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The acid component of jute hemicellulose Rogers, Ian Henry 1958

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THE ACID COMPONENT OF JUTE HEMICELLULOSE by IAN HENRY ROGERS B.Sc, Queens University, Belfast, N. Ireland, 1954 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Chemistry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1958 ABSTRACT Defatted powdered jute vas delignified by chlorite treatment and the hemicellulose extracted vith alkali. The precipitated material vas purified by washing with alcoholic hydrochloric acid and dried by sol-vent exchange. The hydrolysed hemicellulose yielded neutral sugar and sugar acids separated on ion exchange resins. The neutral sugar was identified as D-xylose. The sugar acid fraction contained mainly an aldobiouronic acid proved by the reduction of the methyl ester methyl glycoside with lithium aluminum hydride followed by hydrolysis to consist of D-xylose linked to a monomethyl glucose. This was shown, via its ahilide and osazone, to be 4-0-methyl-D-glucose. Methanolysis of the aldobiouronic acid yielded the methyl glycoside of a uronic acid which on treatment with diazomethane and then with meth-anol ic ammonia gave 4-Oj-methyl-oC-D-glucuronoamide methyl glycoside, after fractional crystallization. Reduction of the aldobiouronic acid methyl ester methyl glycoside with lithium aluminum hydride, followed by methylation and hydrolysis, gave 2,3,4,6-tetra-0_-methyl-D-glucose and 3,4-di-O-methyl-D-xylose. A search for a crystalline derivative of the purified aldobiouronic acid and of its related xylitol compound formed on reduction with pota-ssium borohydride was unsuccessful. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representative. It i s understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia, Vancouver 3, Canada. Date ACKNOWLEDGEMENT I wish to express my sincere thanks to Dr. G.G.S. Dutton for his patience, advice and encouragement in directing this research work. TABLE OF CONTENTS Page Historical Introduction 1 The Identification of Uronic Acids 8 The Acids in Jute Hemicellulose 11 Experimental 22 Jute Hemicellulose 22 Jute Aldobiouronic Acid 24 Bibliography , 40 TABLES Table I - Aldobiouronic Acids of Proven Structure 4 Table II - Aldobiouronic Acids of Uncertain Structure 7 HISTORICAL INTRODUCTION Land plants usually contain several types of polysaccharide mater-ia l other than cellulose which is their main support structure. These may be described as "homoglycans" where only one type of monosaccharide is present in the structure as for instance in starch or inaulin or as "heteroglycans" where more than one type of monosaccharide residue occurs. These latter materials have been classified as hemicelluloses, gums, and mucilages according to their chemical nature and origin and their dis-tribution in nature is very wide since the composition of the polysacc-harides may vary considerably in different parts of any one plant. Poly-saccharides are also obtained from seaweed and from certain types of 1 • I T bacteria and determinations of structure are essential to many branches of science. The function which they f u l f i l is not always clear but i t would appear that the hemicelluloses may act as reserve support struc-tures or reserve food materials depending on their nature and situation in the plant. They occur in wood, bark, leaves, and seed pods whilst the gums are obtained as exudates on the bark of trees or on fruit and are used by the plant as a protective mechanism against bacterial invas-ion or physical damage ( l ) . The mucilages are of widespread occurence but are most commonly found in seeds, where they assist in water stor-age and may "solubilise" insoluble substances such as cellulose ( 2 ) . Many workers have attempted to define these three classes of poly-saccharides. Generally hemicelluloses are regarded as those polysacc-harides which are extractable from holocellulose by aqueous alkali. Plant gums are defined as giving a clear solution in water, whereas mucilages swell but do not dissolve in water. There are exceptions to -2-this definition since tragacanth gum, vhich is a tree exudate and a true gum, is only partly soluble in vater and behaves as a mucilage (3,4). These polysaccharide materials undergo acid hydrolysis to yield hexoses, pentoses, uronic acids and aldobiouronic acids. The methyl pentoses L-rhamnose and L-fucose are also found occasionally and occur in pyranose form as do a l l the other constituents except L-arabinose vhich prefers the furanose ring. The hexoses, uronic acids and xylose have the D-configuration. The plant gums a l l contain D-glucuronic acid except tragacanth gum which contains D-galacturonic acid. The seed mucilages on the other hand nearly a l l contain D-galacturonic acid whilst the hemicelluloses shov more variation and may yield D-gluruconic, D-galacturonic and 4-0-methyl-D-glucuronic acids whilst 3-0_-methyl-i ! glucuronic acid has also been claimed to occur (5) (see later). In the plant gums so far examined D-galactose and L-arabinose are always found whilst D-xylose is also of very common occurence. Less usual constituents are D-mannose, L-rhamnose, and L-fucose whilst D-glucose has not yet been found. The mucilages are a more complex family than the gums and may be classified as neutral, acidic, or sulphate ester (seaweeds only). D-galactose is universally found and L-arabinose is usually present whilst D-mannose, D-glucose, D-fractose and L-rhamnose are less common. D-xylose is commonly found in the acidic mucilages which contain D-galacturonic acid. In the hemicellulose family D-xylose is very common and xylan-polyuronides are often present in woody tissue. The barks of trees often contain galactan-galacturonides whilst D-galactose is also predominant in leaves and pods and occurs together with L-arabinose in green non-lignified plant tissues (6). - 3 -Previous to 1952, the uronic and aldobiouronic acids were separated from the hexoses and pentoses by virtue of the insolubility of their barium salts in aqueous alcohol. In that year their absorption from solution on to weakly basic anion exchange resins was f i r s t utilised by Hough, Jones and Wadman (7) and this is now the normal method for the separation of sugar acids from the neutral sugars. The isolation of uronic acids from aldobiouronic, aldotriouronic, and acids of higher molecular weight is best accomplished by partition chromatography on cellulose columns or on sheets of thick paper whilst mixtures of uronic acids may be separated by fractional distillation of their methyl gly-coside methyl esters in vacuo. The present work deals with the structure of an aldobiouronic acid isolated from jute hemicellulose. Structural determinations on aldo-biouronic acids are of great importance in studying the chemical nature of polysaccharides and no comprehensive table of their structures and sources exists in the literature. Table I is a literature survey of this nature giving the structures of the acids known at present. Table II comprises those acids whose structure has not been completely deter-mined. In so far as i t was possible earlier work which has been super-ceded is omitted. In a few cases aldotriouronic acids have also been isolated and identified. TABLE I Aldobiouronic Acids of Proved Structure Structure Config- Source Refer-uration ence 2-0-(D-glucuronopyranosyl)- o< maize hull hemicellulose 8 D-xylopyranose oC oat hull hemicellulose 9 corn cob hemicellulose 10 wheat bran hemicellulose 11 — chagual gum 12 3-0-(D-glucuronopyranosyl)- sun flower heads 13 D-xylopyranose cX wheat straw hemicellulose 14 ex. pear cell wall 15 wheat leaf hemicellulose 16 4-0-(D-glucuronopyranosyl)- — Kadsura Japonica mucilage 17 D-xylopyranose oC corn cob hemicellulose 10 4-0-(D-glucuronopyranosy1)- P Type II pneumococcus D-glucopyranose polysaccharide 18 P Type VIII pneumococcus polysaccharide 19 2-0-(D-glucuronopyranosy1)- P cherry gum 20 D-mannopyranose damson gum 21 P gum ghatti 22 4-0-(D-glucuronopyranosyl)- Acacia Karroo gum 23 D-galactopyranose — Neem gum 24 6-0-(D-glucuronopyranosy1)- p Acacia Karroo gum 23 D-galactopyranose 1 egg plum gum 27 p wheat straw holocellulose 28 p gum arabic 29 1 black wattle gum 30 Acacia Pycnantha gum 31 — almond tree gum 32 — peach tree gum 33 p gum ghatti 22 - 5 -TABLE I (cont'd.) Structure Config-uration Source Refer-ence 2 - 0 - ( 4 - 0-Methyl-D-glucurono-pyranosyl)-D-xylopyranose oc P «=>< <X ex. white birch hemicellulose white elm hemicellulose western hemlock hemicellulose beechwood hemicellulose black spruce hemicellulose Scots pine hemicellulose aspen wood hemicellulose pinus radiata hemicellulose American beechwood hemicellulose Loblolly pine hemicellulose wheat bran hemicellulose corn cob hemicellulose oat hulls hemicellulose f l a x birchwood holocellulose' sitka spruce hemicellulose 3 4 3 5 3 6 3 7 3 8 3 8 3 9 4 0 4 1 4 2 1 1 4 3 9 4 4 4 5 4 6 3 - 0 - ( 4 - 0-Methyl-D-glucurono-pyranosylJ-D-xjUJpyranose oC Pinus radiata hemicellulose 4-0- (4—O-Methyl-D-glucurono- cx pyranosyl)-D-galactopyranose 6 - 0 - ( 4 - 0-Methyl-D-glucurono- p> pyranosyl)-D-galactopyranose gum myrrh mesquite gum lemon gum grapefruit gum gum myrrh mesquite gum modal gum 4 0 4 7 4 8 , 4 9 2 5 , 2 6 2 5 , 2 6 4 7 4 8 , 4 9 5 0 4 - 0 - ( 4 - 0-Methy1-D-glucurono-pyranosyl)-L-arabofuranose o c lemon gum 5 1 -6-TABLE I (cont'd.) Structure Config-uration Source Refer-ence 4-0-(D-galacturonopyranosyl)• D-xylopyranose • oc Plantago arenaria seed mucilage 52 2-0-(D-galacturonopyrano sy 1-L-rhamnopyranose flax seed mucilage Plantago arenaria seed mucilage 53 54 Plantago ovata seed mucilage 55 okra mucilage 56 slippery elm bark mucilage 57 Karaya gum 58 o< grape juice sterculia setigera gum 59 60 4—0-(D—galacturonopyranosyl)-D-galactopyranose Karaya gum sterculia setigera gum 58 61 -7-TABLE II Aldobiouronic Acids of Uncertain Structure Structure Source Refer-ence (D-glucuronopyranosyl)-D-xylopyranose Prunus serrulata 62 cottonseed hull hemicellulose 63 (4-0-Methylglucuronopyranosyl)-D-xylopyranose Eucalyptus regnans 64 3-( -D-glucuronopyranosyl)-2-deoxy-2-amino-D-galactopyranose cartilage 65 (D-galacturonopyrano sy1)-D-galac t uronic acid sterculia setigera gum 61 (D-galacturonopyranosyl)-D-galactopyra-nose gum jeol 66 (D-galacturonopyranosyl)-D-gluco-pyranose asparagus racemosus tuber mucilage 67 cress seed mucilage 68 (D-galacturonopyranosyl) -L-rhamno-pyranose white mustard seed mucilage 69 cholla gum 70 -8-THE IDENTIFICATION OF URONIC ACIDS The characterization of the hexuronic acids and particularly of their monomethyl ethers is often d i f f i c u l t as their crystalline deriva-tives are rare and often their preparation is quite time-consuming. Three main methods are available for this purpose. Two involve the preparation of derivatives from the acid by direct and indirect means, whereas in the third method the acid is f i r s t reduced to the correspond-ing neutral hexose from which crystalline derivatives can be more readily obtained. Oxidation of the Uronic Acids In 1931, Challinor, Haworth and Hirst (29) isolated 2,3,4-tri-0-methyl-D-glucuronic acid from the hydrolysate of methylated gum arabic and proved its structure by oxidation with HN0o followed by inethanolysis o and disti l l a t i o n to yield the methyl ester of 2,3,4-tri-O-methyl-D-glucaro-1,5-lactone which was similarly derived from 2,3,4-tri-0_-methyl-D-glucose. Similar derivatives of methylated uronic acids have been prepared by many authors following this route. Edington and Percival have synthesised 3,4-di-O-methyl-D-galacturonic acid (71) and proved its structure by oxidation with bromine water followed by esterification to yield the dimethyl ester of 3,4-di-O-methyl-D-galactaric acid. Haworth, Hirst, Isherwood and Jones (72) synthesised 2,3,4-tri-O-methyl-D-mannuronic acid which they identified by oxidation with HN0o followed by treatment with methanolic ammonia to yield the diamide of 2,3,4-tri-0-methyl-D-mannaric acid previously prepared from 2,3,4-tri-0-methyl-D-mannose. A more unusual proof of structure, was used by White (49) who identified 4—O-methyl-D-glucuronic acid by periodate oxidation followed -9-by further oxidation with bromine to yield 2-hydroxy-3-methoxy-L-erythrosuccinic acid. This substance when reacted with a methanolic solution of methylamine gave the crystalline bismethyl amide. Reduction of the Uronic Acids The reduction of uronic acids with LiAlH^ was f i r s t described by Lythgoe and Trippett (73) and by Abdel-Akher and Smith (74) in 1950. To confer solubility in ether or tetrahydrofuran the methyl ester methyl glycoside of the uronic acid was f i r s t prepared and the final product was the methyl glycoside of the corresponding hexose. Since aluminum salts form stroig complexes with free hydroxyl groups i t is now usual to isolate the reduced sugar as its acetate from which the methyl glycoside of the free sugar is easily obtained upon saponification. The reduction of uronic acids with LiAlH^ works very well and gives good yields of the corresponding neutral sugars from which crystalline derivatives may be made. This method is probably the most widely useful means of identi-fication of uronic acids known today. Formation of the Amide of the Methyl Glycoside The use of this direct derivative in characterising uronic acids was f i r s t reported by Smith (75). The methyl ester methyl glycoside of 4-0-methyl-D-glucuronic acid obtained from mesquite gum wad dis t i l l e d in vacuo and treated with a methanolic ammonia solution. Evaporation gave a white solid found to be a mixture of the «*• and /3 forms of the amide and these were easily separated by fractional crystallization. This derivative has since been prepared by many authors, the only objec-tion to its use being a tedious separation of the <x and p forms arising from the optical activity of C . - 1 0 -The Identification of Aldobiouronic Acids It is a well-known fact that the linkage between a uronic acid group and the neighbouring neutral sugar molecule in a polysaccharide is peculiarly resistant to acidic hydrolysis. This is seen from the large amounts of aldobiouronic acids produced upon acidic hydrolysis of hemicelluloses, gums and other polysaccharide materials. The reason for this resistance is not yet understood. Due to their widespread occurrence as products of the hydrolysis of polysaccharides the identification of aldobiouronic acids has become of much importance in structural studies. Unfortunately these materials can very rarely be characterised directly as scarcely any crystalline deriva-tives are known. In a very few cases the amide or methyl ester of a methyl glycoside has been obtained crystalline (29,53,76,77). Smith and Srivastava (78) have obtained the crystalline pentaacetate of 2-0-(<x-D-glucuronopyranosyl)-D-xylopyranose in both its ot and p forms. This is the only occasion on which a xylose containing aldobiouronic acid has yielded a crystalline derivative. It is an object in this present work to search for a suitable derivative of the aldobiouronic acid from jute or of a compound easily obtained therefrom. At the present time aldobiouronic acids are identified by reduction to neutral disaccharides followed by hydrolysis and identification of the products. It is also essential to determine the points of linkage of the constituents by methylation of the neutral disaccharide followed by hydrolysis and identification of the methylated fragments. Such a long procedure for the characterization of aldobiouronic acids requires a lot of time and, i f suitable crystalline derivatives were available, this would be greatly reduced. -11-JUTE ALDOBIOURONIC ACID The f i r s t study of jute hemicellulose vas made by Sarkar, Mazumdar and Pal (79) in 1952. In i t they determined the optimum conditions for its extraction from holocellulose and carried out detailed analyses of hemicelluloses extracted under varying conditions. They found the pure material to contain a monomethyluronic acid and xylose. From methoxy1 content and neutralization equivalent measurements they deduced an equi-valent weight of around 1000. In a further paper based on periodate oxidation studies on the hemicellulose Das Gupta and Sarkar (80) con-cluded that jute aldobiouronic acid probably had the structure 4-0-(3T^0-methyl-D-glucuronopyranosyl)-^ -D-xylopyranose. As further evidence for this structure the same authors in a letter to the editor (5) told how they had reduced the methyl ester methyl glycoside with lithium aluminum hydride and hydrolysed the resulting neutral disaccharide. The , sugars in the hydrolysate were identified as xylose and 3-0-methyl glu-cose by paper chromatography in solvent 5. The constituent sugars present in jute oc-cellulose remaining after alkaline extraction of jute holocellulose have been investigated by Adams and Bishop (81). Formic acid hydrolysis and paper chromato-graphy of the hydrolysate showed the presence of large quantities of glucose and xylose together with a definite amount of arabinose. This may indicate the occurence of chemical bonding between the oc-cellulose and the hemicellulose present in jute fibre. The Indian workers (80) said that they would present final evidence for the presence of 3-0-methyl-D—glucuronic acid in the hemicellulose by the preparation and identification of suitable crystalline derivatives. - 1 2 -This evidence has not been produced and furthermore i t i s d e f i n i t e l y shown i n the present work that no d i s t i n c t i o n can be made by paper chromatography between 3-and 4-0-methyl-D-glucose using solvents 1 to 5.* If 3-0-methyl-D-glucuronie acid r e a l l y i s present i n the hemicellu-lose i t i s the f i r s t time i t has been found i n nature whereas 4-0-methyl-D—glucuronic acid has been found in many sources (34-46). Fur-ther investigation of t h i s question appeared to be desirable. Powdered defatted jute was d e l i g n i f i e d readily by means of the chlorite process of Wise, Murphy and D'Addieco (81), to y i e l d white holo-c e l l u l o s e . This material was extracted with 10$ aqueous sodium hydroxide and the solution neutralised. Upon addition of ethanol the hemicellu-lose was thrown out as a flocculent precipitate. After washing with aqueous alcohol to remove salts the material was dried by solvent ex-change to give a d i r t y white powder i n 16$ y i e l d . A sample of crude hemicellulose was purified by precipitation as in i t s copper salt/alkaline solution. After careful washing the salt was decomposed with alcoholic hydrochloric acid and the pure hemicellulose i. isolated as before (yield 64$). The snow-white powder had ash content 2.33$, and1 was designated hemicellulose I. Another sample of the crude material was suspended for a day i n ethanolic hydrophloric_acid and the hemicellulose on recovery was washed and dried as usual to give a snow-white powder (y i e l d 70$) with an ash content of only 0.28$. This material was designated hemicellu-lose I I . A comparison of the optical rotations, neutralisation equivalents and methoxyl contents of hemicelluloses I and II showed them to be * Key to this i s given on p. 22. -13-i d e n t i c a l . P u r i f i c a t i o n as hemicellulose II was therefore adopted as a better product was obtained. An attempt to extract the hemicellulose d i r e c t l y from jute without d e l i g n i f i c a t i o n was not encouraging. The y i e l d was very poor and the product isolated after p u r i f i c a t i o n by the copper s a l t process had a brown colour. Hydrolysis of the material and examination of the hydro-lysate showed the presence of a trace of glucose which might have derived from cellulose present as an impurity. T r i a l experiments on the hydrolysis of jute hemicellulose showed that 1 Normal sulphuric acid for six to eight hours gave satisfactory results whereas incomplete hydrolysis resulted from the use of 45^ formic acid even after twelve hours on the steam bath. The presence of oligosaccharides in this hydrolysate was demonstrated by further hydrolysis with 1 Normal sulphuric acid when chromatography showed only xylose, uronic and aldobiouronic acids to be present. It was not found possible to avoid the formation of uronic acid which occulted also i n the formic acid hydrolysate. A large batch of hemicellulose was hydrolysed with sulphuric acid and the hydrolysate separated into neutral sugar and sugar acid f r a c -tions using Amberlite IR 120 and Duolite A4 resin ion exchange columns. Chromatography on these fractions revealed only one neutral sugar which appeared to be xylose whereas the acid fraction contained uronic-and aldobiouronic acids. Later a second neutral sugar was found present only i n trace amounts and noticable only on heavily loaded chromatograms. The neutral sugar was obtained c r y s t a l l i n e and was i d e n t i f i e d as D-xylose. This was confirmed by the preparation of the dibenzylidene dimethyl acetal derivative (82). The trace sugar was i d e n t i f i e d only -14-by paper chromatography and ran identically with L-rhamnose in three solvents. The sugar acid mixture was obtained as a syrup which did not crys-t a l l i z e . The methyl ester methyl glycoside formed by treatment with dilute methanolic hydrogen chloride was reduced with lithium aluminum hydride. The reduced sugar was freed from its aluminum complex by acetylation and the acetate extracted in chloroform and deacetylated with alkali gave the neutral disaccharide methyl glycoside as a yellow syrup which did not crystallise. This syrup was hydrolysed with 1 Normal sulphuric acid as before and the hydrolysate examined chromato-graphically was found to contain xylose and a monomethyl glucose which might have been 3-or 4-0-methyl-glucose. Attempts to separate the sugars in the hydrolysate on a cellulose column using solvent 1 were a failure and the separation was finally carried out on sheets of paper using this solvent. The sugars were extracted from the paper in aqueous acetone and obtained as syrups,one of which crystallized on standing. This material was identified as D-xylose and confirmation was again obtained by preparation of the diben-zylidene dimethyl acetal derivatives (82). The other sugar was shown to be a monomethyl glucose by demethylation with hydrogen bromide and examination of the product by paper chromatography. Final and conclu-sive proof of the position of the methyl group was obtained by prepara-tion of the osazone and arilide. Comparison of these derivatives against similar derivatives made from 4-0-methyl-D-glucose revealed that they were identical. It is worthy of note that 4-0-methyl-D-glucose anilide has not previously been reported in the literature. This material was prepared -15-by the standard method for making sugar anilides and the yields vere found to be rather variable since the formation of aniline tars often took place. The compound gave much difficulty in recrystallization from ethyl acetate and the crystals obtained by diluting with a few drops of petroleum ether were l i t t l e better. Eventually a pure product was obtained after prolonged recrystallization. The maximum melting point found was 158-160°C. In a search for -suitable derivatives of 4—O-methyl-B—glucose the dibenzyl mercaptal was also prepared by the method used by Facsu (83) in the preparation of D-glucose dibenzyl mercaptal. The sugar was con-densed with oc-toluenethiol in hydrochloric acid solution in the pres-ence of zinc chloride. After addition of water the acid was removed on Duolite A4 ion exchange resin and the solution evaporated. The dibenzyl mercaptal crystallized as a white solid from ethanol and after three recrystallizations from ethanol the white needlelike crystals had a melting point of 158-159°C. This value did not rise on further crysta-lliz a t i o n . This derivative has been prepared by an indirect route by other workers (84,85) and melting points of 73°C. and 96-98°C. have been reported. It is apparent that their materials were not obtained pure and i t is possible that the mercaptal was obtained as a hydrate. The dibenzyl mercaptal of standard 3-^0-methyl-D-glucose was also prepared as above. This compound did not crystallize from alcoholic solution but was obtained as white platelets from aqueous alcohol. After two recrystallizations a maximum melting point of 65-70°C. was reached. The material was apparently not pure and may have been a hy-drate. The compound decomposed on heating in boiling toluene in the Abderhalden in vacuo in an attempt to remove water of crystallization. -16-This was unfortunate as 3-0-methyl-D-glucose dibenzyl mercaptal has never previously been reported. A sample of jute aldobiouronic acid was hydrolysed in 10$ methan-olic hydrogen chloride for sixteen hours on the steam bath. After removal of hydrogen chloride the mixture containing methyl xyloside, uronic acid methyl ester methyl glycoside and the aldobiouronic acid methyl ester methyl glycoside was partially separated by continuous ex-traction with ether for twenty-four hours. The ether layer was shown to contain methyl xyloside and the uronic acid derivative, whilst the aqueous phase contained a l l three constituents. The ether extract was saponified with 0.15 Normal barium hydroxide and the uronic acid methyl glycoside separated by running the solution through Duolite A4 ion exchange resin when the acid remained on the resin whilst the methyl xyloside was removed. The recovered uronic acid methyl glycoside was re-esterified with ethereal diazomethane and the amide formed by treat-ment with methanolic ammonia solution. A mixture of the <x. and fi forms of the amide methyl glycoside was obtained on evaporation. The white solid material was fractionally crystallized from aqueous ethanol solu-tion to yield the pure oc form after four recrystallizations. This was shown to be identical to an authentic sample of 4-0-methyl-<x.-D—glucur-onamide methyl glycoside (75). A sample of the neutral disaccharide methyl glycoside obtained by reduction of the aldobiouronic acid methyl ester methyl glycoside with lithium aluminum hydride was methylated three times with Haworth's re-agents using dimethyl sulphate and 40$ sodium hydroxide solution. The partly methylated disaccharide was again methylated using Purdie's re-agents viz. silver oxide and methyl iodide. After three such methyl--17-ations the disaccharide was found to be fully methylated. Hydrolysis of the dist i l l e d syrup with 1 Normal sulphuric acid for eleven hours was followed by separation of the methylated sugars on sheets of chromatographic paper in solvent 1. The dimethylxylose obtained as a syrup was oxidized with bromine and on distillation in vacuo white crystalline 3,4-di-0.-methyi-l£-xyl-ono--^ -S -^lactone was obtained. The tetramethyl glucose was not obtained pure and the contaminant appeared to be trimethyl rhamnose. An attempt to prepare the anilide Jof the tetramethyl glucose was successful but the crystalline anilide was not obtained pure due to the smallness of the scale on which i t was made. From this derivative however and paper chromatography i t was almost certain that the tetramethyl glucose obtained was 2,3,4,6-tetra-O-methyl-D-glucose. From the evidence presented in this work i t is concluded that the aldobiouronic acid present in jute must have the structure [A]* It is also evident however that a small amount of a second aldobiouronic acid occurs in which the neutral sugar unit is not D-xylose but L-rhamnose. The xylose unit to which the uronic acid is attached is part of a chain consisting of mainly xylose units with an occasional rhamnose. The length of this chain is not known but i t is built up from blocks con-taining six xylose units to which one uronic acid group is attached. Jute hemicellulose is unusual in that i t contains no arabinose. This sugar is commonly found in hemicelluloses from annual plants. It would appear that rhamnose is involved somewhere in the biosynthetic pathway's of the jute plant. Whilst this thesis was being written Srivastava and Adams (86) published a short note on work which they had carried out on jute -18-hemicellulose. They also showed the presence of 4-0-methyl-D-glucuro-nic acid and ide n t i f i e d the aldobiouronic acid as having structure [A] . Their characterization of the uronic acid was based on the preparation of the osazone of the monomethyl glucose obtained as already described. In addition they also separated an aldotriuronic acid i n c r y s t a l l i n e form and proved i t to be 4-0-methyl-D-glucuronopyranosyl-l,2-oc-D-xylo-pyranosyl-1,4-|3 -D-xylopyranose. This shows that the xylose units i n the hemicellulose main chain are linked 1,4. As already mentioned, the jute aldobiouronic acid used i n th i s work contained appreciable amounts of uronic acid formed by hydrolysis and also some aldotriuronic acid which had not been completely degraded. To obtain a pure sample of aldobiouronic acid, the crude material was applied as heavy streaks on 3 mm. chromatographic paper and separated by chromatography i n solvent 1. The pure acid was obtained as a pale brown glass with a high positive rotation £o<^  ^  + 146.5° (CO.75 i n HgO). This value i s higher than any previously reported for the material and i t i s probably the purest sample yet prepared. c Attempts were made to prepare c r y s t a l l i n e derivatives of th i s pure acid both d i r e c t l y and i n d i r e c t l y . An attempt to prepare the anilide was unsuccessful. Alcohol, which i s normally used as solvent when pre-paring sugar anilides, could not be used here due to the danger of ester formation. The acid would have had to be dissolved i n a polar solvent and none of suitable b o i l i n g point appeared to be available. An attempt to carry out the reaction without a solvent f a i l e d as the j acid did not appear to dissolve in a n i l i n e . In order to overcome the d i f f i c u l t y of ester formation, a sample of aldobiouronic acid was e s t e r i f i e d with methanolic diazomethane. -19-The methyl ester vas reacted i n methanolic solution v i t h p—nitroaniline. The l a t t e r dissolved on gentle warming but the solution turned brown in colour and no p-nitroanilide precipitated. On evaporation a brown syrup was obtained and some unchanged p-nitroaniline. A further sample of aldobiouronic acid was allowed to react with phenylhydrazine i n the same way as for osazone formation. Some amor-phous brownish-yellow material precipitated. This could not have been an osazone as the 2-position on the xylose was blocked by linkage to the CI of the 4-0-methyl-D-glucuronic acid, ^he material must therefore have been a phenylhydrazide salt or a phenylhydrazone or both. Attempts to r e c r y s t a l l i z e this material f a i l e d and no c r y s t a l l i n e compound was obtained after separation on an alumina column. It appeared that the derivative did not possess a sharp melting point as i t always appeared p l a s t i c i n nature. H C 0 OH H 0 C C H OH--20-C H g O X C O . C g H 4 . N 0 2 [ C ] X [D] X = - C O . C H g - C 0 . C g H 4 . N 0 2 y = -H Y = - C H £ Y = -H In an attempt to make a c r y s t a l l i n e derivative i n d i r e c t l y the aldo-biouronic acid vas reduced in alkaline solution by potassium borofeydride. The reaction product vas dissolved i n methanol and boric acid f i l t e r e d o f f . Evaporation of the solution gave a yellov glass vhich was reacted with excess p-nitrobenzoyl chloride i n pyridine solution* After des-troying the excess reagent with sodium bicarbonate, the p-nitrobenzoate was extracted into chloroform and evaporated to y i e l d a golden yellow glass (compound jVj). This material did not c r y s t a l l i s e and was ester-i f i e d with ethereal diazomethane to f a c i l i t a t e i t s separation on an alumina column (compound [cj). Impurities were removed on washing with dry benzene and the ester removed as a pale yellow band on washing with dry benzene containing 2fo ethanol. The product obtained behaved as a glass when heated and melted over a very wide range of temperature. It appeared l i k e l y that the presence of six p-nitrobenzoyl groups i n the molecule was s u f f i c i e n t to prevent c r y s t a l l i z a t i o n due to th e i r large -21-bulk and mutual interference. In a further experiment the xylitol compound obtained as before by borohydride reduction was acetylated with acetic anhydride in the presence of anhydrous sodium acetate. The acetate was extracted with chloroform and evaporated to yield a viscous colourless syrup which did not crystallise on keeping (compound [D]). No further work was carried out on the aldobiouronic acid. It might however have been possible to obtain a crystalline derivative by oxidation with nitric acid for example. Such an approach may prove successful in the future. -22-EXPEBIMENTAL Unless otherwise stated a l l paper chromatography was carried out on sheets of Whatman No. 1 chromatography paper. The developer employed was p-anisidine reagent and the irrigating, solvents were as follows:-1. ethyl acetate 18, glacial acetic acid 3, 45$ formic acid 1, water 4. 2. 1-butanol 40, 95$ ethanol 10, water 49, 0.880 ammonia 1. 3. methyl ethyl ketone/water azeotrope. 4. 1-butanol 5, benzene 1, pyridine 3, water 3. 5. 1-butanol 4, glacial acetic acid 1, water 5, (upper layer only) expressed as parts by volume. A l l evaporations of solvents were carried out under reduced pressure. The jute used in this work was purchased from the Beamiss Bag Company of Vancouver. Jute Hemicellulose Jute sacking was cut into strips and reduced to powdered form in a Wiley m i l l . Portions of powdered jute (100 gm.) were extracted twice with 1200 ml. benzene 50/ethanol 50 in the cold, filtered and dried. The dried jute was delignified readily by the method of Wise, Murphy and D'Addieco (81) using sodium chlorite and acetic acid at 70—80°C. for three hours. The white holocellulose obtained was filtered off and washed with cold water to give yields of around'90$. Jute holocellulose was extracted overnight in 1000 ml. 9.3$ aque-ous sodium hydroxide and filtered. The remaining solid material was again extracted with 500 ml. of alkali for several hours and filtered - 2 3 -as before. The cellulose remnant vas washed well with cold water and dilute acetic acid, and dried. The orange-yellow filtrates were bulked and neutralised with glacial acetic acid to give a straw-coloured solu-tion from which the hemicellulose did not precipitate. Upon addition of two volumes of ethanol jute hemicellulose was thrown out as a curdy precipitate and was isolated by centrifugation. The product was washed several times with 5 0 $ ethanol to remove inorganic salts, then with i n -creasing strengths of alcohol and finally dried by solvent exchange to yield a white powder ( 1 6 gm. from 1 0 0 gm. jute). Purification of Jute Hemicellulose Crude hemicellulose ( 5 gm.) was dissolved in 3 0 ml. 10fe aqueous sodium hydroxide and 5 0 ml. Fehling's solution added. The greenish-blue precipitate was left standing overnight and isolated by centrifugation. After careful washing with alcoholic sodium hydroxide followed by wash-ing with ethanol, the complex was decomposed with dilute ethanolic hydro-gen chloride and washed free of chloride ion with aqueous ethanol before isolation as a white powder by solvent exchange. This material (hemi-cellulose I , 3 . 2 gm.) had ash content 2 . 3 3 $ , [ ° ° ] ^ ~ 4 5 . 1 ° (calculated as ash free C. 0 . 1 5 in 2 N NaOH). N . E . of 9 5 3 indicated 2 1 . 7 $ anhydrou-ronic acid units. A second batch of crude hemicellulose ( 5 gm.) was suspended in 1 5 0 ml. ethanol containing 4 . 5 ml. hydrogen chloride. After standing over-night the suspension was centrifuged and the precipitate washed free of chloride ion as before. After solvent exchange the hemicellulose was obtained as a snow-white powder (hemicellulose I I , 3 . 5 gm.) with ash content 0 . 2 8 $ and [ o c ] ^ ° - 4 7 . 1 ° (C. 0 . 5 in 2 N NaOH). N . E . of 1 0 0 5 indicated 2 0 . 6 $ anhydrouronic acid units. The methoxyl content of -24-3.18$ indicated a mean E. Wt. of 975 or 21.3$ anhydrouronic acid. An attempt to prepare absolutely pure hemicellulose by electrodi-alysis of an aqueous solution of the crude material failed as the pure material vas precipitated in gel form from vhich i t vas found impossible to remove the last traces of vater by solvent exchange. In viev of the higher purity obtained, the main batch of jute hemi-cellulose vas purified by the ethanolic hydrogen chloride method and vas obtained as hemicellulose II. Attempted Direct Extraction of Hemicellulose from Jute A 100 gm. sample of jute vas solvent extracted and then alkali extracted in the usual vay. The hemicellulose vas purified by the copper salt method to yield a brovnish-vhite powder (4.9 gm.). Ash con-tent 6.38$, ~ 47.0° (calculated as ash free C. 1.25 in 2N NaOH). A sample (304 mg.) of this material vas hydrolysed on the steam bath for, sixteen hours vith 1 Normal sulphuric acid and the hydrolysate i neutralised vith barium carbonate, the solution obtained being deionised in an Amberlite IR 120. resin column. The concentrated eluate shoved chromatographic spots corresponding to xylose, glucose, uronic acid, aldobiouronic acid, and oligosaccharides vhen irrigated in solvents 1 and 2 and developed vith p-anisidine spray reagent. The glucose vas present only in trace amounts. Jute Aldobiouronic Acid In a preliminary experiment hemicellulose II (2 gm.) vas hydrolysed vith 30 ml. 1 Normal sulphuric acid for six hours on the steam bath and the acids neutralised vith barium carbonate. The hydrolysate vas deionised in an Amberlite IR 120 resin column and the concentrated -25-eluate shoved spots with Rx values 0.30, 0.69, 1.00 and 1.44 correspond-ing to oligosaccharide, aldobiouronic acid, xylose, and uronic acid when chromatographed in solvent 1. Another t r i a l hydrolysis of hemicellulose II (3 gm.) vas carried out in 100 ml. 45$ formic acid. After twelve hours on the steam bath the formic acid vas removed by evaporation under reduced pressure. Chromatography shoved the same spots as before with a larger amount of aldotriuronic acid (Rx 0.35) apparently due to incomplete hydrolysis. Further hydrolysis of the material in 1 Normal sulphuric acid removed this spot vith a corresponding increase in uronic and aldobiouronic acid. A large batch of hemicellulose II (15.0 gm. expressed as dry weight) was hydrolysed on the steam bath in 400 ml. 1 Normal sulphuric acid and the reaction followed on the polarimeter. After seven hours the rota-tion had reached a steady valie of +57° when the acids were neutralised with barium carbonate as before and deionised in an Amberlite IR 120 resin column. Upon running the eluate through a Duolite A4 resin column the sugar acids were retained and the neutral sugar fraction obtained in solution. The column was carefully washed with water and the sugar acids eluted with 1 Normal sodium hydroxide. Upon returning this eluate to the Amberlite IR 120 column a solution of the sugar acids was obtained. The neutral sugar solution when concentrated under reduced pressure yielded a pale yellow syrup (9.976 gm.) which crystallised overnight. It appeared at f i r s t to contain only xylose when spots were irrigated in solvent 1 but later a true sugar with an Rx value greater than that of xylose was found. The sugar acid fraction when concentrated under reduced pressure -26-gave a yellow glass (3.604 gm.). Spots of this material when irrigated in solvent 1 showed the presence of an appreciable amount of uronic acid and small amounts of triuronic acid besides the main fraction of aldo-biouronic acid. The fraction had OMe 8.05$ (calculated for C . Q H ^ O , , i t is 9.1^) J ° + 124.4° (C. 0.5 in H 20). N.E. 314 (calculated for CjgHggOjj i t is 340). The low values of the inethoxyl vaue and neutral-isation equivalent were attributable to the uronic acid present in the fraction. The recovery of sugars from the hydrolysate was approximately 90$ and the anhydrouronic acid content of 18$ was in fair agreement with the value estimated by other methods. Identification of Neutral Sugars The crystallised neutral sugar fraction was twice recrystallised from methanol to give white prismatic crystals melting point and mixed melting point 143-145°C. with an authentic sample of pure xylose, ^cx-Jjg,^  + 17.0° (equivalent C. 1.0 in HgO). The dibenzylidene dimethyl acetal derivative was prepared by dis-solving a sample of the sugar (686 mg.) in 7 c c . of benzaldehyde -methanolic hydrogen chloride reagent (82). This reagent was prepared by dissolving freshly dist i l l e d benzaldehyde (40 ml.) in a mixture of methanolic hydrogen chloride (20 ml., 2.5N) and methanol (120 ml.). After standing for four days a white crystalline product was obtained. Two recrystallizations from methanol/benzene gave fine white needle-like crystals melting point and mixed melting point 211°C. with a stan-dard derivative made from crystalline D—xylose. A sample of the true sugar was obtained when a sheet of Whatman -27-3 mm. chromatographic paper vas heavily streaked vith the neutral sugar mixture and irrigated in solvent 1. The band of sugar vas extracted into 80$ aqueous methanol and concentrated to give a yellov syrup. Chromatography in solvents 1, 3 and 4 against standard rhamnose and xylose shoved that the unknown sugar vas L—rhamnose. Reduction of Jute Aldobiouronic Acid The aldobiouronic acid (2.210 gm.) vas treated vith 3$ methanolic hydrogen chloride on a steam bath under reflux and the reaction followed on the polarimeter. After five hours a constant rotation of +112° vas attained. The hydrogen chloride vas neutralised with lead carbonate and the precipitate removed after centrifugation. After careful washing of the precipitate with methanol the f i l t r a t e and washings were bulked and evaporated to yield the methyl glycoside methyl ester (2.697 gm.) as a thin brown syrup. Finely crushed lithium aluminum hydride (2.8 gm.) was suspended in 120 ml. dry tetrahydrofuran and refluxed to promote dispersion. A solu-tion of the methyl ester methyl glycoside of the aldobiouronic acid in 50 ml. dry tetrahydrofuran was added gradually to the cooled suspension and the reaction mixture refluxed for thirty minutes. Excess reagent was destroyed by the addition of ethereal ethyl acetate solution and the solvents removed under reduced pressure. Acetic anhydride (50 ml.) and anhydrous sodium acetate (3.5 gm.) was added to the reaction vessel which was heated under reflux for three hours on the steam bath. After evaporating off excess acetic anhydride the inorganic salts were dis-solved in dilute hydrogen chloride and the solution extracted with 250 ml. chloroform in four separate washings. The chloroform extract was -28-freed of chloride ion by washing with water and the solvent removed to give a mobile yellow syrup of the disaccharide methyl glycoside penta-acetate(3.68 gm.). Removal of the acetjl groups was accomplished by saponification in 20 ml. ethanol with 20 ml. 1 Normal sodium hydroxide for one hour on the steam bath. After deionising in Amberlite IR 120 followed by Duolite A4 resin columns the solution of the disaccharide methyl glycoside was evaporated to give a viscous yellow syrup (2.093 gm.). OMe 19.7$ (calc. for C 1 3H 24 0io i s 18*2#)> M D °' + 9 8 , 5 ° (C* 0 , 7 5 i n H2 f l^* Hydrolysis of the Neutral Disaccharide. The disaccharide methyl glycoside (850 mg.) was hydrolysed in 50 ml. 1 Normal sulphuric acid on the steam bath and the reaction followed on the polarimeter. After fourteen hours a steady rotation of +44.5° was attained. The hydrolysate was neutralised with barium carbonate and the precipitate removed by centrifugation and washed free of carbo-hydrate material. The f i l t r a t e and washings were bulked and evaporated to yield a viscous yellow syrup (820 mg.). Paper chromatograms were spotted with this material and irrigated in solvents 1, 2, 3, 4 and 5 using D-xylose, 3-0-methyl-D-glucose, and 4-0-methyl-D-glucose as standards. An excellent separation was achieved in solvent 1 giving two spots Rx 0.98 and 1.24. There was no doubt that the slower yellow spot was xylose but the faster pale brown spot might have been either 3-or 4-0-methyl-D-glucose. These sugars were found to be quite impossible to distinguish chromatographically. Separation of the Sugars in the Hydrolysate Attempts to separate the monomethyl glucose from the xylose using -29-a cellulose column irrigated with solvent 1 were unsuccessful and only a partial separation was achieved. The sugar mixture (500 mg.) was streaked on sheets of Whatman 3 mm. prewashed chromatographic paper and irrigated for sixteen'hours in solvent 1. After drying the sheets the sugar zones were located by spraying strips with p-anisidine reagent. A good separation was achieved and the sugars were extracted from the paper into 80$ aqueous methanol. Evaporation of the extracts yielded two chromatography cally pure sugars. One fraction (120 mg.) had Rx 0.98 in solvent 1 and was apparently D-xylose whilst the other (200 mg.) had Rx 1.23 and was the monomethyl glucose* Identification of D-xylose The extracted sugar crystallised on standing for two days and after treatment with charcoal in aqueous solution and evaporation i t was ob-tained as white prismatic crystals j^-J^ + 22.9° (equil. Literature value + 18.0°). The dibenzylidene dimethyl acetal was made as already described (82) and after recrystallization from benzene/petroleum ether was obtained in the form of white needles melting point amd mixed melt-ing point 211°C. with a standard derivative made from crystalline D-xylose. Identification of L-rhamnose When attempting the separation of xylose and monomethyl glucose on a cellulose column three tubes were obtained containing a sugar running faster than monomethyl glucose in solvent 1. This appeared to be present in only trace amounts and its identification was only carried out by paper chromatography. When examined in solvents 1, 2 and 4 the unknown had Rx values of 1.58, 1.88 and 1.23 respectively and ran side by side -30-with a standard spot of L-rhamnose. Both sugars gave a pale yellow spot with p-anisidine reagent. Identification of 4 Methyl Glucose (a) Demethylation A sample of the chromatographically pure sugar (20 mg.) was heated with 1.5 ml. 40$ hydrobromic acid in a sealed tube in the steam bath for five minutes when the solution in the tube was diluted with 20 ml. water. The hydrobromic acid was removed on Duolite A4 exchange resin and the deionised solution evaporated to give a pale yellow syrup. When examined by paper chromatography in solvent 4 this syrup showed spots coinciding with B—glucose and unreacted monomethyl glucose. (b) Preparation of the osazone A portion of monomethyl glucose (55 mg.) was dissolved in 1.8 ml. water and 0.18 ml. freshly dist i l l e d phenylhydrazine, 12. ml. 20$ acetic acid and 60 mg. sodium bisulphite added. The reaction was carried out in a water bath aat 70-80°C. for two hours when the osazone separated as small yellow crystals which were filtered off and recrystallized twice from aqueous alcohol to yield small yellow needlelike crystals of the osazone melting point 159-160°C. without decomposition. The osazones of standard 3-and 4-0-methyl-D-glucose were prepared in similar fashion. 4—0-methyl-D-glucosazone was obtained as small golden needles from aqueous alcohol melting point 158.159°C. (84) but the 3-methyl analogue (yellow sheaves) did not give a sharp melting point even after many recrystallizations from aqueous alcohol and aqu-eous acetone. The best value obtained was 168-172 C. (values of 164-166°C. (87) and 178-179°C. (88) are reported in the literature). -31-The osazone from jute gave mixed melting point 158-159°C. vith 4-0-methyl-D-glucosazone and 146-164°C. with 3-0-methyl—D-glucosazone. (c) Preparation of the anilide To a sample of monomethyl glucose (99 mg.) was added 2 ml. absol-ute alcohol and 0.1 ml. freshly dist i l l e d dry aniline. After refluxing on the steam bath for one and a half hours the reaction was stopped and the alcohol and excess aniline removed by evaporation in vacuo. The i pale brown reaction product was treated with 20 ml. ethyl acetate under reflux and the extract upon concentration yielded pale yellow crystals of the anilide. After three recrystallizations from ethyl acetate small white needlelike crystals were obtained (15 mg.) melting point 158-160°C. Further recrystallization did not raise this value. A sample of 4-0-methyl-D-glucose anilide was similarly prepared from the standard sugar. It yielded white mushroom like crystals melt-ing point 156-158°C. from straight ethyl acetate solution. This material was d i f f i c u l t to obtain pure and the melting point quoted was the high-est that could be obtained. The mixed melting point for these derivatives' was 156-159°C. (d) Preparation of other standard derivatives In a search for suitable derivatives of standard 3-and 4-0-methyl-D-glucose the dibenzyl mercaptals were prepared. Standard 4-0-methyl-D-glucose (270 mg.) was dissolved in 0.35 ml. hydrogen chloride and 0.33 ml.°<- toluenethiol and 150 mg. granular zinc chloride added to the solution. After placing in the mechanical shaker for six hours a one phase liquid was obtained, and this was d i l -uted with water throwing out a white o i l from solution. The hydrogen chloride was removed on Duolite A4 resin which was filtered off and -32-washed with alcohol. The fil t r a t e and washings were bulked and evapor-ated to yield a thick yellow o i l which solidified overnight. This was dissolved in absolute alcohol and treated with charcoal. After f i l t r a -tion and concentration of the f i l t r a t e white crystals of the mercaptal were obtained (50 mg.). These after three recrystallizations from ab-solute alcohol had melting point 158-159°C. Further recrystallization did not raise this value. An attempt was made to prepare 3-0-methyl-D-glucose dibenzyl mer-captal in similar fashion. The material could not be induced to crys-ta l l i z e from absolute alcohol but a crop of white platelike crystals was obtained after diluting the concentrated alcoholic solution with a few drops of water. These had melting point 06-69°C. before and after recrystallization from aqueous alcohol. Upon drying this material in the Abderhalden over boiling toluene decomposition took place and a small amount of yellow o i l remained. Methanolysis of Jute Aldobiouronic Acid A sample of aldobiouronic acid (3.06 gm.) was treated with 10$ methanolic hydrogen chloride on the steam bath overnight. After neut-ralization with lead carbonate and evaporation of the f i l t r a t e a dark brown syrup (3.07 gm.) was obtained. This showed three spots when chromatographed in solvent 2 the Rx values being 0.23, 1.00 and 2.11. The mixture was continuously extracted into ether for one day and the ethereal and aqueous phases evaporated to jield brown syrups (0.979 gm. and 1.544 gm. respectively). The syrup from the aqueous phase was dissolved in 50 ml. 0.15 Normal barium hydroxide and kept at 60°C. for two hours when the excess alkali -33-was destroyed by the addition of solid carbon dioxide. The precipi-tated barium carbonate was centrifuged off and the f i l t r a t e deionised with Amberlite IR 120 resin. When the eluate was run through Duolite A4 resin a solution of methyl xyloside was obtained. The acids were eluted from the column in 30 ml. 1 Normal sodium hydroxide and this on running through Amberlite IR 120 resin yielded a solution of the acid component which on evaporation gave a brown syrup (0.643 gm.). Chroma-tography in solvent 1 and spraying with a 1$ bromophenol blue indicator solution showed two yellow spots with Rx 0.18 and 0.49 due to aldobio-uronic and uronic acid methyl glycosides. A similar treatment of the ether extracted syrup gave an acid com-ponent (0.431 gm.) Rx 0.53 corresponding to the methyl glycoside of the uronic acid. The methyl xyloside solutions were bulked and evaporated to yield a pale yellow syrup (0.687 gm.) which gave fernlike crystals on keeping. f This material was not further investigated. Characterisation of Jute Uronic Acid The uronic methyl glycoside (431 mg.) obtained above was esteri-fied with ethereal diazomethane and evaporated. The methyl ester methyl glycoside was dissolved in 25 ml. cold saturated methanolic ammonia and allowed to stand for two days. Evaporation gave a white solid which was decolourised with charcoal in methanol solution. Con-centration yielded white mushroomlike crystals melting point 204-213°C. After four recrystallizations from aqueous ethanol small white pris-matic crystals of 4-0-methyl-oc-D—glucuronamide methyl glycoside were obtained melting point and mixed melting point 234.5-236°C. with a stan-dard sample. (Literature value 236°C. (75)). -34-Methylation of Neutral Disaccharide To the neutral disaccharide methyl glycoside (864 mg.) vas added 6 ml. 40$ sodium hydroxide solution followed by 0.5 ml. dimethyl sul-phate. The mixture vas stirred at high speed at a temperature of 45-55°C. and 0.5 ml. portions of dimethyl sulphate added every fifteen minutes for one and a half hours. A further addition of 6 ml. 40$ sodium hydroxide vas then made and the vhole cycle of operations re-peated twice so that the total reaction time was four and a half hours. The whole procedure was repeated twice more so that in a l l nine 6 ml. portions of 40$ sodium hydroxide and 27 ml. dimethyl sulphate were used. The excess dimethyl sulphate was destroyed at the end on heating the mixture to 80°C. for half an hour. After neutralisation to pH 8.9 with sulphuric acid the reaction mixture was continuously ex-tracted with chloroform overnight. Evaporation of the extract gave a yellow mobile syrup (675 mg.) whose infra-red spectrum showed only a weak hydroxyl function. The partly methylated syrup was dissolved in 20 ml. dry methyl iodide and a few grains of Drierite added. Silver oxide (5 gm.) was added in small amounts to the reaction mixture.kept at 50°C. under reflux in a flask fitted with mercury sealed stirrer. After nine hours acetone was added and the excess methyl iodide di s t i l l e d off. The methylated sugar was filtered in acetone solution and the fi l t r a t e returned to the flask for evaporation of the solvent. After three such methylations the product gave an infra-red spectrum showing zero hyd-roxyl function. The methylated sugar was purified by distillation ih vacuo when a pale yellow syrup (517 mg.) was obtained (bath tempera-ture 170-180OC. at 0.01 mm. Hg). Oide content 55.7$ (calculated for -35-C18H34°10 1 8 6 2 » 9 ^ ) [ £ X ] 1° + 108.6° (C. 1.75 in CBClg). The high methpoxyl content found was due to the presence of some rhamnose con-taining disaccharide present as impurity. Hydrolysis of Methylated Disaccharide and Separation of the Methylated Sugars The methylated disaccharide (437 mg.) was dissolved in 25 ml. 1 Normal sulphuric acid and hydrolysed oh the steam bath for eleven hours when a steady rotation of +81.0° was attained. The acid was neutralised with barium carbonate and the precipitate centrifuged off and washed free of carbohydrate. The fi l t r a t e and washings were bulked and evapor-ated to yield a yellow syrup (396 mg.) showing two spots R '0.68 and 0.83 when chromatographed in solvent 11. The spots gave buff and pale yellow colours with p-anisidine reagent, the former resembling 3,4-di-methyl xylose whilst the latter ran identically to 2,3,4,6-tetramethyl glucose in solvents 1 and 2. The mixture of methylated sugars (396 mg.) was streaked on pre-washed Whatman 3 mm. chromatographic paper and irrigated in solvent 1. The zones were located by spraying strips and the sugars were extracted from the dried paper using ether for the tetramethyl glucose and absolute alcohol for the dimethyl,xylose. Evaporation of the extracts gave syrups of tetramethyl glucose (129 mg.) and dimethyl xylose (56 mg.). Identification of 2,3,:4>6-tetra-0-methyl-D-glucose The extracted sugar showed traces of trimethyl rhamnose as impurity when examined on chromatograms. Attempts to crystallise the tetra-methyl glucose from ether solution were unsuccessful and an attempted fractional distillation of the syrup in vacuo did not yield a pure -36-product. Finally a second separation on paper yielded some pure tetra-methyl glucose (26 mg.). The anilide was prepared in the usual way and a few long needle-like crystals were obtained. The material was recrystallised from absolute alcohol and gave melting point 122-132°C. (literature value 138°C. (89)). Owing to the smallness of the sample i t was impossible to recrystallize the material to constant melting point. The mixed melting point' with standard 2,3,4,6-tetra-0-methyl-D-glucose anilide was 130-136°C. J Identification of 3.4-di-0-methyl-D-xylose The dimethyl xylose (56 mg.) was dissolved in 2 ml. water and 20 drops bromine added. The well-stoppered flask was kept for two days in the dark when a spot on paper gave a negative test with p-anisidine spray. The bromine was removed by aeration and" hydrobromic acid neutralised with silver oxide. After centrifugation and washing of the precipitate the bulked f i l t r a t e and washings were treated with hydrogen sulphide for three minutes. After evaporation the product was taken up in a l i t t l e water, centrifuged and transferred to a clean flask. Evaporation yielded a pale brown glass (47 mg.) and on d i s t i l -lation in vacuo white needlelike crystals were obtained (bath tempera-ture 140-160°C. at 0.1 mm. Hg). The lactone (25 mg.) had melting point 58-64°C. and was contaminated with a small amount of colourless syrup. Redistillation gave crystals of 3,4-di-0-methyl-D-xylose- &-lactone melting point 64-66°c. (literature value 64-66°C. (8)). -37-Pure Jute Aldobiouronic Acid Impure aldobiouronic acid (4.65 gm.) prepared in the usual way was heavily streaked on Whatman 3 mm. chromatographic paper at a load of approximately 8 mg. per cm. After irrigation in solvent 1 for six-teen hours the aldobiouronic acid was located by the method of spraying strips. The dried paper was extracted with 80$ aqueous acetone until a faint Molisch test was given by the extract. On evaporation the chromatographically pure aldobiouronic acid was obtained as a brownish-white glass (0.947 gm.) [ot] ^  + 146.5° (C. 0.75 in HgO). The uronic' acid zone was also retained and extracted with 80$ aqueous acetone to yield on evaporation a brownish-yellow glass (0.124 gm.). The material was found to be chromaiographically pure^exJ ^  + 55.0° ~(C. 1.25 in HgO). Attempted Preparation of a Crystalline Derivative by Direct Methods An attempt to make the anilide from the aldobiouronic acid was un-successful since the choice of solvent gave difficulty. The acid did not react with aniline without a solvent being present. A sample of aldobiouronic acid (82 mg.) was esterified with methan-olic diazomethane and evaporated to give a brown syrup. Upon reaction with recrystallized p-nitroaniline (45 mg.) in methanol (2 ml.) a dark brown solution was obtained after heating at 70°C. for fifteen minutes. No crystalline p-nitroanilide was found and when the solution was d i l -uted with water some p-nitroaniline was recovered unchanged. A further portion of acid (71 mg.) was dissolved in 2 ml. water o and reacted at 80 C. with 0.13 ml. distil l e d phenylhydrazine in the presence of 20$ acetic acid (1.4 ml.) and sodium bisulphite (50 mg.). 38-After three hours the mixture was allowed to cool when a yellow-brown solid separated. This material was filtered off and applied to the top of an alumina column after dissolving in the minimum volume of acetone. Excess phenylhydrazine was removed by washing with benzene containing 2$ ethanol. The derivative remained as a brown band at the top of the column and was finally removed in 5$ aqueous alcohol solution. After treatment with charcoal, fi l t r a t i o n and evaporation the material was recrystallised from aqueous alcohol and had melting point 95° to 130°. The indefinite melting point persisted after a further recrystallization and the attempt to obtain a satisfactory crystalline derivative by this method was abandoned. Attempted Preparation of a Crystalline Derivative by Indirect Methods A sample of acid (107 mg.) was dissolved in 2.0 ml. water and made alkaline by the addition of 1.0 ml. 5 Normal sodium hydroxide. After the addition of potassium borohydride (55 mg.) the reduction was allowed to proceed in the cold for two hours when the excess reagent was des-troyed with glacial acetic acid. The solution was deionised with Amber-li t e IR 120 exchange resin and filtered. Evaporation gave a mixture of the xylitol compound with white crystalline boric acid. This mixture was acetylated with acetic anhydride (5 ml.) in the presence of anhydrous sodium acetate (150 mg.) under reflux on the steam bath. After three hours the excess acetic anhydride was dist i l l e d off under reduced pres-sure and the inorganic salts dissolved in dilute hydrochloric acid. The solution was extracted three times with chloroform and the bulked extracts washed free of chloride ion with water. Evaporation yielded a pale yellow viscous syrup (41 mg. Compound [©])• A l l attempts to crystallize the acetate were unsuccessful. -39-A large batch of acid (655 mg.) was reduced with potassium boro-hydride as before and the xylitol compound dissolved in acetone. The boric acid was filtered off and the solution evaporated to yield a yellow glass (623 mg. 1 mole). This was dissolved in dry pyridine (40 ml.) and reacted with 4.23 gm. (12 moles) pure p-nitrobenzoyl chloride on the steam bath under reflux for two hours. After standing overnight a heavy precipitate probably of pyridine hydrochloride was obtained. Excess saturated sodium bicarbonate solution was added to the reaction mixture until no further effervescence was observed and the solution left for three hours. The solution was extracted three times with chloroform and the chloroform extracts washed free of b i -carbonate with water. Evaporation of the extract yielded a yellow glass (1.265 gm. Compound [&])• Only a fraction (800 mg.) of this material was soluble in chloro-form the remainder having been dissolved by pyridine which had carried over in the chloroform extract. This fraction was dissolved in chloro-form and methylated with ethereal diazomethane. Evaporation gave the p-nitrobenzoate methyl ester as a golden yellow glass (800 mg. Compound [c]). A sample of this compound (158 mg.) was applied to the top of an alumina column in chloroform solution (3 ml.) and the column i r r i -gated with 400 ml. dry benzene. This was followed by irrigation with dry benzene containing 2$ ethanol when the p-nitrobenzoate came off as a thin yellow band. When evaporated this gave a yellow material (69 mg.) melting point 97 to 185°C. which was apparently a glass. The ben-zene extract yielded a mixture of p-nitrobenzoate and what appeared to be methyl p-nitrobenzoate present as an impurity melting point 75-80°C. It did not appear that the p-nitrobenzoate of the xylitol compound could be obtained crystalline. - 4 0 -BIBLIOGRAPHY 1 . White, E.V., Tappi Monograph No. 6 . (1948) . 2 . Kirchaner, W. and Tollens, B., Ann., 17JT : 205 (1874~). 3 . O'Sullivan, C , J . Chem. Soc, 1164 (1901). 4 . James, S.P. and Smith, F., J. Chem. Soc, 739 (1945). 5 . Das Gupta, P.C. and Sarkar, P.B., Textile Research J., 24 : 1071 (1954). 6 . Jones, J.K.N, and Smith, F., "Advances in Carbohydrate Chemistry", Vol. 4 . , ed. Pigman, W.W. and Wolfrom, M.L., Academic Press Inc., New York, (1949) p. 243. 7 . Hough, L., Jones, J.K.N, and Wadman, W.H., J . Chem. Soc, 796 (1952), 8. Montgomery, R., Smith, F. and Srivastava, H.C., J . Am. Chem. Soc, :. 78 : 2837 (1956). 9 . Falconer, E.L. and Adams, G.A., Can. J. Chem., 34 : 338 (1956). 10. Whistler, R.L. and Hough, L., J . Am. Chem. Soc, 75 : 4918 (1953). 1 1 . Adams, G.A. and Bishop, C.T., J . Am. Chem. Soc, 78 : 2842 (1956). 12. Hamilton, J.K., Sprietersbach, D.R. and Smith, F., J . Am. Chem. Soc . , i 79 : 443 (1957). 13. Bishop, C.T., Can. J . Chem., 33 : 1521 (1955). 14. Bishop, C.T., Can. J . Chem., 31 : 134 (1953) . 15. Chanda, S.K., Hirst, E.L. and Percival, E.G.V., J . Chem. Soc, 1240 (1951). 16. Adams, G.A., Can. J . Chem., 32 : 186 (1954). 17. Nishida, K. and Hashima, H., J . Agr. Chem. Soc. JAPAN, 13 : 660 (1937). cf. C.A., 32 : 4142 (1938). 18. Hotchkiss, R.D. and Goebel, W.F., J . Biol. Chem., 121 : 121 (1937). 19. Jones, J.K.N, and Perry, M.B., J. Am. Chem. Soc, 79 ; 2787 (1957) . 20 . Jones, J.K.N., J . Chem. Soc, 558 (1939). 2 1 . Hirst, E.L. and Jones, J.K.N., J . Chem. Soc, 1174 (1938). 22 . Aspinall, G.O., Hirst, E.L. and Wickstrom, A., J . Am. Chem. Soc, 77 : 1160 (1955) . -41-23. Char 1 son, A.J., Nunn, J.B. and Stephen, A.M., J . Chem. S o c , 1428 (1955). 24. Mukherjee, S. and Srivastava, H.C., J . Am. Chem. S o c , 77 : 422 (1955). 25. Connell, J . J . , Hainsvorth, R.M., Hirst, E.L. and Jones, J.K.N., J . Chem. S o c , 1696 (1950). 26. Andrews, P. and Jones, J.K.N., J . Chem. S o c , 1724 (1954). 27. Hirst, E.L. and Jones, J.K.N., J . Chem. S o c , 1064 (1947). 28. Roudier, A., Compt. Rend., 237 : 662 (1953). 29. Challinor, S.W., Haworth, W.N. and Hirst, E.L., J . Chem. S o c , 258 (1931). 30. Stephen, A.M., J . Chem. S o c , 646 (1951). 31. Hi r s t , E.L. and Pe r l i n , A.S., J . Chem. S o c , 2622 (1954). 32. Brown, F., Hirst,, E.L. and Jones, J.K.N., J . Chem. S o c , 1677 (1948). 33. Jones, J.K.N., J . Chem. S o c , 534 (1950). 34. Glaudenmans, C.P.J, and Timell, T.E., J . Am. Chem. S o c , 80 : 941 (1958). 35. Gillham, J.K. and Timell, T.E., Can. J . Chem., 36 : 410 (1958). 36. Dutton, G.G.S. and Smith, F., J . Am. Chem. S o c , 78 : 2505 (1956). 37. Aspinall, G.O., Hirst, E.L. and Mahomed, R.S., J . Chem. S o c , 1734 (1954). 38. Garrod, A.R.N, and Jones, J.K.N., J . Chem. S o c , 2522 (1954). 39. Jones, J.K.N, and Wise, L.E., J . Chem. S o c , 3389 (1952). 40. Brasch, D.J. and Wise, L.E., Tappi, 39 : 768 (1956). 41. Adams, G.A., Can. J . Chem., 35 : 556 (1957). 42. B a l l , D.H., Jones, J.K.N., Nicholson, W.H. and Painter, T.J., Tappi, 39 : 438 (1956). 43. Whistler, R.L., Conrad, H.E. and Hough, L., J . Am* Chem. S o c , 76 : 1668 (1954). 44. Geerdes, J.D. and Smith, F., J . Am. Chem. S o c , 77 : 3569 (1955). 45. Saarnio, J . , Wathen, K. and Gustafsson, C , Acta, Chem. Scand., 8 : 825 (1954). -42-46. Dutton, G.G.S. and Hunt, K., J . Am. Chem. S o c , 80 t 4420 (1958). 47. Jones, J.K.N, and Nunn, J.R., J . Chem. S o c , 3001 (1955). 48. Cunneen, J . I . and Smith, F., J . Chem. S o c , 1141 (1948). 49. White, E.V., J . Am. Chem. S o c , 70 : 367 (1948). 50. Parikh, V.M., Ingle, J.R. and Bhide, B.V., J . Indian Chem. S o c , 33 : 119 (1956). 51. Andrews, P. and Jones, J.K.N., J . Chem. S o c , 1724 (1954). 52. Hostettler, F. and JSeuel, H., Helv. Chim. Acta., 34 : 2440 (1951). 53. Tipson, R.S., Christmah, C.C. and Levene, P.A., J . B i o l . Chem., 128 : 609 (1939). 54. Hirst, E.L., Percival, E.G.V. and Wylam, C.B., J . Chem. S o c , 189 (1954). 55. Laidlaw, R.A. and Percival, E.G.V., J . Chem. S o c , 1600 (1949). \ 56. Whistler, R.L. and Conrad, H.E., J . Am. Chem. S o c , 76 : 3544 (1954). 57. G i l l , R.E., Hirst, E.L. and Jones, J.K.N., J . Chem. S o c , 1469 (1939). 58. Hirst, E.L. and Duns tan, S., J . Chem. S o c , 2332 (1953). 59. Buchi, W. and Deuel, H., Helv. Chim. Acta., 37 : 1392 (1954). 60. Hirst, E.L., Hough, L. and Jones, J.K.N., J . Chem. S o c , 3145 (1949). 61. Hirst, E.L., Hough, L. and Jones, J.K.N., J . Chem. S o c , 2332 (1953). 62. Tachi, L. and Yamamori, N., Mokuzai Kenkyu, 4 : 1 (1950). cf. G.A., 45 : 9855 (1951). 63. Anderson, E. and Kinsman, S., J . B i o l . Chem., 94 : 39 (1931). 64. Stewart, CM. and Foster, D.H., Australian J . Chem., 6 : 431 (1953). 65. Davidson, E.A. and Meyer, K., J . Am. Chem. S o c , 76 : 5686 (1954). 66. Mukherjee, S.N. and Chakrauarti, S.C., J . Indian.Chem. S o c , 25 : 113 (1948). 67. Rao, P.S. and Budhiraja, R.P., J . S c i . Ind. Research (INDIA), 10B : 209 (1952). c f . C.A., 47 .10882 (1953). L -43-68. Bailey, K., Biochem. J., 29 : 2477 (1935). 69. Bailey, K. and Norris, F.W., Biochem. J., 26 : 1609 (1932). 70. Sands, L. and Klass, R., J. Am. Chem. Soc, 51 : 3441 (1929). 71. Edington, R.A. and Percival, E.E., J . Chem. Soc, 2473 (1953). 72. Haworth, W.N., Hirst, E.L., Isherwood, F. and Jones, J.K.N., J . Chem. Soc, 1878 (1939)1 73. Lythgoe, B. and Trippett, S., J . Chem. Soc, 1983 (1950). 74. Abdel-Akher, M. and Smith, F., Nature, 166 : 1037 (1950). 75. Smith, F., J. Chem. Soc., 2649 (1951). 76. Jackson, J . and Smith, F., J. Chem. Soc, 74 (1940). 77. White, E.V., J. Am. Chem. Soc, 69 . 2264 (1947). 78. Smith, F. and Srivastava, H.C., J . Am. Chem. Soc, 78 : 2837 (1956). 79. Sarkar, P.B., Mazumdar, A.K. and Pal, K.B., Text. Research J., 22: 529 (1952). 80. Das Gupta, P.C. and Sarkar, P.B., Text. Research J., 24 : 705 (1954).-81. Wise, L.E., Murphy, M. and D'Addieco, A.A., Paper Trade.J., 122 j 35 (1946). 82. Breddy, L.J. and Jones, J.K.N., J. Chem. Soc, 738 (1945). v 83. Pacsu, E., Ber., 58 .1455 (1925). 84. Schinle, R., Ber., 65 : 315 (1932). 85. Munro, J . and Percival, E.G.V., J . Chem. Soc, 873 (1935). 86. Srivastava, H.C. and Adams, G.A., Chem. and Ind. No. 29, 921 (1958). 87. Irvine, J.C. and Scott, J.P., J . Chem. Soc, 564 (1913). 88. Anderson, C.G., Charlton, W. and Haworth, W.N., J . Chem. Soc, 1329 (1929). 89. Wolfrom, M.L. and Lewis, W.L., J . Am. Chem. Soc, 50 : 837 (1928), 


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