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The constitution of the hemicellulose of apple wood Murata, Toyoko Gene 1960

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THE CONSTITUTION OF THE HEMICELLULOSE OF APPLE MOOD by TOYOKO GENE MURATA B.Sc., University of British Columbia, 1958 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 required standard  THE UNIVERSITY OF BRITISH COLUMBIA October, i960  the  In presenting the  this  r e q u i r e m e n t s f o r an  thesis  in partial  advanced degree a t  of B r i t i s h Columbia, I agree that it  freely  agree that for  available  the  f o r r e f e r e n c e and  permission f o r extensive  s c h o l a r l y p u r p o s e s may  D e p a r t m e n t o r by  be  gain  shall  not  Department  of  be  a l l o w e d w i t h o u t my  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a .  shall  study.  I  Columbia  the  of  University  copying of  his representatives.  copying or p u b l i c a t i o n of t h i s  the  Library  g r a n t e d by  that  fulfilment  make  further this  Head o f  thesis my  I t i s understood  thesis for written  financial  permission.  ABSTRACT The hemicellulose Isolated from apple wood (var. Golden Transparent) by alkaline extraction has been shown to contain a Ij.-0-methylglucuronoxylan.  Hydrolysis of the hemicellulose  yielded neutral sugars and uronic acids.  Paper chromatographic  examination of the neutral sugars showed D-xylose to be the major component with small amounts of other sugars corresponding to rhamnose, arabinose and galactose also being present.  Two other  sugars with R values greater than that of xylose were found but f  the identity of these components has not been established.  An  aldobiouronic acid was isolated and characterized as the crystalline acetate of 2-0-(li-0-methyl-«G-D-glucopyranosyluronic acid)-Dxylose. In order to determine the mode of union of the compon ent sugars the polysaccharide was methylated and then hydrolyzed to give 2,3,li-tri-O-methyl-D-xylose, 2 , 3 di-O-methy1-D-xylose, 2-0-(2,3-dl-O-methyl-D-xylopyranosyl)-2,3-dl-0-methyl-D-xylose, a dimethylated lyxose,  2-0-  and 3-0-methyl-D-xylose, 2 - 0 - ( 2 , 3 , 4 -  tri-O-methyl-D-glucopyranosyluronic acid)-D-xylose, and probably a trimethylated rhamnose.  (The dimethylated lyxose isolated i s  not an integral constituent of the native polysaccharide since no lyxose was obtained i n the acid hydrolyzate.  It i s thought  to arise by epimerization of 2,3-di-0-methyl-D-xylose.)  Quanti-  tative analysis of the methylated hemicellulose has shown the t r i - , d i - and monomethyl pentose and 2,3,4-tri-O-methyl-D- d-  glucuronic acid to be present i n the mole ratios of 1:97:21:19, respectively. The methyl ester of the methylated aldobiouronic acid was reduced with lithium aluminum hydride and the resulting neutral disaccharide hydrolyzed.  The cleavage products were  identified as 3-0-me thyl-D-xylose and 2,3, Ij.-tri-0-methyl -D-glucose indicating the uronic acid portion of the molecule to be linked through its reducing end to position 2 of a xylose moiety. Results obtained In this work show that the carbohydrate polymer isolated from apple wood consists of a backbone of approximately 119 anhydro-D-xylopyranose linked by 1,1}.- glycosidic bonds.  The side chains are composed of single units  of If.-0-methyl-D-glucuronic acid which occur at every sixth xylose residue.  A rhamnose unit may perhaps be present as a side chain  too; although i t is not known whether this sugar is an integral constituent of the glucuronoxylan. The general features of the hemicellulose are very similar to glucuronoxylans isolated from other hardwoods and especially resemble white elm and cherry wood in Its structure and high uronic acid content. We are grateful to Dr. C. T. Bishop who presented this work at the lj-3rd conference of The Chemical Institute of Canada, Ottawa, June I960.  ACKNOWLEDGEMENTS  To Dr. G. G. S. Dutton I wish to express my deep appreciation for his excellent guidance and encouragement throughout the course of this work. I also wish to thank Dr. T. E. Timell for samples of 2-0-methyl-D-xylose and of the crystalline acetate of 2-0-(lj.-0-methyl-U>D-glucopyranosyluronic acid)-D-xylose.  TABLE OP CONTENTS Page No. I.  HISTORICAL INTRODUCTION  II.  APPLE WOOD HEMICELLULOSE  23  EXPERIMENTAL  34  III.  . . . .  1  A.  ISOLATION OP APPLE WOOD HEMICELLULOSE . . .  34  B.  HYDROLYSIS OP APPLE WOOD HEMICELLULOSE  35  C.  SEPARATION OP THE ACIDIC COMPONENT OP APPLE  . .  WOOD HEMICELLULOSE D.  36  PREPARATION OP THE CRYSTALLINE DERIVATIVE OP 2 - 0 - (4-0-METHYL-cC-D-GLUCOPYRANOSYLURONIC AC ID)-D-XYLOPYRANOSE  37  1.  The Methyl Ester Methyl Glycoside . .  2.  Methyl 2 - 0 - Methyl  37  (2,3-di-O-acetyl-  4-0-methyl-oC-D-glucopyranosyl) uronate 3,4-Di-O-acetyl-D-xylopyranoside  . .  38  E.  IDENTIFICATION OF D-XYLOSE  38  F.  ACETYLATION OF APPLE WOOD HEMICELLULOSE . .  39  G.  METHYLATION OF APPLE WOOD HEMICELLULOSE . .  40  1.  Methylation I  40  2.  Methylation II  4l  3.  Methylation III  .  42  H.  FRACTIONATION OF THE METHYLATED HEMICELLULOSE 42  I.  METHANOLYSIS OF METHYLATED APPLE WOOD HEMICELLULOSE  J.  46  SAPONIFICATION OF METHYL ESTER OF THE ALDOBIOURONIC ACID  46  Page No. K.  SEPARATION OP THE ACIDIC COMPONENT OP METHYLATED APPLE WOOD HEMICELLULOSE . . . .  L.  1+7  PREPARATION OP METHYL 2-0- (2,3,l+-TRI-0METHYL-D-GrLUCURONOSYL)-3-O-METHYL-DXYLOSIDE METHYL ESTER  1+7  1.  Preparation of Diazomethane  2.  Preparation of Methyl Ester of Methyl  1+7  Glycoside of the Aldobiouronic Acid . M.  t|-T  REDUCTION OP THE METHYL ESTER OP METHYL 2-0(2,3,1+-TRI-0 -METHYL-D-GLUCURONOSYL)-3-0METHYL-D-XYLOSIDE  N.  1+8  HYDROLYSIS OP METHYL 2-0-(2,3,l+-TRI-0-METHYLD-GLUC0PYRAN0SYL-3-0-METHYL-D-XYL0SE 1. 2.  0. P.  . . .  1+9  Identification of 2,3,i+-Tri-0-MethylD-Glucose  50  Identification of 3-0-Methyl-D-Xylose  50  SEPARATION OP THE NEUTRAL COMPONENTS OP METHYLATED APPLE WOOD HEMICELLULOSE . . . .  5l  IDENTIFICATION OP THE COMPONENTS  53  1.  Component 1, Tri-O-Methyl-Rhamnose .  2.  Component 2, 2,3,l+--Tri-0-MethylD-Xylose  3.  Component 3 ,  53  53 l+-0-(2,3-di-0-methyl-D-  xylopyranosyl)-2,3-di-0-methyl-xylose  53  1+.  Component 1+, 2,3-Di-O-Methyl-D-xylose  51)-  5.  Component 5 , Di-O-methyl-Lyxose a. Demethylatlon of Di-o-methyl-  51+  Pentose  51+  Page No. p.  IDENTIFICATION OF THE COMPONENTS (continued) 6.  7. Q.  Component 6, 2 - 0 - and 3-0-MethylD-xylose  55  Component 7, D-Xylose  55  QUANTITATIVE COMPOSITION OF APPLE WOOD HEMICELLULOSE  BIBLIOGRAPHY  .: .  55 58  LIST OF TABLES Page No. TABLE IA FRACTIONAL PRECIPITATION OF METHYLATED APPLE WOOD HEMICELLULOSE II  1*1+.  TABLE IB FRACTIONAL PRECIPITATION OF METHYLATED APPLE WOOD HEMICELLULOSE III  1*5  TABLE II SEPARATION OF METHYLATED SUGARS of APPLE WOOD HEMICELLULOSE  52  TABLE III QUANTITATIVE ANALYSIS of METHYLATED SUGARS of APPLE WOOD HEMICELLULOSE by PHENOL-SULPHURIC ACID METHOD  57  I  HISTORICAL INTRODUCTION Of universal occurrence, hemicelluloses constitute a large group of polysaccharides whose abundance, as far as organic material is concerned, is surpassed only by cellulose. Hemicelluloses, and i n particular xylans, are commercially valuable as a source of furfural and furan derivatives.  They  are said to be of value when present In small quantities i n paper pulps.  Accordingly, where once these carbohydrate poly-  mers were regarded as a nuisance in the pulping of wood, they now represent a r i c h , almost untapped source of raw material. As early as 1891, Schulze (1) obtained, by alkaline extraction of plant material, a group of carbohydrate polymers which were more readily hydrolyzed than cellulose.  Because of  their chemical and physical similarities to cellulose, he designated the group hemicellulose.  In the classic work by  O'Dwyer (2), i n 1926, polysaccharides which were precipitated from alkaline solutions by simple acidification and by addition of ethanol- to the f i l t r a t e were designated as hemicellulose A and hemicellulose B, respectively.  Subsequent investigations  by other workers show inconsistent terminology.  For example,  hemicellulose A has also been described as the fraction extracted with 1$ potassium hydroxide and hemicellulose B as the fraction  2. extracted from the residue with 2h\.% sodium hydroxide (3). With the advent of modern techniques such as paper chromatography and electrophoresis, the f i e l d of polysaccharide chemistry has been vastly augmented.  No standard nomenclature  for these macromolecules i s , as yet, available and c l a s s i f i c a tion of polysaccharides varies with the investigator.  Incon-  sistencies in terminology appear in the literature mainly due to this lack of standard nomenclature and classification. Systematic classification and nomenclature, based on normal carbohydrate nomenclature, has been attempted although It has not been generally accepted.  Hemicellulose have been  divided into three fractions: (1) pentosans, (2) polyuronides, including molecules composed of hexose and/or pentose sugars and uronic acids (the term "polyuronide", is a misnomer since It implies that a substance i s composed of uronic acid residues when, i n fact, the polymer consists of chains of hexoses or pentoses with a few uronic acid units joined through i t s hemiacetal hydroxyl to the other hemicellulosic constituents), and (3) cellulosans consisting of xylan, mannan, and perhaps a "glucan" (1+.).  The term non-cellulosic cell-wall polysaccharide  has been suggested ( 5 ) , but i t is admitted that the term f a i l s because the dividing line between true cellulose and hemicellulose is arbitrary.  Other workers consider this group of  polymers to be a mixture of unmodified glycans composed of either pentose or hexose sugar units and of modified glycans containing one or more glucuronic acids joined to the main chain of the molecule (6).  Thus, for example, i n a classification  such as the latter, the hemicellulose group of polysaccharides  can be classified into (1) true xylans, (2) araboxylans, (3) glucuronoxylans, (ii) araboglucuronoxylans, and ( 5 ) complex xylans. A wide range of molecular sizes and shapes exist among these carbohydrates from different plants and from different parts of the plant.  Due to these differences and  differences in their acidic properties, hemicellulose vary widely among themselves i n solubility.  Consequently, i t is  d i f f i c u l t to prepare a pure molecular type by the fractionation procedures used to date.  It apparently seems, however, that  many tissues contain not more than three or four polysaccharide types which differ primarily In molecular weight. Pretreatment of plant materials i s necessary for the isolation of hemicelluloses. and delignification.  This includes solvent extraction  There are certain plant constituents  which occur in association with the polymeric carbohydrate material which are generally extractable with alcohol-benzene azeotrope and consist of compounds such as l i p i d s , waxes, resins, organic acids, free sugars etc.  (7).  Pectin and pectic substances are sometimes removed prior to hemicellulose extraction especially when dealing with plant materials containing a large amount of these substances, such as the cambium layer of wood or the leaves and stems of plants.  Extraction with 0.$% solutions of ammonium oxalate  removes water insoluble pectic substances i f they are not trapped or chemically bound to plant materials  (8).  Hemicelluloses and lignin occur, in close association with cellulose.  Their apparent function appears to be  that of cementing together cellulose fibers thus imparting strength to the plant tissue.  The presence of lignin in plant  tissue presents a problem since i t retards the isolation of hemicellulose.  This d i f f i c u l t y arises presumably because of  mechanical obstruction or because of, as yet, unknown covalent bonding between the lignin, thought to be polymeric phenylpropane units, and the hemicellulose.  The former is soluble  in alkali and thus hampers the purification of hemicelluloses by extraction with aqueous a l k a l i . Many procedures have been proposed for the removal of lignin from plants.  In one of the earlier methods of deligni-  fication, plant material was extracted with $0% ethanol containing 1% sodium hydroxide ( 9 ) . This method, however, Is not recommended since at boiling temperatures, acid containing polyuronides are degraded (1G). the lignin is retarded at lower temperatures.  the uronic Removal of Several other  techniques have been developed for the selective removal of lignin but the method developed by Jayme (11) and improved by Wise and co-workers (12) is most widely used.  Selective removal  of the lignin i s achieved by treating the wood sample with acidified sodium chlorite followed by subsequent extraction of the hemicellulose with a l k a l i . For deciduous plants, a simpler method of delignification has been developed by McDonald (13) In which 0.1N sodium hydroxide was found to remove a major portion of the alkali  5.  soluble impurities.  The loss of lignin was found to be  approximately constant above an alkali strength of 0.1N so that pentosan removal with IN sodium hydroxide was facilitated. Since the time of Schulze (1),  alkaline extractions  have been the, principal method used for Isolating the hemicellulose from plant materials.  Sodium and potassium  hydroxides of varying strengths have been employed. An alkali concentration of IN is the most commonly used but the ideality of the concentration has been found to differ among different plants.  For example, Wise and co-workers (ill)  showed that the solubility of hemicellulose of slash pine holocellulose increases as the potassium hydroxide concentration of the extracting solution increases with optimum solubilization obtained at  In most cases, concentrations of alkali  higher than 16% result In small additional solubilization of hemicellulose. I n i t i a l subdivision of polysaccharide mixtures have been obtained by fractional precipitation.  Acidification of  alkali extract was found to precipitate, in some cases, a large portion of hemicellulose, termed hemicellulose A (2).  This  procedure takes advantage of the fact that high molecular weight polysaccharides precipitate f i r s t while lower molecular weight polymers and uronic acid containing ones are precipitated by the addition of excess alcohol. Further purifications have been achieved by complexing the hemicellulose in aqueous solutions with copper compounds (15).  Sometimes more efficient separation of polysaccharides  are attained by the fractional precipitation of their acetyl or methyl derivatives  (16-18).  Other workers have used organic  solvents for obtaining fractionation ( 1 9 ) , while s t i l l others have employed inorganic salts, for example ammonium sulphate ( 1 8 , 2 0 ) , selective extraction with aqueous ethanol ( 2 1 ) and precipitation with Getavalon (cetyltrimethyl ammonium bromide) (22).  The above described methods for obtaining hemicelluloses have proved very useful but i t must be borne i n mind that degradation occurs when strong oxidizing agents such as chlorine and chlorite are employed.  Degradation i s  also known to occur i n the presence of strong a l k a l i .  Hence,  polysaccharides obtained by chemical means may not necessarily be that found i n the native polysaccharide material.  Also,  no method i s as yet available for determining, unequivocally, the homogeneity of polymeric fractions although steps have been taken in this direction by the use of electrophoresis (23-27). Acid hydrolysis, followed by qualitative and quantitative  a  nalysis, give an insight into the nature of the sugar  residues present i n the macromolecule and the proportions i n which they are present.  Modern chromatographic techniques  have opened new horizons i n carbohydrate chemistry and chromatographic identification of monosaccharides, after hydrolysis, are identified without the tedious task of synthesizing derivatives, as least for the f i r s t analysis.  Quantitative determina  tion of mono sa:c char ides is also facilitated by chromatography  7.  and colorimetry ( 2 8 ) . In determining the architecture of the macromolecules, applications of various analytical techniques are involved i n cluding methylation, graded hydrolysis, enzymatic degradation and periodate oxidation--all require the use of chromatography. Methylation, a procedure developed approximately sixty years ago, s t i l l proves to be the most powerful weapon for determining the mode of linkage in carbohydrate polymers. Methyl ether derivatives of the polysaccharides are obtained when subjected to Haworth methylations ( 2 9 ) . This involves the treatment of material with dimethyl sulphate and a l k a l i . Following this, reaction with methyl iodide and silver oxide (Purdie methylation) generally produces a completely methylated polysaccharide ( 3 0 ) . Modifications to the above procedures have been made to expedite this step of the analysis ( 3 1 - 3 3 ) .  Hydrolysis of the methylated polysaccharide  and identification of the cleavage products reveal the nature of the linkage in the polymers.  Information with regard to  the number of end groups, linearity, or branched nature is also obtained.  This procedure does not, however, reveal the  sequence of the sugar residues in the molecule. Partial hydrolysis and graded hydrolysis are rapidly becoming useful techniques for determining the order of linkage.  Both chemical and enzymatic depolymerization  have been employed ( 2 6 , 3^-1+0). Much useful information can be derived from the study of periodate on the macromolecules.  End group determinations,  8.  the nature and the number of residues so linked that they are not attacked by periodate, and the proportions of residues giving rise to dialdehydes can a l l be dealt with in this way (ii2). It is practically impossible to completely methylate a l l the free hydroxyl groups present in a large polysaccharide.  Similarly, i t is d i f f i c u l t to determine the  extent of over- or under-oxidation in the periodic oxidation of polysaccharides (lj.2).  In addition to chemical and  enzymatic degradation, data as to the kinds of monosaccharides present are required, this being obtained by complete hydrolysis of the molecule.  It is possible to offset some of  the problems involved in correctly Interpreting the results obtained i f the analytical methods described above are used in conjunction with each other. In this present work a hemicellulose consisting' primarily of D-xylose units was investigated.  Therefore,  works on xylose-containing polysaccharides w i l l be reviewed. Xylans occur i n practically a l l land plants and are said to be present In some marine algae (li3,J|li).  They are  the most abundant of the group of hemicelluloses which include xylans, glucoraannans, mannans, arabogalactans etc.  and are  particularly abundant i n agricultural residues such as corn cobs, corn stalks, grain hulls and stems, and these materials have been extensively investigated.  Amounts ranging from  15-30$ of xylan have been obtained.  Mood xylans are found in  smaller amounts in comparison to annual crops.  Hardwoods are  found to contain 20-25?$ xylan while softwoods contain 7-12$ (US).  Xylans have the general properties of insolubility in water, solubility in alkaline solutions, ease of acid hydrolysis, high negative optical rotation and non-reducing actions toward Fettling s solution. 1  three general classes:  They f a l l into either of  (1) pentosan (2) glycan or (3) hemi-  cellulose because they are composed almost entirely of pentose units, are largely a polymer of unmodified sugars and are extractable by hemicellulose extraction procedures (6). Upon acid hydrolysis, they release a variety of sugars and hexuronic acids, namely, D-xylose, 2-0- and 3-0methyl-D-xylose, L-arabinose, L-rhamnose, 3-0-methylL-rhamnose, D- and L-galactose,  D-mannose, D-glucose,  L-fucose, D-galacturonic acid, D-glucuronic acid and U-O-methyl-D-glucuronic acid.  On the basis of their chemical  constitution, therefore, further subdivision of this large class of polysaccharides may be made.  Thus, the hemicellulose  group of carbohydrate polymers can be classified as (i) true xylans, ( i i ) araboxylans,  ( i i i ) glucuronoxylans, (iv) arabo-  glucuronoxylans and (v) complex xylans (1+6). True xylans are polysaccharides composed solely of anhydroxylopyranose units.  This type of hemicellulose from  land plants is not too common and only two such examples are known, to date, to be completely devoid of other sugar residues, those from esparto grass (I}.7) and tamarind seed (1+8).  10.  Esparto grass, which has been studied extensively, was originally thought to be composed of D-xylose and L-arabinose residues (1+9).  After hydrolysis of the f u l l y methylated  derivative of the xylan, the principal products obtained, i n this early work, were 2,3 di-O-methyl-D-xylose and 2 , 3 , 5 - t r i O-methyl-L-arablnose roughly i n the proportions of 20:1.  The  polysaccharide was, therefore assumed to consist of l,Jilinked D-xylopyranose residues as evidenced by the isolation of the dimethyl pentose.  L-Arabinose units were considered  to exist as terminal non-reducing end groups joined to the main chain of D-xylopyranose units. Further investigations have revealed that L-arabofuranose units are, in fact, not an integral part of the molecule and that rather, one of the hemicelluloses from this plant is actually a true xylan.  Chanda and co-workers (Ji?)  have, by vigorous fractionation of the esparto grass pentosan, isolated a xylan free of other sugar residues.  The ready  formation of an insoluble copper complex (15) by the hemicellulose was taken advantage of.  (This method of fractiona-  tion is gentle enough chemically not to disturb the labile arabofuranosyl linkages should they be present in the molecule.) Re-examination of the pure polysaccharide was shown to contain no detectable amount of L-arabinose.  Investigation of the  arabinose-free material by standard methylation procedures and hydrolysis clearly showed i t to be a true xylan composed of Ifh-f  linked D-xylose units with  a  terminal non-reducing  xylose residue every liO-repeating units.  Isolation of  11.  2-0-methyl-D-xylose as a cleavage product of the methylated xylan indicated that perhaps the side chain xylose was linked 1,3-^5  to the main chain.  A portion of the molecule may be represented as Xyl l  I+- (S - X y l l  p  ,  Xyl l  .  p  1+- f -D-Xylpl—  1  1+- - X y l l  p  p  Another pure xylan of the type i n esparto grass was isolated from tamarind seeds (1+8).  It i s quite similar to the  former i n that a non-reducing end group i s present for every 35> ± 5 D-xylose residues with branching through carbon 3 of the xylose of a linear chain. An interesting xylan of the above type was found to exist i n marine red alga, Rhodymenia palmatta  (1+4).  It i s a  true xylan since on acid hydrolysis only D-xylose Is obtained. However, structural determination Indicated a novel feature of this polysaccharide. bone of  1,1+-/?  In addition to the common linear back-  linked D-xylose residues, isolation of  O-methyl-D-xylopyranose suggested the presence of  2,1+  1,3-|S  di-  linked  D-xylose chain, these being present i n the proportions of i+:l, respectively.  The main chain, containing these two types of  backbone, i s terminated by a xylose unit, the whole chain consisting of 17 xylose units. order i n which the linear molecule.  1,1+.-  Nothing i s yet known concerning the  and 1,3-jS  links are arranged i n the  12. Araboxylans, as the name suggests, are polymers composed of arablnose and xylose residues only.  Such a polymer  was isolated from wheat flour and the L-arablnose was shown conclusively, by Perlin (lj.0), to be an integral constituent of the xylan.  Furthermore, he showed that a l l the L-arabinose  units i n wheat flour pentosan were present as non-reducing endgroups in the furanose form.  Graded hydrolysis of the soluble  pentosan of wheat flour readily liberated three-quarters or; more of the arablnose units leaving a water-insoluble araboxylan. Methylation and periodate oxidation of the pentosan showed that i t consisted of D-xylopyranose units linked l,k-f • From this chain, radiated L-arabofuranose residues attached at the 2 or 3 - positions of the xylose framework. The hemicellulose of the endosperm of wheat (50), gave on hydrolysis of the methylated polysaccharide, 2,3,5-tri-0-methylL-arabinose, 2,3-di-0-methyl-D-xylose, 2-0-methyl-D-xylose and D-xylose in the molar ratio of 3 : 1 9 : 6 : l i .  The "squeegee" fraction  of wheat flour (39) on similar treatment gave the sugars in the ratio of lli:2l4.:7:i\..  Again, a backbone of  xylose units is suggested.  linked anhydro-  In both cases, the hemicelluloses  are of a highly branched nature since comparatively large amounts of D-xylose and 2-0-methyl-D-xylose are obtained.  Side chains  of L-arabofuranose residues emanate from the framework and are joined to positions 2 and/or 3 of the latter.  A high proportion  of arabinose in the araboxylan complex appears to be a general feature of hemicelluloses of wheat grains. The water soluble polysaccharide from rye-flour (51)  13.  was found to be similar to those from wheat with the exception that branching occurred primarily through carbon 3 of the xylose moiety whereas in the wheat pentosans branching was observed through carbons 2 and 3« A study of the arabinose-rich fraction of esparto grass hemicellulose also yielded araboxylan.  In addition to  D-xylose and L-arabinose, however, Aspinall and co-workers  (52)  found that D-glucose and D-galactose were also present in the hydrolyzate of the hemicellulose.  Isolation of 2,3,5  tri-O-  methy1-L-arabinose, 2,3.,-di-0-methyl D-xylose and 2-0-methyl-Dxylose (1:3:1) together with small traces of dlmethylated arabinose, tetra-, t r i - and di-methyl galactose suggest that perhaps mixtures of polysaccharides exist.  This information,  together with the previous isolation of a true xylan (ij.?) i n dicate that esparto grass hemicellulose Is a mixture of at least two and perhaps three different polysaccharide entities. Many polysaccharides of the xylan group contain Dglucuronic acid or U-O-methyl-D-glucuronic acid. Aldobiouronic acids, consisting of a glycosiduronic acid and a sugar are particularly resistant to acid hydrolysis.  Consequently, they  can be isolated by graded hydrolysis of xylans and other acidic polysaccharides.  The mode of linkage between the two moieties  can be established by identification of the hydrolysis products of the methylated derivatives.  Thus, Jones and Wise (53)  isolated the aldobiouronic acid from aspenwood. of the acid proceeded as follows:  Identification  the acid resistant acidic  portion of the hemicellulose was subjected to prolonged acid hydrolysis.  Chromatographic examination revealed the presence  111. of D-xylose and 4-0-methyl-D-glucuronic acid.  Methylation of  the aldobiouronic acid, followed by hydrolysis, yielded  2,3,4~  tri-O-methyl-D-glucuronic acid and 3,4-di-O-methyl-D-xylose. Reduction of the aldobiouronic acid with lithium aluminum hydride  (54>55)  and methylation afforded 2 - 0 - ( 2 , 3 , 4 , 6 - t e t r a -  0-methyl-^ -D-glucopyranosy])-3,4~di-0-methyl-D-xylopyranose. A high positive optical rotation of the disaccharide indicated the glycosidic linkage between the pentose and the hexuronic acid moieties to beoC and the acid present i n aspenwood hemicellulose was therefore 2-0-(I4.-O-methyl-<C -D-glucopyranosyluronic acid)-D-xylopyranose. That the configuration of the glycosidic linkage i n 4-0-methyl-D-glucopyranuronosyl-aldobiouronic acids was, i n fact, oC was proved conclusively by Gorin and Perlin ( 5 6 ) .  Th©  2-0-(li-O-methyl-D-glucopyranosyl uronic acid)-D-xylose was converted to 2-0-(Ii-O-methyl-D-glucopyranosyl)-glycerol and then methylated to yield the hexamethyl ether derivative.  A compari-  son of i t s infrared spectrum and specific rotation with those of the synthesized hexamethyl ethers of 2-0-oC and 2 - 0 - ^ -Dglucopyranosyl-glycerol showed the configuration of the oxidized product to be °C . A crystalline acetate derivative of this acid has been prepared by Timell ( 5 7 ) and the neutral methylated disaccharide has been prepared by Dutton and Smith ( 1 6 ) .  The  {§ anomer of the glycosiduronic acid, has been synthesized and characterized'by Bowering and Timell ( 5 8 ) .  The hexuronic acid,  15.  4-0-methyl glucuronic acid, has been obtained by Gorin (59) by the oxidation of the aldobiouronic acid with lead tetraacetate. The unmethylated aldobiouronic acid, 2-0(oC-D-glucopyranosyl uronic acid)-D-xylose, has been isolated by graded hydrolysis. plants.  Some less common acids have also been found In  Wheat straw (60) and sunflower heads ( 6 l ) studied by  Bishop, and pear c e l l wall (62) studied by Chanda et al were found to contain 3-0(«C-D-glucopyranosyluronic acid)-D-xylose, while New Zealand pine (63) contained the 4- "thyl ether derime  vative, 3-0-(ij.-0-methyl-oC-D-glucopyranosyluronic acid)-Dxylose.  An even more rare glycosidic linkage, 1,4-> was ob-  served In hemicellulose B of corn cob (64) as l|.-0-(«C -D-glucopyranosyluronic acid)-D-xylose. In a l l polysaccharides examined so far, the hexuronic acids have been shown to be linked to the xylan backbone as single unit side chains.  If the glycosiduronic acid were present as  terminal end groups as illustrated" below then the 2,3-di-O-methyl4 - 0 - or 3 , 4 di-0-methyl-2-0-(2,3,4-tri-0-methyl-D-glucuronic acid) -D-xylose would be obtained. D-GpA  1  In a l l cases this was not observed. or  4-D-Xylpl  D-G-pA  1  2-D-Xyl  p  Glucuronoxylans are most commonly found in wood and occasionally in graminae.  The constitutional analysis of the  hemicellulose of wheat straw, which had previously been obtained free from arabinose by extraction with hot 70% aqueous alcohol was shown to consist of approximately 40-45 anhydro-D-xylose units linked 1,4-0  in  a  chain to which a D-glucuronic acid was  16.  attached at carbon 2 of a xylose moiety (21).  Araboglucurono-  xylans have also been isolated from wheat straw as w i l l be seen later. Kapok (65) and milkweed floss (66),  examined by  Timell and co-workers were found to be quite similar except that kapok contained twice as many aldobiouronic acid units as milkweed floss.  Pear cell-wall (62) glucuronoxylan also  showed structural similarities with the exception that the acid fragment was linked to carbon 3 of a xylose unit instead of carbon 2 as in the previous two examples. Hemicelluloses from wood constitute a large proportion of polysaccharides which can be classed as glucuronoxylan. hemicellulose A of European beechwood (Pagus sylvatlca)  Thus,  (67)  gave on hydrolysis D-xylose and U-O-methyl-D-glucuronlc acid. Methylation and periodate oxidation studies showed that the molecule consisted of approximately 70  ~L,k--fi  linked D-xylopyra-  nose units with every tenth unit carrying a uronic acid residue at carbon 2 of a xylose unit.  Norway spruce (68) was found to  consist of 80 ± 5 xylopyranose residues linked i n a similar fashion but with every 5th xylose residue carrying a side chain of [|.-0-methyl-D-glucuronic acid linked through carbon 2. The water soluble fraction of American beechwood (Fagus grandifolia) (69) is a smaller molecule containing ij.5 pentose units glycosidically linked l,k f m  with branching at  carbon 2 and hence the molecule has 2 non-reducing end groups and one reducing end group.  Five units of ij.-0-methyl-D-glucuro-  nic acid are joined as single terminal side chains to the xylose unit of the main structure by 1,2 bonds.  17.  The pentosan fraction from Loblolly pine, studied by Jones and co-workers (70-72) was found to be essentially the same as European beechwood.  Seven xylose residues were present  for one 4-0-methyl-D-glucuronic acid with ten repeating units per molecule.  Branching may occur through position 2 of xylose.  The xylan fraction of hemicellulose of white elm (73,74) consists of a backbone of 1,4"/  linked anhydroxylspura-  nose units with a 4-0-methyl glucuronic acid glycosidically linked 1,2- to the backbone.  It i s essentially a linear chain  composed of 185 pentose residues, every 7th one containing an acid residue.  White birch (75,76) (Betula papyrlfera) has a  similar structural pattern with the exception that every eleventh xylose residue carries a 4-0-methyl-D-glucuronic acid residue.  Sugar maple (77), again shows similar architecture  but i t i s slightly larger, the molecule containing 200 xylose units with every tenth unit carrying an acid side chain. An araboglucuronoxylan Isolated from corn cobs has been extensively studied.  By partial degradation of the pento-  san, Whistler and co-workers have isolated a homologous series of oligosaccharides from xylose to xyloheptaose (78,79) a l l with the normal 1 , 4 - / links; nose (80);  2-0-GC-D-xylopyranosyl-L-arabl-  three aldobiouronic acid fragments, namely, 2 - 0 -  (4-0-methyl-oC -D-glucopyranosyluronic acid)-D-xylose  (8l),  4-0-«-D-glucopyranosyluronic acid)-D-xylose (64), and 2-0( oC-D-glucopyranosyluronic acid)-D-xylose (64); and an aldotriouronie acid, 0-oC -D-glucopyranosyluronic acid ( l , 4 ) - 0 - ^ -  18.  D-xylopyranosyl- (1,1*.)-D-xylose (82).  This and other data ( 8 3 ) ,  show that corn cob xylan has a backbone of l , i H $  linked xylose  residues to which are attached side chains of D-glucuronic acid, 4-0-methyl-D-glucuronic acid, and L-arabinose residues.  Ten  side chains of L-arabofuranose are proposed to exist for every sugar units and one mono-methyl D-glucuronic acid for each 9-11 sugar units. Aspinall and co-workers ( 2 1 ) , in 195>4» obtained a glucuronoxylan by fractionation with hot 70% alcohol which contained only a trace of arabinose.  Examination of the arabinose-  rich fraction of wheat straw hemicellulose ( 8 i i ) , by the same group in 19^6, revealed a structure in which this sugar was an integral part of the polysaccharide and that this same sugar occurred exclusively as end-groups in the furanose form.  These  end-groups are postulated to occur as side chains linked glycosidically through carbon 3 of a xylose residue with the glucuronic acid linked through carbon 2 .  The structural pattern  of this araboglucuronoxylan has been depicted as: D-Xyl l p  -itf-D-Xylpl 3  p  l  V 4-D-Xyl  p  -l^D-Xylpl 3  1  L-Arab  J^D-Xylpl  kF> X y l l 2 p  4fD-Xyl  i f  D-G A p  The polyuronide of wheat straw examined by Adams (3b) was proposed to consist of approximately 32 anhydro-D-xylose units for every 5 anhydro-L-arabofuranose and 3 D-glucuronic acid units. This hemicellulose has the distinction in that the acid fragment is glycosidically linked 1 , 3 - to the backbone ( 6 0 ) .  In order to  p  19.  prove that L-arabinose was indeed an integral part of the molecule and does not originate from an araban, Bishop and Whitaker ( 8 6 ) carried out degradative studies to isolate an oligosaccharide containing arablnose and xylose.  A cellulo-  l y t i c enzyme from mold, Myrothecium verrucaria, was used for depolymerization.  The enzyme preparation was found to hydro-  lyze linear chains of  l,k--f  linked xylose chains and a series  'of oligosaccharides containing D-xylose and L-arabinose (dito heptasaccharides)  were isolated.  A trisaccharide was  characterized as 0-(«C or jtf )-L-arabofuranosyl(l,3)-0-/£ -D(xylopyranosyl-(l,li)-D-xylopyranose ( 3 8 ) .  These results demon-  strate that an araboxylan is present and that isolation of 2-0-methyl-D-xylose arose from branch points in the molecule. Wheat leaf polyuronide is structurally similar to wheat straw polyuronide studied by Adams ( 8 7 ) .  Hemicelluloses of barley  husk ( 8 8 ) and oat-straw xylan ( 8 9 ) also show similarity. Wheat bran, which was fractionated to remove L-arabinose present as araban (34>90), was shown electrophoretically to consist of D-glucuronic acid, D-xylose and L-arabinose ( 3 5 ) . Enzymatic degradation showed the molecules to be linear with 7 - 8 xylose residues per hexuronic acid units.  Isolation of  mono-, d i - , and t r i methylated arablnose indicated the araboglucuronoxylan to be highly branched although the xylan portion of the molecule was essentially linear and similar to those found in other wheat plants. The xylan fraction of western hemlock (Tsuga heterophylla) was studied in detail by Dutton and Smith.  Hydrolysis  20. of the hemicellulose gave L-arabinose, D-xylose, D-galactose, D-glucose, D-mannose and an aldobiouronic acid 4-0-(4-0-methyl• -D-glucopyranosyluronic acid)-D-xylose (91).  The constitu-  tion of the xylan polysaccharide, based on methylation studies (16), reveals a branched structure: D-Xyl l p  W-f*-Xyl lj p  4-0-  4 f -D-Xyl l 3 3 p  Me-oC-DG A  Arab  [k-fi - D - X y l l  k-fi - D - X y l l  p  p  f  From mole ratios obtained by quantitative analysis, the polysaccharide was shown to contain three aldobiouronic acid r e s i dues associated with about 13 xylose units.  This high propor-  tion of uronic acid residues is a distinguishing feature of the hemicellulose of western hemlock.  The pentosan fraction of  European larch (Larix decldua) (92) contains one hundred 1,4linked D-xylose, every f i f t h or sixth xylose residue carrying a terminal 4-0-methyl-D-glucuronic acid linked through position 2 and L-arabinose attached to position 3 of xylose. In addition to D-xylose, L-arabinose and hexuronic acids, other neutral sugars are occasionally present in some hemicelluloses.  These polysaccharides are characterized by  the complexity of their chemical architecture.  These hemi-  celluloses are generally highly branched. Corn hull hemicellulose, which shows promise as an adhesive, thickener or stabilizer is a by-product of the corn milling industry.  It has been a subject of intensive investi-  gation in respect to its chemical constitution and structure  21.  by Smith and co-workers and Whistler and co-workers.  On acid  hydrolysis D-and L-galactose has been obtained besides the usual sugar residues.  Cleavage of the methylated polysaccharide has  yielded the following products,  2,3,5-tri-O-methyl-L-arabinose,  3,5 -di-O-methyl-L-arabinose, 3-0-methyl-L-arabinose,  tetra-O-methyl-D and L-galactose,  2,3,1+,6-  2,3,l+--tri-0-methyl-D-xylose,  2,3-di-O-methyl-D-xylose, 3-0- and 2-0-methyl-D-xylose, 2-0-£,3,l+-, tri-O-methyl-D-glucuronic acid)"3-0-methyl-D-xylose (93). hydrolysis gives a number of neutral and acidic  Graded  oligosaccharides  including 2-0-(oC -D-glucopyranosyluronic acid)-D-xylose (37), 3_0-of -D-xylppyranosyl-L-arabofuranose, O-L-galactopyranosyl (1,1+.)-  O-xylopyranosyl-(1,2)-L-arabinose, l+.-0-( § -D-galactopyranosyl)-/^ D-xylose (93), 1+.-0- f -D-xylopyranosyl-D-xylose and 5-0-(D-galactopyranosyl)-L-arabofuranose  (91+.).  These data show a complex architecture which is unique. Isolation of the tetramethyl galactose indicates that a l l the galactose residues constitute the non-reducing ends, a fact which is supported by the isolation of oligosaccharides containing galactose.  Unlike other hemicelluloses where L-arabinose is  invariably found as non-reducing end groups, a few such units occupy a non-terminal position evidenced by the isolation of monand d i - methylated arablnose.  The oligosaccharides provide evi-  dence that the hemicellulose from corn hulls has a highly branched structure and that a variety of branches emanate from the main xylan chain.  Further, that some of the xylose residue have multiple  branching is indicated by the identification of xylose in the hydrolysis product of the methylated polymer.  22.  The hemicellulose of flax straw (Linum Usitatissimum S  P«)  ( 9 5 , 9 6 ) was shown to contain L-rhamnose.  Hydrolysis of the  methylated polysaccharide produced a dimethylated L-rhamnose so that either a rhamnoaraboglucuranoxylan is present or an araboglucuronoxylan with impurities of rhamnose*  The latter possibili-  ty is illustrated as /J-D-Xyl 1  f Ji-/-D-Xyl 1  i+-^-D-Xyl 1  l ^ - D - X y l 1-  P 1  ij.-0-Me-oC-D-G A P Should the former be the case, then rhamnose is joined 1 , 3 by glycosidic bonds to the main chain in which case this portion of the molecule may be represented as 4-D-Xyl 1 p  3 L-Rha 1 P  i+-D-Xyl 1— p  Wheat straw xylan, studied by Ehrenthal and co-workers ( 8 5 ) , gives as a hydrolysis product of the methylated polysaccharide 2,6-di-0-methyl-glucose besides the mono-, d i - and tri-  methyl xyloses and L-arabinose.  The dimethylated glucose  is either a 1 , 3 and 5 linked glucofuranose or a 1 , 3 and I), linked glucopyranose.  Periodate oxidation followed by reduction of the  resulting polyaldehyde with Raney nickel yielded a polyalcohol which on hydrolysis gave no glucose.  This evidence pointed to  a 3 , 4 " linked glucopyranose unit, the reducing end being free.  22, p^-D-Xyl -l}y-I|.-D-Xyl l  [k. D - X y l p l ^ i i - D - X y l p l  1  p  L-Arab Hl}.-D-Xyl ] f  p  m  p  [li-D-Xyl l]  D - X y l — h D-XylJ  p  Q  3  It is thus seen that the xylans from land plants belong to a general group in possessing an essentially linear molecular chain of repeating xylose units linked glycosldlcally by  l,ii-/?  bonds.  Differences in the structures are reflected  by the proportions and type of side chains present and the mode of attachment of these side chains.  Closely related molecular  species exist i n which variations i n detailed structure are manifested by variations i n molecular size.  Properties of  hemicelluloses vary considerably probably due to the nature of the side chains attached to the main structural backbone and to the extent of regularity or irregularity of branching.  z  II APPLE WOOD HEMICELLULOSE The structures of many hemicelluloses from land plants have been extensively investigated in the last ten years.  Most  of the work has, however, been confined to examination of cereal crops and hardwoods and softwoods of commercial Importance so that in this work the hemicellulose of a fruit tree, apple (var. Transparent Golden), has been studied.  Striking similarities  have been noticed between the polysaccharides of apple wood and cherry wood (97). Finely ground apple wood was rendered free of extractives by continuous extraction with hot alcohol-benzene (1:2) azeotrope (7).  Delignification of the sawdust was accomplished  by shaking the extractive-free material i n 0.1H sodium hydroxide at room temperature.  The holocellulose, containing the total  water-insoluble carbohydrate portion of the plant, was extracted with IN sodium hydroxide according to the method of McDonald (13). Precipitation of the hemicellulose was effected by pouring the alkali extract into an excess of acidified alcohol.  The result-  ing amorphous solid was dried by solvent exchange.  The hemicellu-  lose content of apple wood was found to be 8-12$ of the original material. When the hemicellulose was hydrolyzed with acid there was a marked increase in optical rotation  (W -^° p  M , +41?  ).  23.  2k.  This is generally attributed to the cleavage of ft -glycosidic bonds which are characteristic  of xylans.  Chromatographic examination of the hydrolyzate revealed the polysaccharide to be composed of D-xylose, traces of other sugars and uronic acids.  Separation of the neutral sugars  and acidic fragments was effected by the use of ion-exchange columns.  D-Xylose was found to be the major component of the  neutral fraction other sugars, namely, L-arabinose, D- or Lgalactose, L-rhamnose and two other components with Rf = 0.168 and Rf = 0.297 being present i n trace amounts.  D-Xylose was  characterized by i t s melting point and optical rotation. It i s of interest to note that young apple wood, i n vestigated by Gerhard (95) i n 1929, was shown to contain D-xylose and L-arabinose i n the ratio of 7 s 1 .  Th© intensity of the spots  obtained on paper chromatograms indicated that i n the hemicellulose investigated in this present work such a high ratio of Larabinose was not present. L-Rhamnose has been found in hydrolyzates of wood hemicelluloses but no structural significance has been placed on this desoxy sugar.  Hemicellulose from flax straw ( 9 5 ) , however, has  been suggested to be a rhamnoaraboglucuronoxylan, this suggestion being based on the isolation of a dimethylated rhamnose, 2-i+-di0-methy 1-L-rhamnose, as a cleavage product of the methylated polysaccharide.  A trimethylated rhamnose was isolated from  methylated apple wood hemicellulose but whether L-rhamnose is an integral constituent of the glucuronoxylan i s not known.  The  25. identity of the sugars with  =  0.168  and  0.297  have not been  established. On paper chromatograms, the uronic acid fragments showed three spots corresponding to uronic acid, aldobiouronic acid, and probably aldotriouronic acid.  The aldobiouronic acid  was isolated by streaking the acidic mixture on paper,  irrigat-  ing with ethylacetate: acetic acid; formic acid: water solvent system with subsequent elution of areas of the paper containing the desired acid.  An acetylated derivative was prepared from  the methyl ester methyl glycoside of the aldobiouronic acid according to the method of Timell ( 5 7 ) and the constituent acid characterized as methyl-2-0-  (2,3  di-O-acetyl-li-0-methyl- «C -D-gluco-  pyranosyl) uronate -3,h dl-O-acetyl-D-xylopyranoside  (X)  (I).  26  This acetate shows that the aldobiouronic acid from apple wood hemicellulose is 2-0(i|.-0-methyl- ( -D glucopyranosyluronic acid)0  D-xylopyranose  v  (II).  H  (IT)  The acetylated polysaccharide was prepared with i n tention of fractionating i t and also methylating the f u l l y acetylated derivative. ganic solvents.  Unfortunately the product was Insoluble in o r No further attempt was made to fractionate this  product. The hemicellulose from apple wood was methylated six times by Haworth s procedure and three times by Purdie's method. 1  The resulting product showed the absence^ of a hydroxyl band when examined by infrared.  The methylated polysaccharide was  fractionally precipitated with petroleum ether.  Amalgamation of  six fractions yielded enough material for a qualitative examination of the pentosan.  (Further portions of hemicellulose were  methylated and fractionated.  One fraction from this was used for  27.  quantitative analysis of the methylated sugars obtained after hydrolysis of the f u l l y methylated polysaccharide.)  The methyl-  ated polysaccharide was cleaved with methanolic hydrogen chloride and after the methyl ester of the acidic component had been saponified with barium hydroxide the acidic and neutral components were separated by ion exchange. In order to further confirm the identity of the acidic portion i t was esterified with diazomethane and reduced with lithium aluminum hydride  The neutral disaccharide  (54*55).  glycoside failed to crystallize even on seeding with an authent i c sample.  Hydrolysis of the syrup and subsequent separation  of the mixture on a cellulose-hydrocellulose column yielded 3 - 0 methyl-D-xylose and 2,3,4"fc i-0- " kyl-D-glucose which were r  ine  b  characterized as their crystalline anilides. aldobiouronic acid i s therefore,  The methylated  2 - 0 - ( 2 , 3 , 4 ~ t r i - 0 - m e t h y l - < -D-  glucopyranosyluronlc acid)-3-0-methyl-D-xylose (III). ther substantiates  the aldobiouronic acid as being H H H, O H OH COOH H  H O  (in)  This fur-  (II).  28.  The presence of a free hydroxyl group at position ii of the xylose unit establishes the fact that the xylose moiety is joined to other units- through this position.  Since this parti-  cular xylose residue i s probably linked to other residues through i t s reducing end, the former constitutes a branch point in the molecular complex.  Clearly, the ij.-0-methyl-D-glucur-  onic acid Is attached as a single unit side chain to the 2 position of the xylose moiety by a glycosidic bond because only the 3-0-methyl-D-xylose Is obtained from the methylated aldobiouronic acid of the methylated polysaccharide. The mixture of neutral sugar glycosides obtained from the methanolysis of the methylated apple wood hemicellulose was hydrolyzed and resolved on a cellulose-hydrocellulose column. The mixture was found to contain tri-G-methyl-rhamnose,  2,3»4-  tri-O-methyl-D-xylose, 2,3^di-0-methyl-D-xylose, 2 - 0 - and 3 - 0 methyl xylose, l ± - 0 - ( 2 , 3 - d i - 0 - m e t h y l - D - x y l o p y r a n o s y l ) - 2 , 3 - d i - 0 methyl-D-xylose, and small amounts of D-xylose and di-0-methyllyxose. The fastest running component from the separation was tentatively identified as a trimethylated rhamnose with Rf  0.856.  The 2,3,ii-tri-0-methyl-D-xylose was identified by qualitative chromatography.  The trimethylated xylose gave a spot on a paper  chromatogram which corresponded to the R^. of an authentic sample of 2 , 3 , 4 - t r i - 0 - m e t h y l - D - x y l o s e .  Preparation of a crystalline  anilide derivative of the sugar f a i l e d .  Optical rotation i n -  dicated that the syrup was only 60% pure this probably being the reason for the anilide not crystallizing.  The 2 , 3 - d i - 0 - m e t h y l -  D-xylose istic  was i d e n t i f i e d b y i t s t r a n s f o r m a t i o n t o t h e c h a r a c t e r -  crystalline  obtained That  anilide.  o f t h e 2 - 0 - and 3 - 0 - m e t h y l - D - x y l o s e .  as a m i x t u r e  t h e y were i n f a c t  parison  The monomethyl x y l o s e s were  a mixture  o f chromatographic  was e s t a b l i s h e d b y t h e com-  and e l e c t r o p h o r e t i c m o b i l i t i e s  o f the  and o f a u t h e n t i c samples o f 2 - 0 - and 3 - 0 - m e t h y l - D - x y l o s e .  sample  component w i t h R^ 0 . 7 1 gave on h y d r o l y s i s 2 , 3 - d i -  The  0-methyl-D-xylose.  p o n e n t w i t h t h e same R^ v a l u e , same d i m e t h y l a t e d  (95>) a l s o o b t a i n e d a com-  Geedes a n d S m i t h  xylose.  and w h i c h on h y d r o l y s i s gave t h e  They e s t a b l i s h e d t h e sugar  t o be 1+-0-  ( 2 , 3 - d i - 0 - m e t h y l - D - x y l o p y r a n o sy 1 ) - 2 , 3 - d i - 0 - m e t h y l - D - x y l o s e. this  work, t h i s  c o n s t i t u e n t was o b t a i n e d r  i  +112  , while  M  t h e i r x y l o b i o s e as a s y r u p , previously  that high negative  o f 1% g l y c o s i d i c rotation, is  o f the  linkages.  oC-type.  o f D-xylose  polysaccharide condensation glycosidic  fering  exist  optical  Since  this  with  acid  obtained  I t has been s t a t e d  r o t a t i o n s are i n d i c a t i v e x y l o b i o s e has a h i g h  positive  ( 7 2 ) , have  shown t h a t  condensation  polymerization  of oligosaccharides.  T h u s , when t h e  i s h y d r o l y z e d by a c i d , w i t h the formation  the monosaccharides  under-  o f o l i g o s a c c h a r i d e s which  l i n k a g e s d i f f e r e n t l y p l a c e d , and o f a t y p e  from those  present  '100°.  Jones and co-workers  occurs with the formation  tain  D  Geedes and S m i t h  i t c a n be d e d u c e d t h a t t h e b o n d b e t w e e n t h e two x y l o s e s  on t r e a t m e n t  go  and h a d  O  \tc\  optical rotation  crystalline  In  present  i n the o r i g i n a l polymer.  work, w h e t h e r some g l y c o s i d i c  bonds o f t h e  i n t h e n a t u r a l polymer o r whether  2,3-di-0-methyl-D-x7flose  con-  dif-  In this  « C - t y p e do  some r e v e r s i o n o f t h e  has o c c u r r e d t o produce  an a r t e f a c t i s  30.  not known.  (Oligosaccharides of xylose isolated from corncobs  have ft glycosidic linkages as evidenced by their negative rotations  (78).)  The component leaving the column between 2 , 3 d i - 0 methyl-D-xylose and the mono-methyl xyloses had an R^ of and an optical rotation of  [oc]^  - 2 6 ° (ca) i n methanol.  0.396  Larger  amounts of a similar unknown component, which had an optical rotation of [oc]^  - 3 6 ° in water and "which appeared to be chroma-  tographic ally and electrophore:t;Ically similar to the unknown constituent i n methylated apple wood hemicellulose, were also isolated from cherry wood, ( 9 7 ) studied by Dutton and McKelvey. In the case of cherry wood the sugar was found to have a methoxyl content of 3k'5% which is indicative of a dimethyl pentose.  It  was further shown by these workers that the unknown component was not 2,1).-di-0-methyl-D-xylose since on chromatography the Revalues of the unknown pentose and 2,ii-di-0-methyl-D-xylose were not identical. L  The possibility that this sugar was 3,4- or 3 , 5 "  di-0-methyl-D-xylose was also discarded since the latter sugars w i l l show electrophoretic migration while the unknown pentose showed no migration.  Demethylation of the unknown sugar with  boron trichloride ( 9 8 ) showed the parent sugar to be lyxose. Chromatographic examination of the dimethylated lyxose from cherry wood with an authentic sample of 2,3-di-0-methyl-D-lyxose showed these two sugars to be identical.  A mixed melting point  also indicated that they were Identical.  (The dimethylated  lyxose was not obtained crystalline from methylated apple wood.)  31-.  It is of interest to note that Montgomery and Smith (39) also obtained an unknown dimethyl pentose from wheat flour which crystallized spontaneously but failed to give a crystalline anilide. No lyxose was found by acid hydrolysis of the hemicellulose from either cherry wood or apple wood.  Hence, this  pentose could conceivably arise from epimerization of 2 , 3 - d i 0-methyl-D-xylose to 2,3-di-O-methyl-D-lyxose. The mechanism may be proposed H-C=0  H-C-OH  I  H-C-OCH-, 3 l  C-OCH„  -PJL.  CH3O-C-H  .  ||  0 H  >  CH 0-C-H 3  H-C=0 | .  CH O-C-H 3  I  CH 0-C-H 3  Prentice and co-workers (99), have taken advantage of this principle to prepare 2,4,6-trI-O-methyl-mannose from 2 , 1 + , 6 - t r i O-methyl-glucose, a reaction which involves the epimerization of carbon 2 . Prom the above experimental evidence certain structural features of the hemicellulose can be deduced.  It is clear  that the 2,3,4-tri-0-methyl-D-xylose is derived from terminal xylopyranose units in the polysaccharide and i f the trimethylated rhamnose Is an integral part of the polysaccharide then i t would also occur as non-reducing end groups attached to the linear backbone.  Prom the large amount of 2y3-di-0-methyl-D-xylose  i t can be deduced that the backbone consists primarily of 1 , 4 linked xylose units.  Some of the 3-0-methyl-D-xylose probably  32.  arises due to the hydrolysis of the aldobiouronic acid. ence of a mono-uronic acid supports this assumption.  The pres-  The remain-  ing amounts of 3-0-methyl-D-xylose and the 2-0-methyl-D-xylose may very well arise from branching i n the xylan although in view of the evidence presented by Croon and Timell  (100),  methyl xyloses may be due to incomplete methylation.  the monoIt has pre-  viously been stated that the l+.-0-methyl-D-glucuronic acid is linked by t£ glycosidic bonds to carbon 2 of the xylose moiety. From the quantitative analysis the t r i - , d i - and monomethylated pentoses and uronic acid were found to be present in the mole ratio of (1:97:21:19).  The results obtained Indicate  that the hemicellulose has a chain length of approximately 119 units with a uronic acid residue attached to about every sixth residue as single unit side chain.  From the small amounts of  monomethylated xyloses present one can only conclude that the molecule i s only slightly branched or that i t i s essentially linear. as  The structure of the glucuronoxylan may be represented  IV.  D-Xyl lp  •1^-D-Xyl 1  97  ii-^-D-Xyl 1-  Lj.-0-Me-oC-D-OpA  (IV)  [^-D-Xylp  19  33.  The constitution of the hemicellulose of apple wood is similar, although not identical, to that of other methyl glucuronoxylans isolated from deciduous woods.  These polysaccha-  rides are a l l essentially linear with the exception of possibly American beechwood ( 6 9 ) ,  and contain side chains of 1,2-pC  linked 1+-0-methyl-D-glucuronic acid.  Of the hardwoods studied  a l l contain hemicellulose with 10-11 xylose units per acid side group except white elm  (73,74)>  an  & cherry  7 xylose residues per acid side chain.  (97),  which contain  Thus, an'unusual feat-  ure of apple hemicellulose Is apparent in that i t has a high uronic acid content softwoods.  (6:1),  a general characteristic feature of  Ill EXPERIMENTAL The following solvent systems (v/v) were used to separate sugars on paper chromatograms; (A) ethyl acetate: acetic acid: formic acid:water, 1 8 : 3 : 1 : 4 ; ethanol:water:ammonia, 40:11:19:1;  ( ) n-butanol: B  (c) methyl ethyl ketone-  water azeotrope; (D) n-butanol:acetone:water,  £:4:1.  Whatman  No. 1 paper was used for a l l qualitative separations and Whatman No. 3MM for quantitative separations.  With solvent system  (D) phosphate impregnated papers, which were prepared by dipping papers into a buffer solution of disodium hydrogen phosphate and potassium dihydrogen phosphate (pH = 5 ) were used (101).  Electrophoresis was carried out on Whatman No. 1 paper  in 0.05M sodium borate (pH =9.2)  (102).  Sugars were detected  with p-anisidine trichloroacetic acid and aniline phosphate sprays. (103) A.  ISOLATION OF APPLE WOOD HEMICELLULOSE A log of apple wood was reduced to sawdust in a Wiley  mill.  The sawdust (200 gm. portions) was exhaustively extracted  with hot alcohol-benzene (1:2) for four hours, and air-dried. Delignification of the sawdust was effected by suspending the extracted sawdust in 0.1N sodium hydroxide (1.8 1.) for 24 hours, at room temperature after which the mixture was filtered and the 34.  35. colored liquor discarded.  The residue was washed free of alkali  and then extracted with N sodium hydroxide ( 1 . 8 1.) at room temperature for ii8 hours, according to the method of McDonald ( 1 3 ) . The alkaline extract was collected and the residue washed with water to make up a volume of 2 1 . The combined alkali extract ( 5 0 0 ml. portions) was acidified with acetic acid (25 ml.) and precipitated by the addition of 3 volumes of ethanol.  The light brown colored hemicellu-  lose thus obtained was centrifuged and washed successively with ethanol, diethyl ether, petroleum ether and dried.  A typical  yield from 100 gm. sawdust was 8 gm. of hemicellulose which had L^Jj, - 6 2 °  (c,1.2  In  8$  NaOH), neutralization equivalent of  74-1,  and ash content of k.66%. A second batch of apple wood sawdust was extracted as above except that the mixture of holocellulose and N sodium hydroxide was agitated for I4.8 hours.  This procedure Increased  the yield of hemicellulose to 1 2 $ . B.  HYDROLYSIS OP APPLE WOOD HEMICELLULOSE A solution of hemicellulose  acid  (20ml.)  was heated for  9  (21lj.  mg.) in 2N sulphuric  hours on a steam bath at which time  the optical rotation had changed from [^J -62° to 1<CJ „ + 4 l ° . 0  The  dark colored solution was neutralized with saturated barium hydroxide.  Barium sulphate was removed by centrifugation and the  yellow solution passed through Amberllte IR 120 resin.  The eluate  from the cation resin was passed through Duolite A-ii resin which  36.  selectively absorbed the acidic components.  The columns, i n  each case, were washed u n t i l the eluates gave a negative Molisch test.  The acidic components were liberated from the anion resin  with 2N sodium hydroxide (5 ml.) and passed through a fresh column of Amberlite IR-120.  The eluate containing the acidic  components was concentrated (21+..5 mg.) and examined chromatographically using solvent system A as the irrigating solvent (7 hours).  Three spots appeared on spraying with p-anisidine  trichloroacetic  acid corresponding to uronic acid, aldobiouronic  acid, and probably triouronic acid, R = 1.2,1.0, 0.69, respectx  ively. The eluate containing the non-acidic sugars was evaporated to a syrup (11+1+ mg.).  Chromatographic examination  of the neutral sugars i n various solvent systems (A - D) showed a large spot corresponding to xylose.  In solvent system (D)  faint spots corresponding to arabinose, rhamnose and galactose were obtained while i n solvent system (G) two spots with Rf values of 0.168 and 0.297 also appeared.  C  -  SEPARATION OF THE ACIDIC COMPONENT OF APPLE WOOD HEMICELLULOSE The wet hemicellulose was hydrolyzed i n larger quantity  (ca 30 gm.). It was dissolved i n 2N sulphuric acid (1000 m l . ) , heated for 11 hours, neutralized with barium carbonate and passed through the resin columns as above.  The eluate containing the  acidic components was concentrated i n vacuo at 35-l+0°C. yielding a dark brown syrup (5.81+1+ gm.).  The eluate containing the neutral  sugars was also concentrated i n vacuo (22.11+ gm.).  37.  A portion of the syrupy acidic constituent was streaked on Whatman No. 3VM (lj.6 X 57 cms.) f i l t e r paper which had been previously washed with the irrigating solvent (A). were irrigated for 16 hours.  Strips, 2.5 cm. in width, were cut  off the sheets and developed with p-anisidine acid.  The sheets  trichloroacetic  Only areas containing the desired aldobiouronic acid as  shown by the spray reagent were cut out.  These portions contain-  ing the product were cut into 1 mm. squares, packed i n a column (3.5 X It* cms.) and eluted with methanol u n t i l a negative Molisch test was obtained. vacuo at 35-4-0°C D.  The solution was evaporated to dryness in Yield of the aldobiouronic acid was ll±6 mg.  PREPARATION OF THE CRYSTALLINE DERIVATIVE OF 2-0-(ij-O-METHYLo£-D-GLUCOPYRANOSYLURQNIO ACID)-D-XYLOPYRANOSE 1.  The Methyl Ester Methyl Glycoside The methyl ester of the aldobiouronic acid was  prepared by dissolving the acid (lij.6 mg.) in methanol (15 m l . ) . Methanolic hydrogen chloride was added (15 ml. methanol, 1.6 ml. acetyl chloride) and the solution boiled under reflux for nine hours. Ag  After neutralization with silver carbonate the excess  was removed by passage of hydrogen sulphide gas through the  solution. (119.4  Filtration followed by evaporation yielded a syrup  mg.).  38.  2.  Methyl 2 - 0 - Methyl (2.3-dl-Q-Acetyl-4-0-methyl-oC D-glucopyranosyl) uronate -3»4-Dl-0-acetyl-Dxylopyranoslde The methyl ester methyl glycoside of the aldobiour-  onic acid (119.4 mg.) was dissolved in dry pyridine (15 ml.) and r e d i s t i l l e d acetic anhydride (5 ml.) was added.  After standing  21 hours at room temperature, the solution was poured into ice water (150 ml.) and the aqueous solution extracted with chloroform (ca 130 ml.).  The chloroform layer was washed with ice-cold  10% HG1 (10 X 10 ml.), followed by saturated sodium bicarbonate (3 X 20 ml.) and subsequently with water (1 X 50 m l . ) .  After dry-  ing over magnesium sulphate, the chloroform layer was evaporated to a pale yellow syrup ( 8 9 . 2 mg.) which was dissolved i n boiling diethyl ether. formed. ether.  On cooling the solution, crystals immediately  The white crystals (11 mg.) were washed with cold diethyl M.p. 191-193°C., mixed m.p. 194-196 C.(57). G  Insufficient  material was available for measurement of optical rotation.  E.  IDENTIFICATION OF D-XYLOSE Concentration of the eluate from the Duolite A-4 column  afforded D-xylose as the major component. methanol-water  Recrystallization from  gave crystals of D-xylose, m.p. 143-145°C, mixed  m.p. 143.5-145.5°C.,  +17.7°  (c,  1.69,  water).  The trace sugar, presumed to be rhamnose, was shown to be, in fact, chromatographically identical to rhamnose when run i n solvent system (D). the same sugar.  It was also Identical electrophoretically  to  In both cases, the spots gave a yellow color, on  39.  spraying, which was characteristic rhamnose.  Arabinose and gal-  actose were also identified chromatographically i n solvent (D). The mother liquor, after the majority of xylose had been removed, showed two other components with Rj. = 0.168 and 0.297 when examined chromatographically in solvent (G). P.  ACETYLATION OP APPLE WOOD HEMICELLULOSE Apple wood hemicellulose (1.02 gm.) was dissolved, with  heating, i n dimethyl sulfoxide (110 m l . ) . cellulose was centrifuged off.  The undissolved hemi-  Pyridine (30 ml.) was added, f o l -  lowed by dropwise addition of acetic anhydride (30 ml.).  During  the reaction the mixture was maintained at a low temperature (0°C.) and then allowed to stand 3 days at room temperature.  The  solution was precipitated into hydrochloric acid and ice-water. The acetylated polysaccharide oiled out. The yellow o i l and water mixture was extracted with chloroform, evaporated and reacetylated with pyridine and acetic anhydride.  This was allowed to stand 2ij. hours.  The solution  was then poured into ethereal methanolic hydrochloric acid but no precipitate resulted. was not attempted.  Further isolation of a solid derivative  Failure to obtain the acetylated polysacchar-  ide was probably due to i t s high solubility in dimethyl sulfoxide. A further portion of hemicellulose (10 gm.) was treated in a similar manner as described above except that dimethylformamide was used as the solvent instead of dimethylsulfoxide.  Pre-  cipitation of the solution, containing the acetylated hemlcellu-  lose, into methanolic hydrochloric acid resulted in the precipitation of brownish-yellow flocculent solid (13.4 gm.). Fractionation of the acetylated derivative was not attempted since the dried product was not soluble in organic solvents such as chloroform, acetone and diraethylformamide. G.  METHYLATION OF APPLE WOOD HEMICELLULOSE 1.  Methylation I Hemicellulose (ca 20 gm.) was dissolved, by  mechanical stirring, in 8$ sodium hydroxide (92 m l . ) .  After  dissolution of the hemicellulose sodium hydroxide pellets (31.3 gm.) were added to bring the concentration of the alkaline solution to 30%, The polysaccharide was methylated at 50°C. by the dropwise addition of dimethyl sulphate (300 ml.) and 30$ sodium hydroxide (900 ml.) over a period of 2 hours.  Acetone  was added to control the frothing and to reduce the viscosity. During the methylation procedure, the hemicellulose began to precipitate out.  After addition of the reagents the mixture  was heated i n a boiling water bath for 1 hour to decompose the excess dimethyl sulphate and to d i s t i l l off the acetone.  The  partially methylated polysaccharide precipitated on the sides of the reaction flask so that the liquor was able to be decanted off.  The solid was washed with boiling water.  No attempt was  made at this time to reduce.the content of the inorganic salts occluded onto the solid. The above Haworth methylation procedure was repeated  hi.  5> more times.  After the sixth methylation a portion of the solid  was dialyzed overnight against hot water and then cold water. The aqueous solution was acidified with sulphuric acid and extracted with chloroform.  The chloroform was washed with water  until neutral to litmus paper and evaporated to a dark brown syrup. (Yield, 3.745 gm.) 2.  Methylation II Since the yield of partially methylated poly-  saccharide was too l i t t l e for subsequent methylation and fractionation the procedure was repeated with further quantity of hemicellulose ( 2 6 gm.). After five Haworth methylations, one-half of the product was suspended in ice-cold water and acidified with sulphuric acid to precipitate the polysaccharide. was then made alkaline (pH 8 ) , centrifuged and the extracted with acetone. salt layer separated.  The mixture precipitate  On refluxing the solid, a saturated This was exhaustively extracted with  acetone. The acetone extract, evaporated to dryness, was redissolved in acetone (200 ml.), methanol (60 ml.) and methyl iodide (100 ml.).  Silver oxide (30 gm.) was added over a period  of 3 hours to the refluxing solution.  The mixture was stirred  mechanically for 23-| hours to effect the methylation according to Purdie.  The excess of methyl iodide was recovered by d i s t i l -  lation and the insoluble residue repeatedly extracted with b o i l ing methanol.  Evaporation of the combined methanolic extracts  42.  gave a syrup which was shown to be incompletely methylated when examined by Infra-red  spectroscopy.  After two more Purdie methylations no apparent hydroxyl peak appeared on the infra-red spectrum.  A small  sample of the syrup, dissolved i n chloroform, was poured into an excess of petroleum ether to yield a white powder, OCH3 3 8 . 7 $ . 3.  Methylation III The remaining one-half of the partially methylated  polysaccharide from (2) was dialyzed to remove the occluded i n organic salts.  A small portion of the solid was wetted with a  l i t t l e water and dialyzed against running hot water for 4 days. The aqueous suspension (700 ml.) was evaporated to a small bulk (2^0 ml.), acidified and extracted with chloroform as before. The chloroform layer was evaporated in vacuo to a syrup (3.24-1 gm.). The remainder of the salt enriched material was  similarly treated.  Total y i e l d , 11.15. gm.  The extracted product (11.15 gm.) was dissolved i n acetone and methylated three times by Purdie method, OCR3 3 8 . 7 $ .  H.  FRACTIONATION OF THE METHYLATED HEMICELLULOSE The syrup obtained from the third Purdie methylation II  was dissolved i n chloroform (100 ml.), diluted with diethyl ether to precipitate any inorganic impurities and allowed to stand overnight.  The solution was centrifuged and excess petroleum ether  was added to the mechanically stirred solution until a cloudiness was observed.  The mixture was centrifuged and the o i l obtained  43.  was redissolved in chloroform (5 ml.) and again precipitated into petroleum ether (200 m l . ) .  The white precipitate obtained was  dried by solvent exchange.  The centrifugate,  after precipitation  of the f i r s t fraction, was treated i n a similar manner u n t i l no more polysaccharide precipitated upon further additions of petroleum ether. Table IA.  The results of this fractionation are shown in Table IB shows a similar set of results obtained from  the fractionation of Methylation III. Fractions 2-7 were amalgamated and further structural analysis was conducted.  Since, however, these fractions were not  completely methylated, as shown by methoxyl determinations, fraction 3 from Methylation III was used for quantitative analysis.  TABLE  IA  FRACTIONAL PRECIPITATION OF METHYLATED APPLE WOOD HEMICELLULOSE II  Fraction  Total petroleum ether added,ml.  Weight, gm.  r -j I* ij> in CHCI3  -  $OCH3  1  215  0.2357  2  21+0  O.6180  -52.6  39.9  3  290  0.7354  -54.1  38.6  4  310  0.1586  -51.3  38.6  5  390  0.2954  -5o.o  38.4  6  430  0.1672  -50.6  41.7  7  530  0.4544  -1*4.8  38.9  8  1.2282  37.4  TABLE  IB  FRACTIONAL PRECIPITATION OF METHYLATED APPLE WOOD HEMICELLULOSE III  Fraction  Total petroleum ether added,ml.  Weight, gm.  r -I KJp in CHCl^  $  0GH  -  1  400  0.4720  -39.0  35.2  2  500  0.9351  -55-5  38.3  3  580  1.2851  -57.8  39.1  4  780  0.3657  -52.6  38.5  5  98o  1.0930  -51.6  39.8  6  -  2.6314  ii6.  X  -  METHANOLYSIS OF APPLE WOOD HEMICELLULOSE The methylated hemicellulose from fraction 2-7  (Methylation II)  (2.0186 gm.) was suspended in methanol (25 m l . ) .  Methanolic hydrogen chloride (2 ml. acetyl chloride i n 25 ml. methanol) was added and the mixture refluxed on a steam bath. The methanolysis could not be readily followed polarimetrically but i t was allowed to proceed for 7 hours when the optical rotation seemed apparently constant.  The solution was neutralized  with silver carbonate, f i l t e r e d and the methyl glycosides concentrated to a dark syrup (2.1306 gm.).  J.  SAPONIFICATION OF METHYL ESTER OF THE ALDOBIOURONIG ACID The syrup (2.1306 gm.) was dissolved in barium  hydroxide (25 m l . , saturated at room temperature) and heated for 2 hours at 60°C. to saponify the methyl ester of the acidic component.  The aqueous solution was then continuously extracted  with petroleum ether (200 ml.) for 20 hours.  It was filtered  from traces of solid and passed through a column of Amberlite IR 120 resin to remove the barium Ions.  The column was washed  u n t i l the washings gave a negative Molisch test. The eluate from above was passed through a column of Duolite A-lj. resin which selectively absorbed the acidic component.  The effluent from this column was concentrated to a syrup.  The petroleum ether extract was added to this syrup and the whole reevaporated to constant weight (1.5172 gm.).  This syrup  47.  was subsequently separated and the neutral sugars identified. K.  SEPARATION OP THE ACIDIC COMPONENT OP METHYLATED APPLE WOOD HEMICELLULOSE The acidic component was displaced from the Duolite  A-Ij. column with 2N sodium hydroxide (25 ml.) and passed through a fresh column of Amberlite IR 120.  The eluate and washings  were evaporated to a dark syrup (475.5 mg.).  L.  PREPARATION OF METHYL 2-0-(2.3,4-TRI-0-METHYL-D-GLUCUR ONOSYL)3-0-METHYL-D-XYL0SIDE METHYL ESTER 1.  Preparation of Diazomethane An ethereal solution of p-tolysulfonylmethylnltro-  samide (4 gm. in 23 ml. of diethyl ether) was added dropwise to a solution containing potassium hydroxide (1.1 gm. in 2 ml. water), carbitol (6.5 ml.) and diethyl ether (2 ml.) which had previously been heated to 70-75°G. i n a water bath.  The diazo-  methane produced by this reaction was d i s t i l l e d into diethyl ether (38 ml.) which was cooled to -5°C. 2.  Preparation of Methyl Ester of Methyl Glycoside of Aldobiouronic Acid To the dissolved partially methylated aldobiouronic  14.8.  acid (If 75.5 rag. in 25 ml. methanol), was added one-half of ethereal diazomethane prepared above.  The solution was allowed  to stand overnight after which the excess diazomethane and solvent' were removed by d i s t i l l a t i o n .  The 2-0(2,3,lj.-tri-0-methyl-  D-glucuronosyl) -2-0-methyl-D-xyloside methyl ester (I4.6I1 mg.) gave a positive hydroxamic acid test for esters. M.  REDUCTION OF THE METHYL ESTER OF METHYL 2-0-(2.3.1i--TRI-0METHYL-D-GLUCUR0N0SYL)-3-0-METHYL-D-XYL0SIDE The ester (I4.6I4. mg.) was dissolved In dried tetrahydro-  furan (75 ml. dried by d i s t i l l a t i o n from lithium aluminium hydride).  A solution of lithium aluminium hydride was prepared  by refluxing for 1 hour finely ground hydride (500 mg.) and dry tetrahydrofuran (20 m l . ) .  The solution containing the ester was  added slowly and at room temperature to the reducing agent and then the whole refluxed for 1 hour after the addition was complete.  Excess hydride present was decomposed by the addition of  ethereal ethyl acetate followed by dilute acetic acid.  The mix-  ture was evaporated to dryness and acetylated without attempting to separate the reduced material. Anhydrous sodium acetate (500 mg.) and r e d i s t i l l e d acetic anhydride (15 ml.) were added to acetylate the reaction mixture.  Simultaneous cleavage of the inorganic addition com-  plex and acetylation of the disaccharide was achieved by heating the mixture for 3 hours on a steam bath. anhydride was removed by d i s t i l l a t i o n .  The excess acetic Dilute hydrochlorl c acid  49.  (20 ml.) was added to dissolve the inorganic salts and the acidic solution extracted with chloroform (125 ml.).  The chloroform  layer was washed with water until free of chloride ion and then evaporated to dryness, to yield a thick clear syrup (363 mg.). Deactylation was effected by the addition of methanolic sodium hydroxide (6 ml. of IN sodium hydroxide in 10 ml. methanol) and heating under reflux for 1 hour on a steam bath.  De-  ionization and purification of the glycoside disaccharide was effected by passage of the solution through columns of Amberlite IR 120 and Duolite A-4 respectively. The clear neutral syrup (344 mg.) failed to crystall i z e even after seeding with an authentic sample of methyl 2-0(2,3,4-tri-0-methyl-D-glucopyranosyl)-3-0-methyl-D-xyloside. N.  HYDROLYSIS OP METHYL 2-0-(2,3,4-TRI-0-METHYL-D-GLUGOPYRANOr, SYL) -3-0-METHYL-D-XYL0SIDE The syrupy disaccharide (186 mg.) from above was dis-  solved in methanol (10 ml.) containing acetyl chloride (0.5 ml.). After refluxlng for 5 hours on a steam bath the solution was neutralized with silver carbonate.  Extraction of the mixture .  with ethyl acetate followed by f i l t r a t i o n and evaporation of the extracted liquor yielded the methyl glycosides as a syrup (202.5 mg.). The methyl glycosides were hydrolyzed in N sulphuric acid (5 ml.) for 7 hours on a steam bath.  Sulphuric acid was  removed by deionization with Duolite A-4 resin.  Concentration  of the eluate yielded the hydrolyzed products as a syrup  5o.  ( 1 2 6 mg.).  Paper chromatograms and electrograms revealed spots  identical to 2,3,4-tri-0-methyl-D-glucose  and 3-0-methyl-D-  xylose as the major components and a trace of what was assumed to be incompletely hydrolyzed material.  The remainder of the  syrup was added to a cellulose-hydrocellulose column by dissolution of the syrup with a l i t t l e irrigating solvent (C).  The  eluate was collected ( 1 2 ml. per fraction) at 20-minute intervals. 1.  Identification of 2 , 3 , 4 Tri-O-Methyl-D-Glucose Concentration of the solutions of tubes 1 1 - 1 7  gave a syrup ( 3 7 . 2 mg.) which was chromatographically  identical  to 2,3,4-tri-0-methyl-D-glucose,  ethyl ace-  M 1  tate).  +  73.1°(c,  0.7,  i>  J  The anilide was prepared by dissolving the syrup ( 3 7 . 2  mg.) in ethanol ( 1 ml.), adding r e d i s t i l l e d aniline ( 0 . 2 ml.) and refluxing for 3 hours.  The excess solvent and aniline were  evaporated in vacuo and the residue left to crystallize.  Re-  crystallization from ethyl acetate-petroleum ether yielded white needle-like crystals, m.p. Ii4-1-XL|.3 G. Literature value, 0  11L5-HI6 C  (104).  G  2.  Identification of 3-0-Methyl-D-Xylose Amalgamation of the contents of tubes  60-80  afforded a syrup ( 2 3 . 6 mg.) which failed to crystallize.  The  anilide of 3-0-methyl-D-xylose which was prepared i n a similar manner as above, had m.p.  133-134°C,  from ethyl acetate-petroleum ether.  after recrystallization  51. 0.  SEPARATION  OF THE NEUTRAL COMPONENTS OF METHYLATED APPLE  WOOD HEMICELLULOSE The methyl g l y c o s i d e s (1.357 gm.) were h y d r o l y z e d i n N sulphuric acid  (60 ml.) under r e f l u x f o r 8 hours.  The s o l u -  t i o n was n e u t r a l i z e d w i t h barium carbonate, c e n t r i f u g e d and the r e s i d u e e x t r a c t e d w i t h methanol.  C o n c e n t r a t i o n o f the c e n t r i -  fugate and washing y i e l d e d a brown syrup (1.156 gm.). The h y d r o l y z e d monosaccharides  (1.01+2) were d i s s o l v e d  i n methyl e t h y l ketone-water azeotrope (1.5 ml.) and p l a c e d on a c e l l u l o s e - h y d r o c e l l u l o s e column and i r r i g a t e d w i t h the same solvent.  One hundred f r a c t i o n s were c o l l e c t e d at 10 minute i n -  tervals.  Further 100 f r a c t i o n s were c o l l e c t e d a t one-half hour  intervals.  The p o s i t i o n s o f the sugars were determined by  p l a c i n g 5 spots o f s o l u t i o n from each tube on paper and spraying with p-anisidine t r i c h l o r o a c e t i c acid.  Where approximate  break's occurred the r e s p e c t i v e tubes were c o n c e n t r a t e d and i n v e s t i g a t e d f o r p u r i t y by paper chromatography sis.  and e l e c t r o p h o r e -  F r a c t i o n s c o n t a i n i n g c h r o m a t o g r a p h i c a l l y pure  were amalgamated. Table I I .  samples  The r e s u l t s of the s e p a r a t i o n are shown on  52.  TABLE  II  SEPARATION OP METHYLATED SUGARS of APPLE WOOD HEMICELLULOSE  Component 1  Tube Hunger 6-9  Weight, mg. 17.0  10  2  3  4  Identity Tri-O-methyl-rhamnose 1  11-13  21.9  111.-15  34.7  16-22  56.0  23-31  32.6  32-53  637.6  +•  2  2,3,4-Tri-O-raethyl-Dxylose 2  +  3  Methylated xylobiose 3  +  4  2 , 3 - D ! - 0 - m e thyl-D-  xylose  5  54-59  22.0  60-75  23.0  76-108  5  Di-0-methyl-lyxose  10.1  6  109-113  15.6  7  I6O-I85  8.6 859.1  Recovery 8 3 $  4.+  2 - and 3-Monomethyl-Dxylose D-Xylose  53.  P.  IDENTIFICATION OF THE- COMPONENTS Component 1, Tri-0-Methyl Rhamnose  1.  This fraction obtained in very small quantity gave a spot, on paper chromatography, with a large R^ Value of 0.856.  This was assumed to be a tri-0-methyl rhamnose.  Fur-  ther investigation into the exact identity of this sugar was not conducted. 2.  Component 2, 2,3,4 Tri-O-Methyl-D-Xylose This fraction, also obtained i n small quantity,  was found to be chromatographically identical to 2 , 3 , 4 - t r i - 0 methyl-D-xylose.  The optical rotation of the syrup  I'd,  5 . 6 8 ° ( c , O.li in methanol) indicated that i t was impure. anilide failed to crystallize.  ca The  The neutral sugar was regener-  ated by heating the anilide in hot water and adding a l i t t l e Amberlite IR 120 resin (105).  Optical rotation of the recovered  syrup (8.5 mg.) was found to be [«c] +11.2 (c, 0.5 i n methanol), J>  G  Indicating that i t was s t i l l only 60% pure. (Methanol).  Theory  M^+18.5  0  No further attempt was made to identify the sugar  as a crystalline derivative. 3.  Component 3 , 4-0-(2,3-Di-O-methyl-D-xylopyranosyl)2,3-Di-O-methyl-D-xylose The syrup obtained from this fraction had an  of 0.71 and an optical rotation of 112°(c, 3 . 1 , methanol).  It  crystallized readily, on evaporation of the solvent, to long feathery needles.  However, a l l attempts to recrystallize the  51+.  substance from various solvents f a i l e d .  (M.p. 8 3 . 0 - 8 3 . 5 ° C ) .  Hydrolysis of a small portion of this material and subsequent chromatography showed i t to yield only 2,3-di-0-methyl-D-xylose. h»  Component 1+, 2 , 3 Di-O-methyl-D-xylose This component (R 0.1+95) was found to be the f  major constituent of the methylated hemicellulose.  It failed  to recrystallize from ethyl acetate-petroleum ether and was therefore identified as the crystalline anilide, m.p. 1 2 2 - 1 2 3 ° P . 5.  Component 5 , Di-0-methyl Lyxose This fraction (R^ 0 . 3 9 b ) which was present i n only  a small amount has not been completely elucidated but appears to be a di-methylated pentose.  A similar fraction was obtained  from cherry wood hemicellulose ( 9 7 ) .  It has Gc] - 2 6 ° (ca) i n n  methanol. a.  Demethylation of DI-0-methyl Pentose Boron trichloride (redistilled) was d i s t i l l e d  into a receiver containing a solution of dimethyl pentose ( 9 . 6 mg.) in dried dichloromethane (3 m l . ) .  The solution was kept  at -78°C. for 30 minutes and then allowed to come to room temperature.  It was then allowed to evaporate overnight under an-  hydrous conditions. aqueous methanol.  The remaining residue was dissolved i n Electrophoresis of the demethylated sugar  gave a spot Identical to that of D-lyxose.  Paper chromatography  of the demethylated component In solvent (D) also indicated the presence of D-lyxose.  55.  6.  Component 6, 2-0 and 3-0-Methyl-D-xylose This fraction appeared to be homogeneous on paper  chromatograms but was readily shown by electrophoresis to be a mixture of 2- and 3-0-methyl-D-xylose.  No further attempt was  made to separate the two mono-methyl xyloses. 7.  Component 7> D-Xylose Concentration of this fraction yielded a very  small amount of syrup which did not crystallize.  Chromato-  graphic identification indicates i t may be D-xylose due to incomplete methylation. Q.  QUANTITATIVE'COMPOSITION OF APPLE WOOD HEMICELLULOSE For the quantitative analysis of methylated poly-  saccharide, fraction 3 from Methylation III was used, [oC] ,-57.8°(CHCl ), J  3  0CH 39.1$. 3  Fraction 3 was hydrolyzed as before and the neutral sugars separated on Whatman No. 1 paper using solvent system (C).  Guide strips were cut out and sprayed with aniline phos-  phate to locate the sugars. tri-, water.  Appropriate zones, containing the  d i - and monomethyl pentoses were cut out and eluted with The concentrations of the sugars were determined using  the phenol-sulphuricacid method (28). prepared by McKelvey (97) were used. shown in Table III.  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