1 POLYSACCHARIDE INVESTIGATIONa by JOHN LEONARD SNYDER A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF CHEMISTRY. We accept t h i s Thesis as conforming to the standard required from candidates f o r the Degree of MASTER OF SCIENCE Members of the Department of Chemistry THE UNIVERSITY OF BRITISH COLUMBIA September, 1952 ABSTRACT Mesquite gum, sapote gum A and sapote gum B have been subjected to p u r i f i c a t i o n and hydrolysis. Chromato-graphic examination of each hydrolysate has revealed the constituent sugars. The three gums have been methylated and chromatographic examination of hydrolysates from these methylated derivatives has indicated the methylated sugars present. Methylated mesquite gum yielded 2 , ^ , 5-trimethyl-L-arabinose, 3 ,5-dimethyl-L-arabinose, 2,4-dimethyl-j-D-galactose and 2 , 3 , 4-trimethyl-D glucuronic a c i d . Sapote gum A gave 2 , , 4-trimethyl-D-xylose, 2 , 3 , 4-trimethyl-L-arabinose, 3^monomethyl-D-xylose and some 2 , ^ , 4 - t r i m e t h y l -D-glucuronic acid. These re s u l t s are i n agreement with published data. A second sample of sapote gum, designated as sapote gum B, has been shown to contain d i f f e r e n t methylated sugars. Both gums were obtained from the same source. Among the components of sapote gum B 2 , 2 , 4-trimethyl rhamnose, 2, 3 , 5-trimethyl arabinose and 2 , ^ , 4-trimethyl xylose-have been detected. The presence of 2 ,4-dimethyl-D-galactose, 2 , 3 , 4 -trimethyl-D-galactose and 2,'j,,4-trimethyl-D-glucuronic acid has also been shown. The difference i n the methylated pro-ducts obtained from the two samples of sapote gum indicates a d i s s i m i l a r i t y of structure. Both gums were thought to be produced by the same species, but i n view of the above re-su l t s t h i s does not appear l i k e l y . ACKNOWLEDGMENT. I would l i k e to express my appreciation f o r the guidance and encouragement given by Professor G.G.S. Button during the course of t h i s work. Sincere thanks are also extended to the National Research Council f o r a summer research grant and to the Consolidated Mining and Smelting Company for a fellowship. TABLE OF CONTENTS. Page I. INTRODUCTION 1 I I . HISTORICAL A. Mesquite gum 8 B. Sapote gum 10 C. Methods 11 I I I . EXPERIMENTAL 1. Examination of the Three Gums A. Mesquite gum ... ... ... 16 B. Sapote gum A 16 C. Sapote gum B ... • 16 2. Composition of Crude Sapote Gum B ..... ... 17 3 . Methylation of the Gums A. Mesquite gum 18 B. Sapote gum A • 19 C. Sapote gum B 20 4 . Chromatographic Examination of the Native Gums 21 5. Chromatographic Examination of the Methylated Gums 22 6. Chromatographic Examination of the Reduced Methanolysis Syrups 23 7. Methanolysis of the Free Acids of the Three Methylated Gums 24 8. Examination of the Glycosidic and Uronosidic Components of the Methanolysis Syrups ... 26 9 . Components of Sapote Gum B 28 10 . Separation of the Methylated Sugars from Mesquite Gum by a Cellulose Column ... ... 31 11. I s o l a t i o n of the Uronic Acids from Native . Mesquite Gum and Native Sapote Gum B . ... 33 IV. DISCUSSION .36 V. BIBLIOGRAPHY ... 42 TABLE OF CONTENTS. (Continued) Page VI. APPENDIX Table I 46 Table I I 47 Table I I I 48 Table IV. ... 49 Table V .• .. - 50 Table VI 51 Table VII 52 Table VIII 53 I. .INTRODUCTION. Among the enormous number of...plant polysaccharides the plant gums {U» "29 ,'.,.49) which ..contain uronic acid residues, and are thus,.better known as polyuronides, are of wide occurrence. These gums are .produced by plants and trees i n response to i n j u r i e s to the bark. The gum flows from the wound and on e xposure to the a i r s t i f f e n s and forms a horn-like covering which aids i n preventing the invasion of rots and other parasites. This sealing action of the gum also prevents the loss of nutrients and other v i t a l materials. The wound whether naturally or a r t i f i c i a l l y caused i s sealed i n the same manner. Rep-resentative examples of these gums are gum arabic, damson, gum, lemon gum, gum tragacanth, cherry gum, and mesquite gum. Polyuronides which form a heterogeneous group of complex substances have a common character i n that upon acid hydrolysis they y i e l d pentoses, hexoses and hexuronic acids. In the past most investigations dealt with the com-po s i t i o n of the gums. More recent studies are directed to the establishment of structure. S u f f i c i e n t information on the components, t h e i r ring form and the methods by which they are linked together may lead to a f u l l e r under-standing of photosynthesis and perhaps indicate how such diverse substances as rubber, l i g n i n , terpenes, chlorophyll, anthocyanins and c e l l u l o s e are b u i l t up from carbon dioxide and water. The problem of the manner by which the gums are produced i s fundamental i n carbohydrate chemistry. I t was once thought that insects or other invaders were the cause of gum formation. Recently i t has been proposed that the hexoses could lead to pentoses by a simple oxidation-decarboxylation mechanism. However, t h i s could not occur at the polymeric l e v e l without disruption of the gum molecule. Structural studies may provide useful knowledge concerning t h i s problem. The gums are worthy of study i n another res-pect i n that they resemble the ba c t e r i a l polysaccharides which are becoming important 1in chemotherapy. U t i l i z a t i o n of the gums to a greater extent i n d u s t r i a l l y can only be improved • by a more complete knowledge of t h e i r composition and structure. The majority of the gums as t h e i r name would indicate become sti c k y , gummy or gelatinous when moistened. They are high molecular weight amorphous substances which are soluble i n water or alkal i n e solutions from which they can be pre-c i p i t a t e d by the addition of alcohol. They do not reduce Fehling's solution, but treatment with hot mineral acid causes hydrolysis l i b e r a t i n g free reducing sugars. The gum molecule i s usually produced as a s a l t , but methyl esters have been found as well as methyl ethers. The separation of the complex mixture of clos e l y related substances which i s obtained by the hydrolysis of a polysaccharide presents problems not unlike those met with i n protein chemistry. Not only must the nature of the constituent sugar be established but also the isomeric form and the method of linkage between i n d i v i d u a l sugar molecules as we l l as the order i n which they are arranged. Simple hydrolytic technique does not serve to indicate the s t r u c t u r a l form of a polysaccharide. In the syrups r e s u l t -ing from a hydrolysis the hydroxyl groups of the sugars are not able to be d i f f e r e n t i a t e d and there i s no way of determining which hydroxyl group of a given sugar was concerned i n the glycosidic l i n k between i n d i v i d u a l residues. Various techniques have been developed f o r the;purpose of masking the free hydroxyl groups present i n the native polysaccharide without causing any s t r u c t u r a l changes during the procedure. The group used as a masking or blocking agent must be resistant to subsequent treatment, i t should not cause too great an increase i n the molecular weight and the methods available f o r introducing i t should be convenient. Acetylation f u l f i l l s some of these s t i p u l a t i o n s , but as the acetyl group i s l a b i l e i n the presence of acid i t i s subject to removal during subsequent mineral acid hydrolysis. N i t r a t i o n may be a:. i s r e a d i l y converted to the free sugar by hydrolysis. Using t h i s technique Lythgoe and Trippet ( 4 2 ) i d e n t i f i e d the uronic acids obtained from methylated g l y c y r r h i n i c acid, the acid saponin of l i q u o r i c e root. I t has also been used by Hough, Jones and Wadman ( 36 ) to demonstrate 4-methoxy-D-glucuronic acid i n gum myrrh, by Chanda, Hirst and Pe r c i v a l ( 1 5 ) to show that 2 , 3 , 4-trimethyl-D-glucuronic acid i s obtained by the hydrolysis of methylated pear c e l l wall xylan. Adams ( 1 ) used sodium borohydride to demonstrate phe presence of the same methylated uronic acid i n hydrolysates of methylated wheat straw hemicellulose polyuronide. The use of chromatographic techniques and synthetic ion-exchange resins as well as the application of l i t h i u m aluminum hydride i n problems of carbohydrate chemistry has considerably s i m p l i f i e d the task of separating and characterizing polysaccharide hydrolysates. In the investigation of sapote gum B, an attempt has been made to u t i l i z e these new techniques. - 16 -I I I . EXPERIMENTAL. 1. Examination of the Three Gums. A. Mesquite gum. This gum was obtained i n the form of a b r i t t l e r e s i n ; many of the pieces were worm-shaped and i t exhibited various shades of amber. Pieces of bark, leaves, twigs and sand were the p r i n c i p l e inclusions. Selected samples d i s -solved completely i n water, forming a moderately viscous solution from which the contaminants could be screened out. The gum was re a d i l y precipitated from aqueous solutions by the addition of alcohol to y i e l d a f i n e white powder. The p u r i f i e d gum was insoluble i n organic solvents. B. Sapote gum A. Sapote gum A was received i n the form of large nodules which could only be broken with d i f f i c u l t y . The broken pieces were l i g h t amber i n colour and s l i g h t l y opaque. There was very l i t t l e contamination by bark eta i n the nodules. Selected samples dissolved rather r e a d i l y and com-p l e t e l y i n water to y i e l d a pale amber solution which was le s s viscous than either mesquite gum or sapote gum B. The gum was precipitated by the addition of alcohol giving a white f l u f f y powder. The p u r i f i e d gum was insoluble i n organic solvents. C. Sapote gum B. This gum was obtained i n small pieces of no d e f i n i t e - 17 -shape. It was e a s i l y broken giving clear fragments of v a r y i n g shades of amber. The gum dissolved slowly i n water producing a rather viscous solution with a p u t r i d odour. Insoluble matter could be screened out i n theform of a clear water insoluble j e l l y and a brown resinous powder. The gum was r e a d i l y precipitated by alcohol to y i e l d a white powder. The j e l l y was insoluble i n excess water i n which i t swelled, considerably. Sodium hydroxide dissolved the j e l l y quite r e a d i l y from which i t could be precipitated, i n the form of a white powder, by the addition of acid alcohol. The p u r i f i e d gum and j e l l y were insoluble i n organic solvents. 2. Composition of Crude Sapote Gum B. The crude gum (25 gm.) was dissolved i n water (300 ml.) and centrifuged. After decanting the supernatant, the sed-iment was washed twice with water. From the combined super-natant and washings the soluble gum was precipitated by the slow addition of excess alcohol to the s t i r r e d solution. The sediment i n the centrifuge cups was t r i t u r a t e d twice with 5% sodium hydroxide (20 ml) and f i l t e r e d through a medium f r i t t e d glass funnel. The f i l t e r e d solution was roughly neutralized with ice-cold 5% hydrochloric acid and the dissolved j e l l y p r ecipitated by the addition of excess alcohol. Reprecipitation of the j e l l y i n the same manner produced a f i n e white powder. The r e s u l t s of t h i s separation are given i n Table I. - 18 -3. Methylation of the Gums. A. Mesquite gum. Selected pieces of crude mesquite gum (100 gm.) were dissolved i n water (100 ml.) and screened to remove bark, chips, etc. The f l a s k and screen were washed with a d d i t i o n a l water (50 ml.) and the combined solutions placed i n a f l a s k f i t t e d with two dropping funnels and a powerful s t i r r e r . Dimethyl sulphate (300 ml.) and 30% sodium hydroxide (600 ml.) were added dropwise and simultaneously over a period of four hours with vigorous s t i r r i n g , the reaction being maintained at 10-20°C. Acetone was added from time to time to reduce v i s c o s i t y and foam. After the addition of the rea-gents, s t i r r i n g was continued f o r an hour to insure complete hydrolysis of the dimethyl sulphate. The reaction mixture was l e f t to s e t t l e into two phases and the lower aqueous phase was siphoned off leaving the insoluble p a r t i a l l y methylated sodium s a l t of the gum i n the reaction f l a s k . After four treatments -methylation was v i r t u a l l y complete and the product was placed i n cellophane bags, dialyzed against running water u n t i l n e utral, f i l t e r e d and evaporated to a syrup under reduced pressure. The syrup, was added i n a fin e stream to an excess of cold 0.2N sulphuric acid, warmed to 25°C. and centrifuged. After washing the precipitate neutral with water i t was d i s -solved i n chloroform, dried over anhydrous magnesium sulphate, and evaporated to a syrup under reduced pressure. Pure dry - 19 -ether was added (75%) to p r e c i p i t a t e incompletely methylated material and the f i l t e r e d solution evaporated under reduced pressure to give a white to pale yellow r e s i n ; Y i e l d 7 8 % ; MeO, 39.2%; [ ^ - I Q 0 + ^ 1 0' M e t h a n o l ) « T n e r e s u l t s of several t r i a l s are shown i n Table I I . B. Sapote Gum A. The gum (75 gm.) was dissolved i n water (350 ml.) and f i l t e r e d through cheese c l o t h . Ethanol was added with s t i r r i n g u n t i l no more precipitate appeared and the mixture l e f t to s e t t l e . The supernatant was decanted and the pre-c i p i t a t e d gum washed twice with ethanol, a i r dried and d i s -solved i n water (75 ml.). The solution was methylated four times using dimethyl sulphate (150 ml.) and 30% sodium hydroxide (300 ml.) f o r each treatment. The material did not separate on standing and was precipitated each time by the careful addition of ice-cold 6 N . sulphuric a c i d . After d i a l y s i s and f i l t r a t i o n the product was evaporated under re-duced pressure to a syrup and added slowly to an excess of 0 . 5 N . sulphuric acid, warmed to 3 0°C, centrifuged, washed and dissolved i n methanol. The methanol solution was dried by d i s t i l l a t i o n , f i l t e r e d and evaporated to dryness under re-duced pressure. The product, a tough white p l a s t i c , was.in-soluble i n chloroform but soluble i n methanol, ethanol and water; Yield 44%, MeO, 35.2%. - 20 -C. Sapote gum B. A solution of sapote gum B (130 gm.) i n water (1 1.) was treated with excess ethanol and centrifuged. The sediment was washed twice with ethanol, dried, dissolved i n water (100 ml.) and methylated. After the f i r s t treatment with dimethyl sulphate (300 ml.) and 30% sodium hydroxide (600 ml.) the reaction mixture was neutralized by the cautious addition of ice-cold 6N. sulphuric acid. The neutralized material was then evaporated to a paste on a steam bath and remethylated. The mixture r e s u l t i n g from the second methylation was s i m i l a r l y neutralized and then dialyzed, f i l t e r e d and evaporated under reduced pressure. This procedure was followed f o r the t h i r d and fourth methylations. After the f i n a l treatment the mix-ture was dialyzed u n t i l neutral, f i l t e r e d and evaporated to a syrup under reduced pressure. This syrup was added slowly to an excess of s t i r r e d , cold 0.5N sulphuric acid. No pre-c i p i t a t i o n occurred u n t i l the mixture was warmed to 30°C. The precipitated gum acid was centrifuged, washed, taken up i n chloroform, dried with anhydrous magnesium sulphate and f i l t e r e d . Removal of the solvent under reduced pressure gave a yellow r e s i n , Yield •> 70%, MeO, 37.4%, [ Methanol). The r e s u l t s of a series of t r i a l s are given i n Table I I I and I V . 4 . Chromatographic Examination of the Native Gums. Samples of each of the three gums (1 gm.) were dissolved i n 0.5% sulphuric acid (150 ml.) and sealed i n heavy glass tubes. The tubes were then heated i n flowing steam f o r f o r t y -eight hours, cooled and opened. Excess acid was neutralized with s i l v e r carbonate and the s i l v e r sulphate removed by f i l t r a t i o n . The f i l t r a t e s were treated with hydrogen sulphide, f i l t e r e d , decolorized with activated carbon, f i l t e r e d and evaporated to t h i n syrups under reduced pressure. Small drops of these syrups, together with known sugar samples, were placed on the st a r t i n g l i n e of paper chromatograms using Whatman ffl paper and the chromatograms were then developed i n a vapour - 22 -t i g h t c y l i n d r i c a l tank with a solvent composed of n-butanol-ethanol-water, 4 : 5 : 1 , containing a trace of ammonium hy-droxide. After forty-eight hours the papers were a i r dried and sprayed with various reagents. Mesquite gum produced three spots corresponding to uronic a c i d , galactose and arabinose i n color and po s i t i o n . Sapote gum A produced three spots corresponding to uronic acid, arabinose and xylose while sapote gum B produced f i v e spots corresponding to uronic acid, galactose, arabinose, xylose and rhamnose. Chromato-grams developed with methyl ethyl ketone-water azeotrope or n-butanol-acetic acid-water, 5:1^4, served to confirm these r e s u l t s . 5. Chromatographic Examination of the Methylated Gums. samples of each of the methylated gums (1 gm.) were d i s -solved i n methanol (50 ml.) and made 5% i n hydrogen chloride. The solutions were sealed i n tubes and heated i n flowing steam f o r forty-eight hours. After cooling the sealed tubes i n an acetone-dry ice mixture they were opened and the excess hy-drogen chloride was removed by d i s t i l l a t i o n under reduced pressure. Residual a c i d i t y was neutralized by s i l v e r carbon-ate and the neutral solutions f i l t e r e d . Hydrogen sulphide was passed into the mixtures to remove excess s i l v e r and the p u r i f i e d solutions were f i l t e r e d through activated carbon. The syrups obtained by evaporation of the f i l t r a t e s were d i s -solved i n 0.5% hydrochloric acid (25 ml.) and refluxed f or D - 23 -nine hours to hydrolyze the glycosides. Neutralization of excess acid with s i l v e r carbonate, removal of residual s i l v e r by hydrogen sulphide followed by treatment with activated carbon and evaporation under reduced pressure gave syrups. These syrups were spotted on paper chromatograms and de-veloped f o r eighteen hours with n-butanol-ethanol-water, 5*.4*1. The dried chromatograms were then sprayed with various reagents and the presence of several methylated sugars i n each hydrolysate was noted. The r e s u l t s of these separations are given i n Table V. 6 . Chromatographic Examination of the Reduced Methanolysis Syrups. Each of the gums (2 gm.) was subjected to methanolysis by 5fo methanolic hydrogen chloride (75 ml.). The glycosidic mixtures were neutralized, p u r i f i e d and evaporated to syrups under reduced pressure. These syrups were dried over phosphorus pentoxide i n a vacuum f o r twenty-four hours. The anhydrous syrups were dissolved i n sodium dried tetrahydro-furan (20 ml.), l i t h i u m aluminum hydride (1.5 gm.) was added to each solution, they were refluxed for two hours and l e f t over-night. The excess hydride and sugar-salt complex was decomposed by the cautious addition of ice-cold N. sulphuric acid i n excess. The a c i d i c solutions were neutralized with s o l i d barium carbonate, f i l t e r e d and evaporated to dryness under reduced pressure. Extraction of the residues with - 2 4 -chloroform followed by evaporation of the solvent gave the reduced methylated glycosides of mesquite gum (1.9 gm.), sapote gum A (1.7 gm.) and sapote gum B (1.8 gm.). These syrups were refluxed f o r eight hours with 0.5% hydrochloric acid (15 ml.) followed by ne u t r a l i z a t i o n , p u r i f i c a t i o n and evaporation under reduced pressure. Chromatographic ex-amination of the re s u l t i n g syrups showed that a l l the uronic acids had been reduced and each gum contained a new component, RQ .80-.82, corresponding to 2,3,4-trimethyl-glucose. 7. Methanolysis of the Free Acids of the Three Methylated Gums. The methylated gums (30 gm.) were dissolved i n methanol (200 ml.) and made 4% i n hydrogen chl o r i d e . These solutions were refluxed with s t i r r i n g f o r eight hours. Small samples (2 gm.) were removed from time to time f o r chromatographic examination. After removal of excess acid by d i s t i l l a t i o n under reduced pressure, and neu t r a l i z a t i o n of residual a c i d i t y by s i l v e r carbonate the solutions were p u r i f i e d and evaporated under reduced pressure to syrups. These syrups were dried by repeated d i s t i l l a t i o n of chloroform. The re s i d u a l chloroform was then removed under reduced pressure. Exhaustive extraction of the dried syrups with l i g h t b o i l i n g petroleum ether removed the acid sensitive methyl pentosides. Mesquite gum gave 14.5 gm. sapote gum A gave 9.0 gm. and sapote gum B gave 11.5 gm. of petroleum ether soluble extract. The petroleum ether insoluble portions were dissolved i n methanol (150 ml.) and the solutions - 25 -were made 5% i n hydrogen chloride. After a second hydrolysis i n sealed bottles at IOO°C. f o r eight hours i n a shaking-hydrogenator the excess acid was d i s t i l l e d cff under reduced pressure. Neutralization of residual a c i d i t y followed by p u r i f i c a t i o n and evaporation under reduced pressure gave 17.5 gm. of syrup from mesquite gum, 20.5 gm. from sapote gum A and 25.5 gm. from sapote gum B. The two glycosidic f r a c t i o n s of each gum were combined, dissolved i n O.3N barium hydroxide solution (250 ml.) and warmed t o 65°C. f o r three hours to hydrolyze the uronoside esters, a f t e r which the excess base was removed by passing i n carbon dioxide. The solutions were f i l t e r e d , treated with decolorizing carbon and evaporated under reduced pressure to syrups which were dried by d i s t i l l a t i o n with chloroform followed by storage i n a desiccator over phosphorus pentoxide for forty-eight hours. Mesquite gave 30.5 gm., sapote A gave 31.5 gm. and sapote B gave 39.0 gm. of dried syrup. The three syrups were mixed with dry ether, refluxed, d i l u t e d with dry ether u n t i l no more precipitate formed and then an excess was added. After s e t t l i n g , the supernatants were decanted through a coarse grade f r i t t e d - g l a s s funnel. The residues were repeatedly washed with ether and rinsed onto the funnels. The precipitated barium s a l t s were dried over phosphorus pentoxide. i n a vacuum and the ether solutions were evaporated under reduced pressure. Mesquite gum gave 6 gm. of barium s a l t s arxh24.8 gm. - 26 -of glycosides. Sapote gum A gave 15.0 gm. of barium s a l t s and 16 g. of glycosides. Sapote gum B gave 15.5 gm. of barium s a l t s and 21.9 gm. of glycosides. £. Examination of the Glycosidic and Uronosidic Components of the Methanolysis Syrups. The glycosidic portion (25 gm.) of mesquite gum was fractionated by vacuum d i s t i l l a t i o n using a ten centimetre vacuum jacketed Vigreaux. column. The barium s a l t s (21 gm.) obtained from mesquite gum were refluxed for twenty-four hours with Sfo methanolic hydrogen chloride (150 ml.). F i l t r a t i o n followed by d i s t i l l a t i o n and n e u t r a l i z a t i o n of the excess acid gave a clear solution which on evaporation under reduced pressure yielded a syrup (5.5 gin.). This syrup was d i s t i l l e d i n the same manner as the^ glycosidic por-t i o n . A sample (0.5 gm.) of each f r a c t i o n obtained by the d i s -t i l l a t i o n of the glycosidic and uronosidic portions from mesquite gum was refluxed f o r three hours with 0:5% hydro-ch l o r i c acid (5 ml.) to hydrolyze the glycoside or uronoside to the free sugar. Excess acid was neutralized with s i l v e r carbonate, residual s i l v e r was removed as the sulphide and aft e r treatment with activated carbon each solution was ev-aporated under reduced pressure to a syrup. Spots of the syrup from each f r a c t i o n ..were placed on the s t a r t i n g l i n e of paper chromatograms and developed with d i f f e r e n t solvent mixtures. Spraying of the chromatograms.with di f f e r e n t - 2 7 -reagents, comparison with known sugar samples and measure-ment of the H Q values served to indicate the various sugars i n the f r a c t i o n s . The r e s u l t s of t h i s procedure are con-tained i n Table VI. The glycosidic portion (13 gm.) of sapote gum A was d i s t i l l e d i n the same manner as described f o r mesquite gum. The barium s a l t s (15 gm.) were converted to the uronoside esters by refluxing f o r twenty-four hours with 8% methanolic hydrogen chloride (200 ml.). Barium chloride (3 . 7 gm.) was f i l t e r e d off and the neutralized solution evaporated to a syrup (12.0 gm.). This syrup was dried by d i s t i l l a t i o n with chloroform and exhaustively extracted with dry ether. Evaporation of the ether solution under reduced pressure gave a t h i c k pale brown syrup (10 gm.) which was fractionated by vacuum d i s t i l l a t i o n through a s i x centimetre vacuum jacketed Vigreaux column. Each f r a c t i o n obtained by these d i s t i l l a t i o n s was examined chromatographically. The r e s u l t s are shown i n Table VII. The glycosidic portion (18 gm.) of sapote gum B was fractionated as described for mesquite gum. Treatment of the barium s a l t s (15 gm.), obtained from t h i s gum, with 8% methanolic hydrogen chloride (200 ml.) f o r twenty-four hours, followed by f i l t r a t i o n to remove barium chloride (3.1 gm.), n e u t r a l i z a t i o n and evaporation gave a syrup (12 gm.). This syrup was dried, exhaustively extracted with ether and the ether evaporated to y i e l d a pale brown syrup (10 gm.), 8.5 gm. - 28 -oi" which was fractionated by vacuum d i s t i l l a t i o n through a s i x centimetre vacuum jacketed Vigreaux column. Chromato-graphic examination of the f r a c t i o n s from these d i s t i l l a t i o n s indicated the methylated sugars present. The r e s u l t s are given i n Table V I I I . 9 . Components of Sapote Gum B. Fractions 1 and 2, Table V I I I , were mixtures of 2,3,4-trimethyl-xylose and 2,3,5-trimethyl-arabinose, i d e n t i f i e d by t h e i r R Q values and color reactions with various spray reagents as wel l as by comparison with authentic samples. A t h i r d component, R Q 1.02, was also present, and thought to be 2,3,4-trimethyl-rhamnose because of i t s high R Q value and colour reactions. Chromatograms run i n three d i f f e r e n t s o l -vent mixtures f a i l e d to resolve these components. Neither the mixture composed of the glycosides nor a mixture of the free sugars could be induced to c r y s t a l l i z e . The mixture of the free sugars ( I gm.) was stored i n the dark with bromine (2 ml.) and water (20 ml.) u n t i l i t became non-reducing. Excess bromine was removed by aeration and r e s i d u a l a c i d i t y was neutralized by s i l v e r carbonate. The solution a f t e r f i l t r a t i o n , p u r i f i c a t i o n and evaporation was d i s t i l l e d (100°C, .2 mm.) to give a syrup (.7 gm.) which did not c r y s t a l l i z e . Treatment of t h i s syrup with saturated methanolic ammonia f o r three days followed by evaporation of the solvent gave a - 2 9 -syrup which could not be c r y s t a l l i z e d . Another portion of the free sugars (0.5 gm.) from fractions 1 and 2 was re-fluxed with fre s h l y d i s t i l l e d a n i l i n e (0.5 ml.) i n absolute ethanol (10 ml.) f o r three hours. Removal of the solvent gave a syrup which could not be c r y s t a l l i z e d . Fractions 4-7, Table VI I I , were mixtures containing p r i n c i p a l l y dimethyl and trimethyl hexoses together with some dimethyl pentose. The free sugar mixture (.5 gm.) from t h i s group of fra c t i o n s was refluxed with f r e s h l y d i s -t i l l e d a n i l i n e ( . 6 ml.) i n absolute ethanol (15 ml.) f o r three hours. Removal of the solvent gave a f e l t - l i k e c r y s t a l l i n e mass. R e c r y s t a l l i z a t i o n from ethanol gave white matted needles, m.p. 165-166°C; MeO, 31.1, 31.3%. A sample (5 mg.) of t h i s a n i l i d e was warmed with d i l u t e hydrochloric acid (1 ml.). Chromatographic examination of t h i s solution produced only one spot R & . 6 3 - . 6 9 . Reported f o r 2,3,4 t r i -methyl-D-galactose, R Q .64j a n i l i d e m.p. 168°C. (12)j MeO, 31.3%. From the mother liq u o r a small amount of material m.p. 206°C.j MeO, 22.4% was obtained. Reported f o r 2,4-di-methyl-D-galactose a n i l i d e m.p. 215°C (55) J MeO, 21 .9%. Treatment of the free sugar mixture (1 gm.) with phenyl-hydrazine hydrochloride (2 gm.) and anhydrous sodium acetate (3 gm.) i n water (15 ml.) at 85°C. f o r twenty minutes pro-duced an o i l which did not c r y s t a l l i z e . Fraction 8, Table V I I I , c r y s t a l l i z e d completely on - 30 -standing. Chromatographic examination showed a large spot R Q . 4 4 together with a small amount of material R^ .53. Recrys t a l i z a t i o n of a sample from methanol-ether-light pet-roleum mixture gave small white needle clusters m.p. 166-166.5°C. Reported f o r Q -methyl-2,4-dimethyl-D-galactoside m.p. 165°C. (55). Hydrolysis of the galactoside (1 gm.) with 0.5% hydrochloric acid followed by p u r i f i c a t i o n and c r y s t a l -l i z a t i o n from acetone-ether-light petroleum furnished small white beads m.p. 101-103.5°C.; R Q .44. Reported f o r 2 ,4 -dimethyl-(3-D-galactose m.p. 100-103°C. (10);'R Q .41. The free sugar mixture (.5 gm.) from f r a c t i o n 8 was refluxed with f r e s h l y d i s t i l l e d a n i l i n e (.5 ml) i n absolute.ethanol (15 ml.) f o r three hours. Removal of the solvent, followed by re-c r y s t a l l i z a t i o n from acetone furnished an a n i l i d e m.p. 213°C. } sublimation point 155-l60°C; MeO, 22.1%; [O(]Q 0, -55° (.1, ethanol). Reported f o r 2 ,4-dimethyl-D-galactose a n i l i d e m.p. 215°C. (55)j MeO, 21.9%. Fractions 9 and 10, Table VIII were composed of methylated uronic acid contaminated with some aldobiuronic acid and methyl hexose. The syrup (.5 gm.) was treated with methanolic ammonia (5 ml.) f o r one week. Removal of the solvent furnished a c r y s t a l l i n e amide which was r e c r y s t a l l i z e d from methanol-ether-l i g h t petroleum mixture. M.p. 180°C, MeO, 50.0, 49.9%. Reported f o r 2^3 ,4-trimethyl-methyl-D-glucuronoside amide m.p. 184°C. (55); MeO, 5-0.0%. A second sample of the syrup - 31 -(.5 gm.) was dissolved i n pure dry ether (5 ml.). Lithium aluminum hydride (.5 gm.) was added and the mixture re-fluxed for two hours. Decomposition of the mixture with ice-cold IN..sulphuric acid followed by extraction with ether and evaporation of the ether solution gave a syrup (.07 gm.) which did not c r y s t a l l i z e . Treatment-of t h i s syrup with 0.5% hydrochloric acid for f i v e hours followed by n e u t r a l i z a t i o n and p u r i f i c a t i o n gave a small amount of syrup, which showed the presence of 2,3 ,4-trimethyl-D-glucose, H Q .85 as wel l as 2 ,4-dimethyl-D-galactose and 2,3 , 4-trimethyl-D-galactose when examined on paper chromatograms. Fractions 11, 12, and 13'., Table ¥111, contained aldo-. biuronic acids and traces of a l l other components. These fr a c t i o n s have not been further examined. 10. Separation of the Methylated Sugars from Mesquite Gum by a Cellulose Column. Methylated mesquite gum (13 gm.) was dissolved i n methanol (100 ml.), made 5% i n hydrogen chloride and refluxed f o r ten huurs. The syrup (16 gm.) obtained a f t e r n e u t r a l i z a t i o n , p u r i f i c a t i o n and evaporation was dissolved i n 0.3N. barium hydroxide (150 ml.) and kept at 65°C. f o r s i x hours. Carbon dioxide was passed i n to remove excess barium hydroxide and the material f i l t e r e d . Evaporation under reduced pressure yielded a syrup (16 gm.) which was dried by d i s t i l l a t i o n with chloro-form. Ti t u r a t i o n of the syrup with excess dry ether removed - 32 -uronic acid as the insoluble barium s a l t (3 gm.). Evapor-ation of the ether solution gave a syrup (11 gm.). Hy-d r o l y s i s of the glycosidic syrup with 0.5% hydrochloric acid (100 ml.) f o r s i x hours followed by n e u t r a l i z a t i o n of excess acid by s i l v e r carbonate, removal of excess s i l v e r with hydrogen sulphide and evaporation, produced a mixture of the free sugars (10 gm.). Extraction of t h i s syrup with a mixture of n-buranol-petroleum ether 65-110°C., 7'3, gave an insoluble residue (2 gm.)'and a f t e r evaporation a syrup (8 gm.). This syrup was placed on a column of powdered cellulose prepared according to Hough, Jones and Wadman (34) and eluted with n-butanol-petroleum ether 65-110°C, 7;*3> saturated with water and containing a trace of ammonia. Fractions of the eluate (5 ml.) were collected every f i v e minutes and every tenth f r a c t i o n was evaporated and examined on paper chromatograms. Fractions containing pure compounds were combined and evaporated under reduced pressure to give from tubes 1-110 2,3,5-trimethyl-L-arabinose (1.6 gm.), from tubes 116-232 2,3-dimethyl-L-arabinose (3 gm.) and from tubes 236-300 an unidentified dimethyl pentose (0.5 gm.). Tubes 304-540 did not contain any sugar. The insoluble residue remaining af t e r the n-butanol-petroleum ether extraction c r y s t a l l i z e d on standing. I t was exhaustively extracted with b o i l i n g ether and the residue dissolved i n methanol-ether-light petroleum. From the ether extract by the addition of l i g h t petroleum and storage i n the r e f r i g e r a t o r , f i n e star-shaped c r y s t a l s were obtained m.p. 85-86.5°C., R Q . ;-42. The methanol-ether-light petroleum solution, after storage i n the r e f r i g e r a t o r f o r one month, deposited white wart-like c r y s t a l s m.p. 137-138°C, . 3 4 . These compounds have not been further examined. 11. I s o l a t i o n of the Uronic Acids from Native Mesquite Gum and Native Sapote Gum B. Crude mesquite gum (50. gm.) was dissolved i n 0.5N. sulphuric acid (1 .5 1.) and refluxed on a hot plate, the ( i n i t i a l value), + 3.15° (12 hours), +' 3.09° (15 hours), + 3.12° (19 hours), + 3.13° (21 hours). After twenty-two hours the dark solution was treated with activated carbon and f i l t e r e d through kieselguhr. The clear pale yellow solution . was neutralized by the portion-wise addition of Amberlite IR-4B. This mixture of solution and r e s i n was poured into a column (9 x 140 cm.) containing a small bed of fresh resin to remove residual a c i d i t y . The r e s i n was l e f t to s e t t l e and the solution allowed to percolate through the column followed immediately by water u n t i l the eluate was non-reducing. The sugar free r e s i n was placed i n a large beaker (4 1.) and 3'N. sulphuric acid added i n portions with s t i r r i n g u n t i l a s l i g h t excess was present. After s t i r r i n g f o r a further half-hour the r e s i n was f i l t e r e d o f f and washed u n t i l the f i l t r a t e was non-reducing. hydrolysis being followed The combined f i l t r a t e s were t i t r a t e d with barium acetate solution u n t i l just sulphate free, f i l t e r e d and evaporated under reduced pressure, to a dark brown syrup (4 gm.)• This syrup was refluxed with 8% methanolic hydrogen chloride f o r ten hours, neutralized and evaporated to a syrup (3.8 gm.). Treatment with methanolic ammonia (25 ml.) f o r one week gave a syrup which did not c r y s t a l l i z e on evaporation of the solvent. The material was dissolved i n absolute methanol and acetone added to produce a s l i g h t cloudyness. Storage of the mixture i n the r e f r i g e r a t o r for three weeks induced c r y s t a l l i z a t i o n . R e c r y s t a l l i z a t i o n from the same solvent mixture gave the amide (1.5 gm.) of a methyl glucuronoside as thick p l a t e l e t s m.p. 237°C; MeO, 27.7%. Reported for the amide of 4-methyl-oL -methyl-D-glucuronoside m.p. 236°C. (56); MeO, 28.05%. No Q form of the compound could be obtained from the mother liquors; only additional small amounts of the cL form. Once precipitated sapote gum B (50 gm.) was treated i n the same manner as mesquite gum and the hydrolysis followed hours), + 1.90° (3 hours), + 1 .99° (13 hours), + 2.00° (14 hours), + 2.05° (15 hours), + 2.17° (18 hours). After twenty hours the solution was decolorized with activated carbon and f i l t e r e d . Treatment of the solution with Amberlite IR-4B as described f o r mesquite gum changed the rotation from + 2v02° to + 0 . 9 2 ° . The r e s i n was washed + 1.62° (1 hour), + 1.70° (2 sugar free and the uronic acids displaced by the addition of a s l i g h t excess of d i l u t e sulphuric a c i d . After f i l -t e r i n g and washing,the combined f i l t r a t e s were t i t r a t e d with barium acetate to remove excess sulphuric acid and the pre-c i p i t a t e d barium sulphate removed by f i l t r a t i o n . Evaporation of the solution under reduced pressure gave a dark syrup (4.8 gm.). This syrup was treated with methanolic hydrogen chloride followed by methanolic ammonia as described f o r mesquite gum. Attempts to obtain c r y s t a l l i n e compounds from the product were unsuccessful. - 3 6 -IV. DISCUSSION. The methylation of the three gums considered here was performed i n the usual manner with dimethyl sulphate and sodium hydroxide. However,, during the procedure differences were observed i n t h e i r behavior towards these reagents. Mesquite gum, even a f t e r the f i r s t treatment., gave an insoluble curd which r a p i d l y separated and floated to the top of the spent reagents making i t s removal and remethylation a simple procedure. Sapote gum A and sapote gum B on the other hand did not behave i n t h i s manner. Even a f t e r three treatments these gums could only be recovered e f f i c i e n t l y from the spent rea-gents by ne u t r a l i z i n g the excess base present. This procedure i s an acceptable one provided precautions are taken to pre-vent hydrolysis of the product. Two other methods f o r the recovery of the p a r t i a l l y methylated product were t r i e d . The f i r s t involved saturating the spent reagents with ammonium sulphate to sa l t out the gum. The product could be recovered by t h i s treatment as a thick syrup which was scooped from the surface of the mixture. This method was unsuitable because the separation of the product was incomplete and sa t i s f a c t o r y y i e l d s could not be obtained. The second method involved d i a l y z i n g the reaction mixture and evaporating the neutral dialysate under reduced pressure a f t e r each treatment. The procedure was very time-consuming since the dialysate usually - 37 -amounted to four l i t r e s . Some degradation of the material could also occur during the repeated concentrations. During the methylation of mesquite gum i t was observed that the product, a f t e r the f i r s t treatment, could be more e a s i l y recovered i f the reagents were added very slowly and the mixture s t i r r e d very vigorously. This would indicate that these conditions contribute to the achievementof a rather high methoxyl content on the f i r s t methylation, permitting suitable y i e l d s and reducing the manipulation d i f f i c u l t i e s to a minimum. The hydrolysis procedures employed were the customary methods used i n carbohydrate chemistry. Once again, however, the three gums exhibited differences i n t h e i r reactions, whereas methylated mesquite gum was almost f u l l y hydrolyzed by r e f l u x i n g f o r eight hours i n b o i l i n g methanolic hydrogen chloride, methylated sapote gum A and sapote gum B were s t i l l incompletely hydrolyzed a f t e r eight hours i n methanolic hy-drogen chloride at 100°C. This resistance to hydrolysis of these gums w i l l have to be overcome i f a quantitative e s t i -mation of the components i s to be achieved. More dra s t i c conditions of hydrolysis may do t h i s , but since the sugars are sensitive to acid, degradation of the fragments may pro-h i b i t t h e i r use. Since the resistant fragment usually i n -volves a uronic acid residue a possible solution may be.found by reducing t h i s a c i d and hydrolyzing the more l a b i l e o l i g o -saccharides obtained. - 38 -In common with other methylated polysaccharides such as methylated starch, c e l l u l o s e and xylan, the three methylated gum acids exhibited a decrease i n water s o l u b i l i t y with an increase i n temperature. A cooled solution of the methylated gum when warmed precipitated the polysaccharide, a temperature increase of about ten degrees causing p r a c t i c a l l y complete p r e c i p i t a t i o n . This phenomenon has been explained as a reversible hydration and dehydration of the dissolved molecules. The gum ether forms a hydrate with water i n the cold which undergoes cleavage on heating. The degree of poly-merization and methylation influence t h i s behavior. Unmethy-lated gum possesses many free hydroxyl groups and i n the absence of moisture these can hydrogen bond amongst themselves giving r i g i d i t y to the structure. As water i s introduced the hydrogen bonding occurs between the water molecules and the hydroxyls of the gum, progressively decreasing the r i g i d i t y of the gum. Substitution of the hydroxyls by ether groups reduces the hydrophilic nature of the gum and since the bond between the ether and the water i s eas i l y ruptured, heating produces a water insoluble dehydrated molecule. The products i s o l a t e d from mesquite gum by d i s t i l l a t i o n and chromatography corresponded with those reported i n the l i t e r a t u r e . Small amounts of unidentified components have also been obtained. These compounds may not have any s i g -nificance i n the major repeating unit of the structure. They - 39 -are p o s s i b l y i n c o m p l e t e l y methylated fragments or the r e s u l t o f s m a l l i d i o s y n c r a s i e s i n t h e polymer. Sapote gum A a l s o gave products corresponding t o those a l r e a d y r e p o r t e d i n t h e l i t e r a t u r e . Besides the two t r i m e t h y l pentoses and the monomethyl pentose, Dr. E. V. White, i n a p r i v a t e communication, has r e p o r t e d the i s o l a t i o n o f two methylated g l u c u r o n i c a c i d s from t h i s gum methyl et h e r . T h i s would i n d i c a t e t h a t the u r o n i c a c i d i n t h i s p o l y s a c c h a r i d e does not occupy o n l y a t e r m i n a l p o s i t i o n , as i n mesquite gum, but has a second f u n c t i o n i n the s t r u c t u r e of t h i s gum. The r e -p e a t i n g u n i t of t h i s gum then would appear to c o n t a i n arabo-pyranose and x y l o f u r a n o s e r e s i d u e s i n t e r m i n a l p o s i t i o n s . Some of these w i l l be l i n k e d to x y l o s e which s e r v e s as a p o i n t of branching i n the r e p e a t i n g u n i t . The s i x d i f f e r e n t methylated sugars d e t e c t e d i n h y d r o l y s a t e s of sapote gum B do not g i v e s u f f i c i e n t i n f o r m a t i o n f o r t h e de-velopment of a complete s t r u c t u r e . More d e f i n i t e p r o o f s o f the t h r e e t r i m e t h y l sugars i n t h e mixture from the lower b o i l i n g f r a c t i o n s i s r e q u i r e d , s i n c e these compounds have only been shown by chromatographic methods, and w h i l e t h i s c e r t a i n l y g i v e s a good i n d i c a t i o n of t h e i r i d e n t i t y , i t i s not c o n c l u s i v e . I t i s a l s o t r u e t h a t while these s i x compounds r e p r e s e n t t h e b u l k of the components t h e r e are s t i l l s mall amounts of m a t e r i a l which have not yet been i d e n t i f i e d . P r e c i s e a n a l y t i c a l d a t a on the amounts of the v a r i o u s methylated sugars i s a l s o r e q u i r e d . - 40 -This information can be obtained by application of paper chromatographic methods once pure specimens of each component have been obtained. The r e s u l t s obtained here do, however, permit certain conclusions to be drawn concerning the structure of t h i s gum. The i d e n t i f i c a t i o n of the uronic add component as 2,3,4-trimethyl-D-glucuronic acid indicates that the uronic acid i n the native gum occupies a terminal p o s i t i o n . Whether i t i s a methylated or an unrnethylated acid or both cannot be said. I f some uronic a c i d occupies a position other than terminal, i t cannot be present to any extent since only the trimethyl derivative was detected. If we accept the i d e n t i t y of the three trimethyl sugars of the lower b o i l i n g f r a c t i o n s , then these too can be given a position i n the structure of the gum. The arabinose would occupy a terminal position and occur in the furanose configur-ation. S i m i l a r l y the xylose would occupy a terminal p o s i t i o n but possess the pyranose configuration. The rhamnose possess-ing apyranose configuration would likewise occupy a terminal p o s i t i o n . The galactose f r a c t i o n of the gum, the most abundant f r a c t i o n , was found to be dimethyl and trimethyl galactose, both compounds occurring i n the pyranose configuration. The dimethyl galactose offers p o s s i b i l i t i e s f o r linkage through i t s f i r s t , t h i r d and sixth hydroxyl groups, whereas the t r i -methyl galactose can be linked only through i t s f i r s t and - 41 -s i x t h . This gum then l i k e many other polyuronides, possesses a highly branched repeating u n i t . I t may be that a branched galactan chain forms the back-bone of the repeating u n i t , and attached to t h i s at the appropriate carbon atoms are chains composed of the other sugars. These chains w i l l not be very long since very l i t t l e dimethyl pentose has been detected i n the hydrolysates. The uronic a c i d , also occupying aterminal p o s i t i o n , w i l l be linked to the galactan back-bone, possibly a l t e r n a t i n g i n position with the sugar chains. VI. APPENDIX -46 -TABLE I THE GROSS COMPOSITION OF SAPOTE GUM B Material Sample A Sample B Crude gum 25.0 gm. 25.0 gm. Bark and chips .4 gm. .3 gm. Dry j e l l y 4.9 gm. 5.8 gm. Recovered gum 19.0 gm. 18.3 gm. - 47 -TABLE I I THE METHYLATION OF MESQUITE GUM T r i a l Yield % Methoxyl % 1 3 . 5 3 3 . 3 2 3 0 . 38 . 1 3 1 0 . 38 .8 4 52. 3 3 . 6 5 6 0 . 3 3 . 5 6 7 8 . 3 9 . 2 7 8 2 . 3 7 . 6 8 $1. 2 8 . 2 9 1 0 3 . 3 7 . 9 - 48 -TABLE I I I METHOXYL INCREASE DURING METHYLATION OF SAPOTE GUM B Number of Treatments Methoxyl % 0 2.6 1 23.6 2 3 4 . 4 3 3 5 . 3 4 3 7 . 2 5 3 7 . 5 6 7 4 0 . 4 ± M e t h y l a t i o n 1-5 w i t h dimethyl sulphate and sodium hydroxide, 6 - 7 w i t h methyl i o d i d e and s i l v e r oxide. - 49 -TABLE IV THE METHYLATION OF SAPOTE GUM B T r i a l Y i e l d % M e t h o x y l % 1 10. 37.0 2 34. 37.1 3 4 0 . 33.0 4 4 8 . 33.2 5 6 0 . 37.0 6 33. 37.9 TABLE V CHROMATOGRAPHIC EXAMINATION OF THE METHYLATED GUM HYDROLYSATES RG Mesquite gum Sapote gum A Sapote gum B .08 Aldobiuronic acid .12 Aldobiuronic acid .20 Aldobiuronic acid .24 Trimethyl glucuronic acid Trimethyl glucuronic, acid Trimethyl glucuronic acid .36 Methyl hexose • 44 Dimethyl galactose Monomethyl xylose Dimethyl hexose .33-.SO .81 Trimethyl arabinose Dimethyl pentose. Trimethyl hexose .82 Dimethyl arabinose . 9 4 - 1 . 0 2 Trimethyl arabinose Trimethyl xylose Trimethyl pentoses TABLE VI THE FRACTIONATION OF METHYLATED MESQUITE GUM AFTER METHANOLYSIS' Fraction Y i e l d gm. B.p. ° C , (0.02 mm.) MeO % 7) 2 0 'Y D Components 1 4.5 0-55 52.9 1.4378 2,3,5-Trimethyl arabinose, traces of 3,5 dimethyl arabinose. 2 4.0 55-75 46.5 1.4451 3,5-0imethyl arabinose, traces of 2,3,5-t n m e t h y l arabinose. 3 6.5 90-100 45.6 1.4480 3,5 Dimethyl arabinose, traces of 2,3,5-trimethyl arabinose and 2,4-dimethyl 1 galactose. v. 4 2.5 100-110 47.9 1.4510 3,5-Dimethyl arabinose, with 2,4 dimethyl 1 galactose and 2,3,4-trimethyl glucuronic a c i d . 5 4.5 130-140 crystalline 2,4 Dimethyl galactose with 2,3,4 trimethyl glucuronic acid and 3,5 dimethyl arabinose. 6 1.0 140-170 th i c k syrup 2,3,4 Trimethyl glucuronic a c i d with 2,4 dimethyl galactose and aldobiuronic a c i d . Pot res-idue 7.0 170- Unhydrolyzed material, sugar and traces of a l l other components. ft Data given are f o r the methyl glycosides TABLE VII THE FRACTIONATION OF METHYLATED SAPOTE GUM A AFTER METHANOLYSIS Fraction Y i e l d gm. B.p. °C. (0.02 mm.) -h 20 n D U]D20S* (5, water) Components 1 7.1 55-60 1.4429 +95.6° 2,3,4-Trimethyl arabinose and 2,3,4-trimethyl xylose. 2 4.7 60-105 1.4578 +61.00° - 2,3,4-Trimethyl arabinose and 4-monomethyl xylose. Pot res-idue 1.0 105- +11.64° 4-Monomethyl xylose with traces of uronic a c i d . 3 2.25 80-90 1.4570 +92.20° 2,3,4-Trimethyl glucuronic acid, with aldobiuronic acid and 4-monomethyl xylose. 4 .15 90-100 2,3,4-1'rimethyi glucuronic a c i d with aldobiuronic acid and 4-monomethyl xylose. 5 2.55 100-110 1.4611 +118.80° 2,3,4-Trimethyl glucuronic a c i d with aldobiuronic acid and 4-monomethyl xylose. 6 .35 110-170 1.4601 Aldobiuronic acid with traces of 2,3,4-trimethyl glucuronic acid and 4-monomethyl xylose. 7 3.15 120-160 glass +115.34° Aldobiuronic acid with traces of 2,3,4-^ trimethyl glucuronic acid and 4-monomethyl xylose. Pot res-idue 1.50 160- Aldobiuronic a c i d , sugars and unhydrolyzed material. ft Data given are f o r the methyl glycosides. I n i t i a l values. / Fraction 1-2 from the ether soluble glycosidic portion. Fraction 3-7 from the ether insoluble uronosicid portion. ) / c TABLE VIII THE FRACTIONATION OF METHYLATED SAPOTE GUM B AFTER METHANOLYSIS' A Fraction Y i e l d gm. B.p. °C. • (0.02 mm.) ' s 20 7} D (5 , water) MeO % Components 1 4 .1 45-47 1.4354 -51 .00° 59.2 2 , 3 , 5-Trimethyl arabinose, 2 , 3 , 4 -trimethyl xylose and 2 , 3 , 4 -trimethyl rhamnose. 2 0.9 50-65 1 . 4 4 0 8 +3.26° 54.5 2 , 3 j 5-Trimethyl arabinose and 2 , 3 , 4-trimethyl xylose. 3 1.2 65-75 1.4526 +3.96° 46.6 Dimethyl pentose, 2 ,4-dimethyl galactose and 2 , 3 , 4-trimethyl galactose. 4 1.1 75-6*5 1.4559 +30.28° 46.9 2 , 3 , 4-Trimethyl galactose and 2,4-dimethyl galactose. 5 1.5 85-95 1.4598 +66.80° 47.1 2 , 3 , 4-Trimethyl galactose and 2,4-dimethyl galactose. 6 0 .7 95-105 1.4600 +84.62° 47 .2 2 , 3 , 4-Trimethyl galactose and 2,4-dimethyl galactose. 7 0 . 8 105-125 1 . 4 6 3 0 +80.54 45 .1 2 , 3 , 4-Trimethyl galacto se, 2,4-dimethyl galactose and an unidentified component. / Fraction 1-8 from the ether soluble glycosidic portion. Fraction 9 - 1 3 from the ether insoluble uronosidic portion. TABLE VIII (continued) Fraction Y i e l d gm. B.p. °C. ( 0 . 0 2 mm.} 7l f [ A g 0 * * ( 5 , water) MeO % Components 8 - 4 . 7 1 2 5 - 1 3 0 c r y s t a l -l i n e +107 .72 4 2 . 1 2,4-Dimethyl galactose and the un i d e n t i f i e d 1 component. Pot res-idue 1 . 9 1 3 0 - glass + 8 2 . 9 6 ° 2,4-Dimethyl galactose, unidenti-f i e d component, and decomposed material. 9 0 . 6 7 0 - 9 5 1 . 4 4 7 9 + 6 3 . 9 0 ° 5 7 . 9 2 , 3 , 4-Trimethyl glucuronic acid and traces of aldobiuronic acid A. 1 0 2 . 0 9 5 - 1 1 0 1 . 4 4 8 2 + 8 6 . 9 6 5 4 . 7 2 , 3 , 4-Trimethyl glucuronic acid aldobiuronic acid A and some 2 , 4 -dimethyl galactose. 1 1 2 . 7 110-125 1 . 4 5 9 0 + 8 5 . 7 4 4 7 . 5 Aldobiuronic acid A with traces of 2 , 3 , 4 - t r i m e t h y l glucuronic acid and 2 , 4-dimethyl galactose. 12 1 . 7 125 -145 1 . 4 7 3 7 + 6 6 . 2 6 4 2 . 0 Aldobiuronic acids A and B, 2 , 4 -dimethyl galactose and traces of 2 , 3 , 4 trimethyl glucuronic acid. 13 0 . 8 1 4 5 - 1 9 0 1 . 4 7 8 1 + 9 1 . 0 0 ° 3 8 . 9 Aldobiuronic acids B and A, with traces of 2 , 3 , 4 - t r i m e t h y l glucuronic acid and 2 , 4-dimethyl galactose. 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