United States Air Force, the sponsoring agency being t h e Aeronautical Research Laboratory of t h e Wright Air Development Center, Air Research and Development Command. (2) D . J. Cram and H. Steinberg, THISJ O U R N A L , 76, 2753 (1954). Qudrat-i-Khuda I. and A. K. R a y , J. I n d i a n C h e m . Soc., 16, (3) (a) & 518 (1939); (b) M. Qudrat-i-Khuda and A. Mukherjee, i b i d . , 16, 532 (1939); (c) N. D . Zelinskii and H. V. Elagina. Comfit. rend. m a d . s c i . , U . R . S . S . , 49, 568 (1945). or C. A , , 40, 6058 (1946).
which was reduced catalytically to provide l-aminospiro [4.4]nonane2 (17) which was dimethylated with formaldehyde and formic acid4 to give l-dimethylaminospiro[4.4]nonane (VI). This material was converted to the amine oxide VI1 which on pyrolysis gave l-spiro[4.4]nonene (1'111) .5 The same olefin was obtained in better yield through the Hofmann elimination reaction, the infrared spectra and physical properties of the two olefiii samples being identical. Olefin VI11 was characterized by quantitative catalytic hydrogenation to the known saturated spiro [&.4]nonane (IX) ,6 Bromination of olefin IT11 with N-bromosuccinimide gave an unstable oil which could not be purified, and which was converted directly to 3-dimethylamino-l-spiro[4.4]nonene. Xlkylation of this material led to the quaternary compound XI which when heated with silver oxide gave diene 111. This material was thermally unstable and when distilled a t atmospheric pressure appeared to dimerize to give the (4) H. T . Clarke, H. B. Gillespie and S.Z . Weisshaus, T H I S J O U R 66, 4571 (1933). ( 5 ) For references t o this cis-elimination reaction see: (a) A. C .
NAL,
Cope, T. T. Foster and P. H. Towle, THISJ O C R N A L , 71, 3939 (1949); (b) D . J. Cram, i b i d . , 74, 2137 (1952); A. C . Cope, R . A . Pike and i'. Spencer, ibid., 75, 3212 (1953); D. J. Cram and J . E. IZIcCarty, ibid., 77, in press (1955). (6) N. N. Chatterjee, J . I n d i a n Chem. SOL.,14, 259 (1937).
