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The hemicelluloses of western red cedar (Thuja plicata Donn) : the synthesis of 4-O-β-D-Galactopyranosyl-L-rhamnopyranose Funnell, Norman A. 1973

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THE HEMICSLLULO3E3 OF WESTERN RED CEDAR (Thuja p l i c a t a Donn); THE SYNTHESIS OF 4_p_-^ -r>- GALAC TOPYRA NOSY]>L-RHAKNOPYRAiIOSE BY ' NORMAN A. FUNNELL B.Sc, The U n i v e r s i t y of B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL PULPILME1-TT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of CHEMISTRY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1975 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed'without my w r i t t e n permission. Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada i i Interest i n carbohydrate chemistry i n t h i s laboratory focuses both on the s t r u c t u r a l e l u c i d a t i o n of polysaccharides and on the synthesis of r e l a t e d disaccharides. Thus i t i s appropriate that . Part. I of t h i s thesis deals with studies on the hemicelluloses of western red cedar (Thuja p l i c a t a Donn), while Part I I describes the synthesis of a disaccharide, 4-0 -p-D^galactopyranosyl-L-rhamhose, which i s required f o r b a c t e r i a l polysaccharide s t r u c t u r a l studies being c a r r i e d out i n t h i s laboratory. i i i ABSTRACT PART I The heraicelluloses of western red cedar (Thuja p l i c a t a Donn), a member of Family Cupressaceae, were i s o l a t e d by a l k a l i n e e x t r a c t i o n . Following p u r i f i c a t i o n , methylation and periodate oxidation techniques were employed i n the s t r u c t u r a l studies. The GGM-.type hernicelluloses appear to be very s i m i l a r to those of. other softwood species of Family Pinaceae. However, very l i t t l e arabinose'is present i n the xylan, which i s also moderately branched. These features may prove to be c h a r a c t e r i s t i c of Family Cupressaceae. The use of c i r c u l a r dichroism of a l d i t o l acetates was developed as a non-destructive technique f o r a s c e r t a i n i n g the co n f i g u r a t i o n a l s e r i e s of a sugar. I t may also be used to a s c e r t a i n the composition of a mixture of two sugar d e r i v a t i v e s , one being c h i r a l , the other a c h i r a l . PART II The disaccharide 4-0j-p-Pj-gaIactopyranosyl-L-rhamnopyranose was synthesized by a H e l f e r i c h r e a c t i o n i n 6 0 per cent y i e l d by condensation of 2,5»4»6-tetra-0-acetyl-o(-B-galactopyranosyl bromide with methyl 2 , 5-0-isopropylidene-o<-L-rhamnopyranoside. The disaccharide was characterized by i t s a l d i t o l acetate. Methylation and periodate oxidation afforded proof of stru c t u r e . i v TABLE OF CONTENTS Page ABSTRACT i i i TABLE OF CONTENTS i v LIST OF FIGURES. . . . . . . . o . . . • 0 . . v i i LIST OF TABLES . . . . . . . . . . . . . o . . «• v i i i ACIQ'TOV/LIkDGLiiilL'jTt^» • • o o o © « o » » « o © » © • < > • © • • • • • • • l.'x. PART I INTRODUCTION • . . . 2 DISCUSSION o 6 1. I s o l a t i o n o o 6 2. Hydrolysis « 1 2 3. Methylation 1 2 4 . C i r c u l a r Dichroism. 16 5. Periodate Oxidation 1? 6 . Reduction of the a c i d i c xylan . . . . . . . . 2 2 EXPERIMENTAL . . . . . . . . . . • • • o o . o . o o o . o o o . . . 25 1. General Methods . . . . . . . . . . . . . . . . . . . . . . 25 2. Molecular Weight Determinations . . . . . . . . . . . . . . 2 6 C i r c u l a r Dichroism Measurements . . . . . . . 27 4 . I s o l a t i o n of Western Red Cedar Holocellulose 27 5. Extracetion of Western Red Cedar Holocellulose with A l k a l i . 28 6 . f r a c t i o n a t i o n of the Potassium Hydroxide Extract. . . . . . 28 7. Attempted F r a c t i o n a t i o n Using Ion-Exchange Chromatography . 2 9 8. P u r i f i c a t i o n of Arabinoglucuronoxylan . . . . . . . . . . . 3 0 9. Methylation of Arabinoglucuronoxylan . . 3 2 V Page. 10o Characterization of Component Sugars . . . . . . . . . . . 3 3 1 1 . Periodate Oxidation of Arabinoglucuronoxylan . . . . . . 37 1 2 . I s o l a t i o n and I d e n t i f i c a t i o n of the A l d o t r i o u r o n i c A c i d . . 37 13" Reduction of xylan . . „ . . . . o . . . . 38 1 4 » P u r i f i c a t i o n of Galactoglucomannan . . . . . o , 39 15 » Methylation of Galactoglucomannan o . . . . . . 41 1 6 . Characterization of Component Sugars 41 1 7 . Periodate Oxidation of Galactoglucomannan 41 • 18o P u r i f i c a t i o n of Glucomannan. -. 43 1 9 . Methylation of Glucomannan 43 20. Characterization of Component Sugars 4 4 2 1 . Periodate Oxidation of Glucomannan 45 RESULTS AND DISCUSSION . 46 Summary of Results . 46 Discussion of Results „ 47 BIBLIOGRAPHY . . . . . . . . . . . . 51 PART I I INTRODUCTION 55 DI3CUS3I01I . . . . 58 1. Preparation of the reducing sugar intermediate 58 2. Preparation of the non-reducing sugar intermediate. . . . . 60 3. Condensation of the two intermediates . 0 . . 6 1 4 . Removal of the p r o t e c t i n g groups o o . o . . < > 6 2 5» Characterization and proof of structure • • • • « . . . . . 63 vi Page 60 Conclusion^ » 0 0 . ° . . . . . . . 67 EXPERIMENTAL . . . . . . . . 68 1. General Considerations . 0 . 0 0 . . 6 8 2. Methyl at-L-Rhamopyrano side (l_) . 69 J. Methyl 4-0-Acetyl-2,3-0-isopropylidene-^L-rhamjaopyranoside (2_) 69 4. Methyl 2,3-0-Isopropylidene-e(-L-rhamnopyranoside (_3_). . . . . . 70 5. 1 ,2,3,4>6-Penta-0-acetyl-^-D-galactopyranose (A) , 7 2 6 . 2 , '3,4»6-Tetra-0-acetyl-o(-D-galact6pyranosyl Bromide (_5_) . . . . 72 7. Methyl 4-0-(2, 3 , 4 , 6 -Tetra-0-acetyl - ^ - r>-galactopyranosyl)-2, 3- -0-isopropjrlidene-<^-L-rhamnopyranoside —(6_) 74 8. Methyl 4-0-(2,3,4,6-Tetra-0-acetyl-^-B-galactopyranosyl)-ol-L-rhannopyranosi&e . . ~ . 75 9 . 4-0-(2,3,4,6-Te tra-0-ace ty1 D-galactopyranosy1)-1,2,3-tri-0-aceiryl-of-L-rhamnopyranoside (ff^ f 0 • 75 10. 4-0-^D-Galactopyranosyl-L-rhamnopyranose (£) . . "]6 11. Methyl 4-0^-B-Ga.lactopyranosyl-^-L-rhamnopyranoside (10) . . . 76 12. 4-0-^D-Galactopyranosyl-L-rhaianitol (12) 78 BIBLIOGRAPHY « . . .•><>. c . . . . 80 v i i LIST 0? FIGURES Figure Page PART I 1 I s o l a t i o n of Crude Hemicelluloses . . . . „ 9 2 P u r i f i c a t i o n of Crude Kylan <>• 10 3 P u r i f i c a t i o n of Crude GGM -10 4 P u r i f i c a t i o n of Crude Gli. 11 5 Periodate Oxidation Mechanism 20 6 Periodate Oxidation of GGM and C-M 21 7 Periodate Oxidation of Xylan. . . 23 8 Gas-Liquid Chromatogram of A l d i t o l Acetates of Methylated Xylaaa . . . . . . . 34 9 S i m p l i f i e d Mass Spectrum of 2 ,3 ,5-Tri-O-methyl-L-arabinose. . 35 PART I I 1 4-C-^-D^telactppyranosyl-L-rhamnopyranose / . . 57 2 Preparation of Reducing Sugar . . . . . . . o . . . . . . . . 59 3 Preparation of Kon-Reducing Sugar • 59 4 Synthesis and Characterization of 4-£-^~i^Galactopyranosyl-L-rhanmopyranose o . T . . .""o 6 4 5 Periodate Oxidation of Methyl 4-0-^-D-Galactopyi ,ano3yl-c^-L-rhamnopyranoside. ...<><>•. . . T . "~. • 6 6 6 Methyl 4 - 0-Acetyl - 2 , 3 - 0-isopropylidene^-L-rhaninopyranoside . 71. 7 Methyl 2, 3-O-isopropylidene^-L-rhannopyranoside 75 v i i i LIST OP TABLES Table . Page 1 Physical Properties of Hemieellulose Fr a c t i o n s 31 2 Methylated Sugars from Xylan and Carboxyl-Redueed Xylan as A l d i t o l Acetates. ..-<, 36 3 Methylated Sugars from Galactoglucomannan and Glucomannan . 42 ix ACKNO'.VLEDffllENTS I wish ib thank Dr. G.G.S. Dutton, who guided t h i s work and who offe r e d many h e l p f u l suggestion. In ad d i t i o n , I must thank everyone i n the laboratory: Mr. Y.M. Choy and Mr. M.T. Yang f o r discussions on experimental matters dealing with polysaccharide s t r u c t u r a l a n a l y s i s , and.Ms. G.M. Bebault and Mr. J.M. Berry f o r suggestions about experimental procedures concerning disaccharide synthesis. I must also thank Mr. T. Randall f o r c a r r y i n g out the gel f i l t r a t i o n experiments. PART I. THE HEMICELLULOSES OP WESTERN RED CEDAR (Thuja p l i c a t a Donn) 2 INTRODUCTION Trees, both angiospenns and gymnosperms, are composed of four major components- inorganic and organic e x t r a c t i v e s , l i g n i n , c e l l u l o s e and hernicelluloses. E x t r a c t i v e s , while i n general comprising l e s s than one per cent of the dry wood weight, impart s p e c i a l q u a l i t i e s to wood. For example, i n western red cedar (Thuja p l i c a t a Donn), the presence of the fungicide t h u j a p l i c i n ( 1 ) gives red cedar exceptional decay r e s i s t -ance. Lignin, which makes up about 30 per cent of the weight of red cedar ( 2 ) , acts as a bonding agent i n wood. I t i s a three-dimensional polymer c o n s i s t i n g of phenylpropane residues (3), but i s of undetermined t o t a l structure and s i z e . C e l l u l o s e , a high molecular weight j3-D-(l->4)-l i n k e d glucan, i s the sing l e most important component of the tree i n terms of the c h a r a c t e r i s t i c s of the wood of a given species. Approx-imately 50 per cent of the dry weight of wood i s c e l l u l o s e ( 2 ) . F i n a l l y , there are the hernicelluloses, a group of r e l a t i v e l y short chain heteropolysaccharides, which can be divided i n t o three basic classes*, xylans, glucomannans and galactoglucomannans. Hernicelluloses, comprising 15 to 30 per cent of the dry wood weight, may be defined as that p o r t i o n of the t o t a l wood polysaccharides which are soluble i n , and extractable by d i l u t e a l k a l i (4). Hernicelluloses of t e r r e s t r i a l p l a n ts are composed of very few sugars, p r i n c i p a l l y I)~ xylose, D-mannose, D - 6 i I u c o s e > D-galact-ose, L-arabinose, 4-0-methyl-D-glucuronic a c i d and D-glucuronic acid# The chemistry of hernicelluloses has been reviewed extensively (4,5)» 5 The hemicelluloses of the gymnosperms d i f f e r from those of angiosperms i n that the former contain s u b s t a n t i a l q u a n t i t i e s of a p a r t i a l l y O-acetylated galactoglucomannan (GG1vI) of two types plus an arabino - (4 -0-methylglucurono)xylan, whereas the l a t t e r contain almost e x c l u s i v e l y an O-acetylated (4-0_-methylglucurono) xylan (4)« The two types of softwood GGM, which d i f f e r chemically only i n t h e i r galactose content, possess markedly d i f f e r e n t s o l u b i l i t y p r o p e r t i e s , and hence may be considered as two d i s t i n c t classes of hemicelluloses. The s o - c a l l e d glucomannans ( G M ) , which contain about one per cent galactose, are much l e s s soluble i n d i l u t e potassium hydroxide s o l u t i o n than the s o - c a l l e d GGM's, which contain about 20 per cent galactose (6). The hemicelluloses of softwoods are, i n most species studied, mostly of the GGM type, with a small amount of xylan. However, i n Sequoia sempervirens, a member of Family Taxodiaceae, there i s a very high xylan content (7). Family •Taxodiaceae i s considered to be more hig h l y evolved than Family Pinaceae ( 8 ) , to which v i r t u a l l y a l l commercially important species which have been studied, belong. An important exception i s western red cedar, a member of Family Cupressaceae, which i s also considered to be 1 more highly evolved than Family Pinaceae. Thus i t was of i n t e r e s t to see i f the hemicelluloses of western red cedar were of a structure c l o s e r to those of Pinaceae-type gymnosperms, or to those of the more highly evolved angiosperms. In the determination of the structure of polysaccharides, several approaches may be taken. I n i t i a l l y , a t o t a l a cid-catalyzed cleavage of the polymer to i t s constituent monosaccharides i s c a r r i e d out, followed 4 by determination and q u a n t i t a t i o n of the amounts of each sugar. Then, c l a s s i c a l l y , a l l free hydroxyls i n the polymer are e t h e r i f i e d . to methyl groups. Subsequent t o t a l h y d r o l y s i s w i l l l i b e r a t e a s e r i e s of p a r t i a l l y methylated sugars which w i l l have unblocked, or f r e e , hydroxy1 groups only where points of linkage i n the o r i g i n a l polymer occurred. This information may be supplemented by periodate cleavage, followed by. h y d r o l y s i s and product a n a l y s i s , of the polymer. Since the periodate ion w i l l cleave only v i c i n a l hydroxyl groups, t h i s technique gives confirmatory information to methylation studies about the point of linkage i n the polymer. A t h i r d method of a n a l y s i s i s to carry out a p a r t i a l h ydrolysis of the polymer, using mild a c i d i c conditions i n which only c e r t a i n g l y c o s i d i c linkages- those which are most a c i d - l a b i l e - w i l l be cleaved. Analysis of the r e s u l t a n t oligosaccharides by methylation, periodate oxidation, and h y d r o l y s i s can give a d d i t i o n a l information about f i n e structure, i . e . ordering of sugar u n i t s . This l a s t technique i s only of r e a l use i f there e x i s t s some ordered structure to the polymer- i f i t has a "repeating u n i t " . Such has been found to be the case i n b a c t e r i a l polysaccharides (see eg. 9)» Plant hernicelluloses, however, have been found to be both d i f f u s e i n molecular weight, and a l s o l a c k i n g of any o v e r a l l order ( 5 ) . Therefore, t h i s l a s t technique was not used i n the present study. Based on the information gathered by these techniques, one can generally propose a structure f o r the polysaccharide under i n v e s t i g a t i o n . Continuing the program of study i n t h i s laboratory (10,11) of the hernicelluloses of western Canadian softwoods, an a n a l y s i s of the gross 5 structure of those of western red cedar was c a r r i e d out. i t was also of i n t e r e s t to see i f i t would be possible to use the structure of the hernicelluloses as a chemotaxonomic key to the evolutionary p o s i t i o n of western red cedar. While there has been some work c a r r i e d out previously on western red cedar hernicelluloses by Hamilton and Partlow (12), no complete study of them has been conducted. These workers studied only the GM i n d e t a i l , f i n d i n g i t to be a t y p i c a l softwood glucomannan, c o n s i s t i n g of glucose and mannose residues i n the r a t i o of 1 to 2.5, plus one per cent galactose, which they considered to be due to a contaminating GGM. They concluded from t h e i r work that the GM was e s s e n t i a l l y l i n e a r , had a random ordering of glucose and mannose, and d i d not contain any galactose. No work was c a r r i e d out on the structure of the GGM or the xylan. 6 DISCUSSION 1 e I s o l a t i o n In order to determine the structure of wood hemicelluloses, they must f i r s t be removed from the wood. This i s not an easy task. Whether or not they are chemically bound to the l i g n i n of the v/oody c e l l i s a point of discussion; however, there i s no question that they are very strongly p h y s i c a l l y bound. Since one would l i k e to obtain a p u r i f i e d hemicellulose f o r a n a l y s i s i n a state as close as possible to that i n which i t occurred i n the tree, a problem i s apparent. While some work has been done i n v o l v i n g the use of neutral solvents such as dimethyl sulfoxide (DMSO) or N,N-dimethyl formamide (BMP) f o r e x t r a c t i o n of hemicelluloses (16), the y i e l d s are generally low. Usually, the approach has been to d e l i g n i f y the wood, then a l k a l i - e x t r a c t the h o l o c e l l u l o s e . This procedure i s followed bearing i n mind the f a c t that a l k a l i - l a b i l e groups, such as esters, w i l l thus be l o s t . In a d d i t i o n , a l k a l i n e p e e l i n g reactions (14)> y i e l d i n g saccharinic acids, with concomitant decrease i n the degree of polymerization (D.P.), may a l s o occur. Unfortunately, two f u r t h e r problems a r i s e . During the d e l i g n i f -* Holocellulose may be defined as that p o r t i o n of the wood remaining a f t e r solvent extraction, to remove e x t r a c t i v e s , and complete d e l i g n i f i c a t i o n (4)» T h e o r e t i c a l l y , i t contains a l l c e l l u l o s e and hemicelluloses i n an unaltered state. i c a t i o n step, which i s c a r r i e d out, i n general, by a chlorine bleaching process, some degradation of polysaccharide materials may occur. D i f f e r e n t bleaching agents have been proposed to t r y to overcome t h i s , i n c l u d i n g chlorine (15), chlorine dioxide (16) and peroxyacetic a c i d ( 1 7 ) « In the present study, the method of Wise and coworkers (18) was used, employing an aqueous s o l u t i o n of a c e t i c a c i d and sodium c h l o r i t e . This method has the advantages of causing a minimum of degradation while l e a v i n g only about three to four per cent l i g n i n . The second problem encountered i s to resolve each hemieellulose, once i s o l a t e d i n crude form, from the others. As mentioned i n the introduction, the GM may be obtained i n a high state of p u r i t y by u s i n g aqueous sodium hydroxide, rather than aqueous potassium hydroxide, to extract i t ( 6 ) . However, the separation of xylan from GGM, both of which are extracted by potassium hydroxide s o l u t i o n , i s more d i f f i c u l t . A number of schemes have been proposed ( 1 9 - 2 1 ) , based on p h y s i c a l d i f f e r e n t i a t i o n . Despite the f a c t that the xylan i s a c i d i c and the GGM n e u t r a l , there has not been much success i n making use of t h i s chemical d i f f e r e n c e , f o r instance by the use of ion-exchange chromatography ( 2 2 ) . S i d d i q u i and Wood ( 2 3 ) have recently reported the use of DEAE-cellulose i n the carbonate form as a chromatographic medium f o r f r a c t i o n a t i o n . However, attempts to f r a c t i o n a t e western red cedar xylan and GGM using t h i s system f a i l e d e n t i r e l y , and gave only a separation of degraded c e l l u l o s e (high D.P.) and hernicelluloses low D.P.). Thus the system was f u n c t i o n i n g on a p h y s i c a l phenomenon, molecular weight, and not on the chemical d i f f e r e n c e s between the xylan and GGM and c e l l u l o s e . The success of S i d d i q u i and Wood appears to r e s u l t f o r the same reaaon, as 8 they separated an amylose-type polysaccharide from a mixture of hemicelluloses. S i m i l a r l y , the use of a Dowex anion-exchange r e s i n (22) was unproductive. The separation and p u r i f i c a t i o n of the xylan and GGM were therefore c a r r i e d out by the method of Timell (19), which i s l i k e l y the most widely accepted procedure f o r obtaining pure GGM f r a c t i o n s . D e t a i l s are described i n the experimental sec t i o n . The o v e r a l l schemes f o r i s o l a t i o n and p u r i f i c a t i o n of the hemicelluloses are shown i n Figures 1-4. Even when i s o l a t e d i n what i s believed to be a pure state, i t i s d i f f i c u l t to prove r i g o r o u s l y the absolute homogeneity of a polysacchar-ide. Techniques such as electrophoresis, gel f i l t r a t i o n chromatography, ion-exchange chromatography, and sugar a n a l y s i s a l l may be used as c r i t e r i a of p u r i t y . As mentioned above, the use of an anion-exchange r e s i n f a i l e d to separate a known mixture of a c i d i c and neutral hemicelluloses. 'This points out a p o t e n t i a l e r r o r i n i n t e r p r e t a t i o n of r e s u l t s ; the presence of two peaks proves the presence of two d i f f e r e n t compounds, whereas the presence of a single peak only i n d i c a t e s , and does not prove, the homogeneity of the ma t e r i a l . This problem i s f u r t h e r compounded i n the case of plant polysaccharides as they have very wide molecular weight ranges, which may or may not a r i s e because of d r a s t i c i s o l a t i o n procedures. Thus the f a c t that the p u r i f i e d ' western red cedar xylan and GGM f r a c t i o n s gave sin g l e maxima on gel f i l t r a t i o n chromatography only i n d i c a t e s homogeneity. The technique of analyzing polysaccharide hydrolysates by paper 9 H i l l e d Wood Methanol e x t r a c t i o n Soluble e x t r a c t i v e s Holocellulose 2 4 $ KOH Insoluble 17$daOH/4?B b o r i c a c i d Insoluble Degraded c e l l u l o s e Soluble Ethanol p r e c i p i t a t i o n GM Soluble Extracted wood Chl o r i t e d e l i g n i f i c a t i o n L i g n i n Soluble Ethanol/acid GGM + X a) C h l o r i t e d e l i g n i f i c a t i o n b) Ethanol c) IO73 KOH P Ba(OH), Insoluble aj 5 0 $ EOAc b) Ethanol GGM. Figure 1. I s o l a t i o n of Crude Ilemicelluloses 10 17$ NaOH./4/S boric' a c i d Insoluble d i s c a r d Soluble 5^ Ba(OH), Insoluble a) 50$ HOAc b) Ethanol Figure 2. P u r i f i c a t i o n of Crude Xylan. Soluble a} Ethanol b) 507'i HOAc c) Ethanol GGM-. 10/, KOH Insoluble] a) 5O7S b) Etha •3 HOAc anol GGM Soluble Ethanol GGM. Insoluble Soluble. Jjo Ba(OH), Insoluble aj 50$ HOAc b) Ethanol GGI' *2 a) 10? b) Jfo 10$ KOH Ba(OH), aj 507b HOAc bj Ethanol Soluble Ethanol GGI M 5 GGM, Figure 3. P u r i f i c a t i o n of Crude GGM 11 GM Chl o r i t e d e l i g n i f i c a t i o n Ethanol 10$ Ka0H/4$ b o r i c a c i d Insoluble d i s c a r d Soluble j/o Ba(OH), Soluble disca r d Insoluble a) - 5 0 $ HOAc b) Ethanol 1 0 $ NaOH/4^ b o r i c a c i d Insoluble d i s c a r d Soluble % Ba(OE), Soluble d i s c a r d Insoluble Figure 4 . P u r i f i c a t i o n of Crude GM 12 chromatography i s useful i n f o l l o w i n g q u a l i t a t i v e l y the p u r i f i c a t i o n of a polysaccharide, and coupled with q u a n t i t a t i o n of the sugar r a t i o s by g a s - l i q u i d chromatography (g . l . c . ) provides good evidence f o r the p u r i t y o f a polysaccharide. Because of the very n o n v o l a t i l e nature of carbohydrates, they must be converted to a suitable v o l a t i l e d e r i v a t i v e before i n j e c t i o n into the g a s - l i q u i d chromatograph. Common d e r i v a t i v e s used are t r i m e t h y l s i l y l (TM3) ethers of the sugars ( 2 4 ) , t r i f l u o r o a c e t -ates of the sugar a l d i t o l s ( 2 5 ) , or a l d i t o l acetates ( 2 6 ) . In t h i s i n v e s t i g a t i o n a l l sugar r a t i o determinations were c a r r i e d out using a l d i t o l acetates. This method has a d i s t i n c t advantage over the use of other d e r i v a t i v e s * only one peak per sugar i s obtained, and thus q u a n t i t a t i o n i s much s i m p l i f i e d . 2. Hydrolysis There have been several a c i d systems proposed f o r the .... '•. h y d r o l y s i s of polysaccharides (27). Of these, the two most common are s u l f u r i c a c i d and formic a c i d . These systems, however, have the disadvantages of r e l a t i v e d i f f i c u l t y of removal and/or of causing degradation of hydrolyzed sugars. In 1967, Albersheim and coworkers (28) proposed the use of t r i f l u o r o a c e t i c a c i d (TPA) as an a l t e r n a t i v e a c i d f o r h y d r o l y s i s . TPA has the advantages over other a c i d systems of causing a minimum of degradation' and of being e a s i l y removed by evaporation. In t h i s study a l l hydrolyses were- c a r r i e d out using TFA as advanced by Albersheim. 3. Methylation In polysaccharide and oligosaccharide a n a l y s i s , the technique of methylation has been employed f o r over 70 years. The method, o r i g i n -1 3 a t i n g with Furdie and Irvine ( 2 9 ) and Eaworth (50)> has been used " because the methyl ether f u n c t i o n a l i t y i s one of few blocking groups which can survive a subsequent t o t a l a c i d h y d r o l y s i s . The methods of Haworth and Purdie complement each other i n that the former i s c a r r i e d out i n aqueous s o l u t i o n while the l a t t e r is.done i n organic solvents. Thus polysaccharides may be p a r t l y methylated by an i n i t i a l Haworth methylation using sodium hydroxide and dimethyl s u l f a t e , then f u l l y methylated by repeated a p p l i c a t i o n s of Purdie's technique using s i l v e r oxide i n r e f l u x i n g methyl iodide. 'These methods remained v i r t u a l l y unchanged f o r many years u n t i l a p r o t i c solvents, such as DMF and DMoO, were developed. In 19^4, Hakomori ( 3 1 ) published the d e t a i l s of what has come to be recognized as the most important development i n the methylation technique, Using the base methyl s u l f i n y l carbanion, termed "dimsyl ion", formed by re a c t i o n of DHSO with sodium hydride, i n combination with methyl iodide, t h i s method generally brings about a high degree of s u b s t i t u t i o n i n only one treatment. In add i t i o n , the procedure i s e a s i l y c a r r i e d out and gives high y i e l d s . The steps i n the methylation procedure are shown below. 2 0 0 2 ) CH,-S-CH "Na + + ROH —> C E 2-S-CH, + HD"Na + 3g 2 33 5 3) RO~Na+ + CHjI --> ROCH3 + Nal Sandford and Conrad ( 3 2 ) , in- an examination of a b a c t e r i a l polysacc-haride, expanded the experimental d e t a i l s given by Hakomori, and i t i s 14 t h e i r procedure which i s generally followed. In the present study an important f a c t arose regarding determina-concentration t i o n of the = . Hh of the dimsyl ion. Sandford and Conrad suggest that i t be determined by withdrawing an a l i q u o t and t i t r a t i n g i t with aqueous mineral a c i d . Thus i t i s a c t u a l l y hydroxyl ion that i s being t i t r a t e d , and not dimsyl ion, because of the r e a c t i o n N a + C H o - S - C H ^ ~ + H 90 — > C H , - 3 - C H , + O H " 2 II 5 2 3 || 3 0 0 I f the sodium hydride used has been opened to the atmosphere or i f traces of water get into the r e a c t i o n mixture, an unknown amount of sodium hydroxide may be present (11), which i n such a t i t r a t i o n w i l l concentration add to the apparent s^ren^ A of the dimsyl ion. I f , however, the dimsyl ion i s t i t r a t e d under dry nitrogen against formanilide, with t r i p h e n y l -methane as i n d i c a t o r (33)> only the dimsyl ion w i l l react, as the . hydroxide ion i s not s u f f i c i e n t l y basic to remove.a proton from e i t h e r formanilide or triphenylmethane. Consistently lower r e s u l t s were obtained f o r the dimsyl ion concentration by t h i s method as compared to aqueous t i t r a t i o n , i n some cases the d i f f e r e n c e being as great as 50 per cent. A second point which arose during the methylation of the glucomannan was the e f f e c t of incomplete s o l u t i o n of the p o l y s a c c h a r i d e ' i n DM30 p r i o r to a d d i t i o n of dimsyl ion. While both GGM and xylan were deionized p r i o r to methylation, the GM was not. The presence of barium ions, from the preceding p u r i f i c a t i o n step, prevented complete s o l u t i o n of the GM and the f i r s t Hakomori methylation d i d not r a i s e the methoxyl content 15 s u f f i c i e n t l y (about 30 per cent) to make the polysaccharide soluble i n methyl iodide. A f t e r a Haworth methylation, however, the i^olysaccharide became soluble i n KISO and a second Hakomori treatment brought about e s s e n t i a l l y complete methylation. For a neutral polysaccharide such as the GM, the a p p l i c a t i o n of a second Hakomori methylation causes no harm, whereas f o r an a c i d i c polysaccharide such as the xylan, degradation may occur during the second methylation ( 3 4 ) hy means of ^ - e l i m i n a t i o n or by d i r e c t r e a c t i o n . Therefore, i f required, a second methylation on an a c i d i c polysaccharide i s u s u a l l y done by means of the milder Purdie's reagents. For both the GGM and xylan, one methylation treatment y i e l d e d e s s e n t i a l l y f u l l y methylated m a t e r i a l . Once a f u l l y methylated polysaccharide has been obtained, i t i s generally hydrolyzed to give i t s constituent p a r t i a l l y methylated monosaccharides, which are then analysed and characterized i n some manner. In t h i s study, the techniques of paper chromatography, 16 comparative retention times on g.l.c, mass sp e c t r a l a n a l y s i s and demethyla.tion to c r y s t a l l i n e parent a l d i t o l acetates A/ere used. Preliminary i d e n t i f i c a t i o n of the hydrolyzate by means of paper chromatography was followed by g . l . c . of the a l d i t o l acetate d e r i v a t i v e s , i n c l u d i n g q u a n t i t a t i o n . C o l l e c t i o n of samples from the g . l . c . (35) and comparison of t h e i r mass spectral fragmentation patterns with authentic standards, a procedure developed by Lindberg , ( 3 6 ) , gave v i r t u a l l y unambiguous i d e n t i f i c a t i o n . In some cases samples were de methylated by means of boron t r i c h l o r i d e (37)> reacetylated and f u r t h e r character-iz e d as the c r y s t a l l i n e peracetylated h e x i t o l . While the g . l . c . column (3 per cent ECI'ISS-M on Chromosorb W) employed i n the a n a l y s i s of p a r t i a l l y methylated a l d i t o l acetates separates most a l d i t o l s e a s i l y , i t does not separate the a l d i t o l acetates of 2 , 3 , 4 > 6-tetra - 0-methyl-D-glucose and 2,3>4j6-tetra-0-methyl-l}-mannose. These two sugars represent the terminal non-reducing group of GM's and GGM's and both sugars may or may not be present i n the same hemicellulose ( 4 ) - Recently, 3 e b a u l t and coworkers (38) have noted that these two sugars may be resolved by means of sugar acetates using the same g . l . c . column as f o r a l d i t o l acetates. This technique s i m p l i f i e s the a n a l y s i s of these two sugars, which otherwise must be done separately, f o r example by means of methyl glycosides on two d i f f e r e n t g . l . c . columns (11). 4. C i r c u l a r Dichroism One important aspect of the a n a l y s i s of a polysaccharide i s the determination of the co n f i g u r a t i o n a l s e r i e s , e i t h e r D or L, to which each sugar belongs. This assignment i s often neglected, and a sugar i 3 assumed to be D or L because of previous studies done on s i m i l a r types 17 of polysaccharides. C l a s s i c a l l y , the assignment has been made by means of o p t i c a l r o t a t i o n of a s o l u t i o n of the sugar. Unfortunately, t h i s technique requires r e l a t i v e l y large samples, which nay not be a v a i l a b l e i n some s t r u c t u r a l studies. Recently, i t has been reported (39) that i t i s p o s s i b l e to assign e m p i r i c a l l y c o n f i g u r a t i o n a l s e r i e s based on the sign of the c i r c u l a r dichroism (c.d.) spectrum of the acetoxy chromo-phore of a l d i t o l acetates. The technique requires only a milligram or l e s s of material, such as may conveniently be c o l l e c t e d by preparative g . l . c . I t i s applicable to any c h i r a l a l d i t o l acetate. Thus f o r glucose, mannose and arabinose, measurement of the c.d. spectrum of the a l d i t o l acetate of each sugar and comparison with those of authentic standards w i l l determine unambiguously the c o n f i g u r a t i o n a l s e r i e s . For galactose and xylose, which as a l d i t o l acetates are a c h i r a l , use of p a r t i a l l y methylated a l d i t o l acetates w i l l provide the same r e s u l t . The technique i s a l s o u s e f u l i n determining the percentage of a c h i r a l sugar i n the presence of one which i s a c h i r a l . Thus i n the h y d r o l y s i s of the methylated xylan, the amount of c h i r a l 2-0-methyl-I)-xylose and a c h i r a l 3-0-methyl-I>-xylose i n a mixture of the two as a l d i t o l acetates was determined. These two sugars do not separate as a l d i t o l acetates on the g . l . c . columns employed. 5. Periodate oxidation The use of metaperiodate ion as an oxidant dates from.the observation of Malaprade i n 1928 (40) that 1,2-glycols are s p l i t , with each hydroxy! function being oxidized to a carbonyl group. 18 I i -C-OH -C=0 RTO. + | —> + EIO + E-0 4 -C-OH -C=0 -> i i Depending on the nature of the hydroxyl groups, whether primary or secondary, e i t h e r formaldehyde or formic a c i d may be produced. I f , however, only one bond can be oxidized by periodate ion i n a sugar moiety due to blocking of adjacent hydroxyl groups, the g l y c o l w i l l simply be oxidized to a dialdehyde, and w i l l not be cleaved out of the 0 0 The mechanism of the re a c t i o n involves a c y c l i c t r a n s i t i o n s t a t e . This was f i r s t postulated by Criegee (41) and i s i l l u s t r a t e d : i n Figure 5. The mild conditions employed and the near qu a n t i t a t i v e nature of the rea c t i o n (42) make i t appealing f o r s t r u c t u r a l polysaccharide studies. The technique i s employed as a mean3 of confirming methylation data, or as an a l t e r n a t i v e to methylation (43) • Depending on the poin t of linkage of the various sugars, both the amount of periodate ion consumed during the oxidation and the h y d r o l y s i s products, w i l l d i f f e r . •C lc\ dies, r.rX £41-2 0 I) 2 H 2 Q * H I Q . ^ H,fO« 2) 3) 4) - C - O H - C - O H H s l O e -• C - 0 | 0 5 H 4 C - O H - G - O i - C - O ' - C - O I O s H + I - C - O H • H 2 0 - C - O I -c-o > 0 4 H 3 + H p -c=o - C = Q • H . O - H I O . Figure 5. Periodate Oxidation Mechanism A l t e r n a t i v e l y , advantage may be taken of the f a c t that g l y c o s i d i c bonds are much l e s s l a b i l e to a c i d than a c e t a l linkages, and a study of the products r e s u l t i n g from a mild a c i d h y d r o l y s i s of the oxidized material w i l l y i e l d s t r u c t u r a l information (44)• In the present i n v e s t i g a t i o n only the former procedure was followed. . For both the GGM and GM, both of which are l i n k e d through the hydroxy1 group of C-4 of glucose and mannose, only one mole of periodate per sugar residue w i l l be required. On reduction and t o t a l h y d r o l y s i s , e r y t h r i t o l w i l l be l i b e r a t e d from the main chain, while g l y c e r o l w i l l be derived from the side chain galactose and the non-reducing end group. This i s i l l u s t r a t e d i n Figure 6. 21 * F i g u r e 6. P e r i o d a t e O x i d a t i o n of GGM and GM 22 For the xylan, the products are d i f f e r e n t . Since the main xylose chain i s l i n k e d through the hydroxyl group of C-4> the presence of a side chain of L-arabinose or 4 - 0-methyl-])-glucuronic a c i d attached through e i t h e r C-2 or C-3 w i l l prevent any oxidation taking place. Thus one of the products i s o l a t e d a f t e r oxidation, reduction and hy d r o l y s i s w i l l he xylose i t s e l f o This i s shown i n Figure 7» 6 . Reduction of the a c i d i c xylan The presence of a uronic a c i d residue i n a polysaccharide s t a b i l i z e s the linkage, to a c i d h y d r o l y s i s , of the a c i d to i t s aglycone. In a t o t a l a c i d h y d r o l y s i s , therefore, not a l l of t h i s bond w i l l cleave, with an aldobiouronic a c i d being formed. In the case of the xylan the r e s u l t i s the formation of the aldobiouronic a c i d 2-0-(4-0-methyl-«<-r>-glucopyranosyluronic acid)-D-xylopyranose, and also the corresponding a l d o t r i o - and aldotetrauronic a c i d s . Thus estimation of the amount of uronic a c i d a f t e r h y d r o l y s i s i s complicated. I f , however, the uronic a c i d moiety i s reduced p r i o r to h y d r o l y s i s , the d i f f i c u l t y i s overcome as the bond i n question i s no longer so a c i d - r e s i s t a n t . Several methods to do t h i s have been developed, e i t h e r i n v o l v i n g reduction of the methyl e s t e r of the a c i d by metal hydrides (45>46) or by reduction of the free a c i d with diborane ( 4 7 ) . Recently, Dutton and Kabir ( 4 8 ) have made use of a method, developed by Rees and Samuel ( 4 6 ) , i n v o l v i n g reduction of the propionated methyl ester by l i t h i u m borchydride i n tetrahydrofuran (THF). I n i t i a l propdonylation with propionic anhydride, followed by es t e r -i f i c a t i o n with diazomethane and reduction with l i t h i u m borohydride, y i e l d e d a carboxyl-reduced xylan with no carbonyl i n f r a r e d absorption. 2 3 KO 0 H . OH \ OH OH pOOH J HjCO \£I_i/^ EOH2C ^ p^— OH OH 10, ^/F~OH CII OH 2 ethylene glycol OH HO " CHgOH glycerol HO KO OH D-xylose OH 1>-xylose « IZO(\EL,OE -3-0-nethyl-&- glycerol erythronic atrid KO CHgOH glycerol Figure 7» Periodate Oxidation of Xylan 24 Subsequent a n a l y s i s of the hydrolysate by g . l . c . gave r e s u l t s i n agreement with the equivalent weight determination as c a r r i e d out by t i t r a t i o n . 25 EXPERIMENTAL 1. General Me thods Melting p o i n t s were taken between glass s l i d e s on a Fisher-Johns apparatus and are uncorrected. Optical r o t a t i o n s were measured with a Perkin-Elmer model 141 polarimeter at 2J + 1°. G a s - l i q u i d p a r t i t i o n chromatography was c a r r i e d out using an F and M 720 instrument equipped with dual thermal conductivity detectors. Peak areas were determined with an Inf o t r o n i c s CR3-100 e l e c t r o n i c i n t e g r a t o r . The f o l l o w i n g columns and operating conditions were usedj 8' x ^ " Jf/o ECN33-M on 60-70 mesh Chromosorb W (non-acid washed) with a helium flow rate of 75 ml/min» For a l d i t o l acetates the program was 170° f o r 10 min, then to 220° a t 2°/min; f o r aldose acetates 150 to 210° a t 2°/min; f o r periodate oxidation products 150 to 180° a t 2°/min. For the a n a l y s i s of the carboxyl-reduced xylan, an 8' x ^ " 5/o butanediol succinate column on 80-100 mesh Diatoport 3 with a helium flow rate of 75 ml/min operated at 210° isothermally was used; f o r the methylated carboxyl-reduced xylan, 145 to 210° a t 2°/min. A l l sugar analyses were by g . l . c . of a l d i t o l acetates. Paper chromatographic separations were c a r r i e d out on Whatman #1 paper using the f o l l o w i n g solvent systems: A, ethyl acetate-pyridine-water (4*1 »1) f o r 24 h; B, butanone-water azeotrope f o r 5-8 h; C, butanol-ethanol-water (4»1t5) f o r 24 h; D, ethyl a c e t a t e - a c e t i c acid-formic acid-water (18I3I1»4) f o r 24 h. Separations 26 were v i s u a l i z e d using s i l v e r n i t r a t e i n acetone f o r non-reducing compounds (49) a n d p_-anisidine hydrochloride spray f o r reducing sugars (50). Rg i n d i c a t e s with respect to 2,3,4»6-tetra-0-methyl-]>-glucose. A l l evaporations were c a r r i e d out under reduced pressure below 40°. Infrared, spectra were run i n carbon t e t r a c h l o r i d e s o l u t i o n at a concentration of about 10$. A l l hydrolyses were done with t r i f l u o r o -a c e t i c a c i d (2 N_) at 100° f o r 4 h unless otherwise noted (28). Samples f o r mass spectral a n a l y s i s were c o l l e c t e d i n c a p i l l a r y tubes from the gas chromatograph (35). Demethylations were c a r r i e d out u s i n g boron t r i c h l o r i d e (37)« Samples c o l l e c t e d from the g . l . c . were eluted with dichloromethane i n t o a si n g l e neck f l a s k immersed i n a dry i c e -acetone bath. Boron t r i c h l o r i d e gas was then admitted from a l e c t u r e b o t t l e . A drying tube was placed on the f l a s k ; i t was allowed to stand at -78° f o r 1 h then slowly warmed to room temperature and l e f t standing overnight. Methanol was cautiously added and dichloromethane and excess boron t r i c h l o r i d e were removed by several evaporations under reduced pressure with methanol. Solutions of d i m e t h y l s u l f i n y l anion used f o r methylations (31»32) were assayed as described f o r the methylation of the xylan. 2. Molecular ',/eight Determinations Molecular weight determinations were c a r r i e d out using gel permeation chromatography (51)• A polyethylene column (1.15 x 86 cm) of Bio-Gel P-300 (Bio-Rad Laboratories), treated with dichlorodimethyl-s i l a n e and equipped with a suction pump, was used. Sodium chloride (M) was used as eluant. Carbohydrate material was assayed by the phenol-s u l f u r i c a c i d method (52). Both xylan and GGM samples gave broad 27 maxima, i n d i c a t i n g a wide d i s t r i b u t i o n i n molecular weights. Values obtained are weight-average molecular weights obtained by c a l i b r a t i n g the column with a s e r i e s of dextrans. 3» C i r c u l a r Dichroism Measurements C i r c u l a r dichroism measurements were c a r r i e d out using a JASCO J-20 instrument, by observing the acetoxy chromophore of a l d i t o l acetates. Samples c o l l e c t e d from g . l . c . i n c a p i l l a r y tubes were weighed by d i f f e r e n c e and eluted d i r e c t l y i n t o the c.d. c e l l with 0.10-0.15 nil of d i s t i l l e d a c e t o n i t r i l e . The region 250 to 200 nm was scanned with the p r i n c i p a l maximum being about 213 nm. This procedure applied to both standards and samples from hemicellulose hydrolyzates. Comparison of the sign and magnitude of the maximum of standard and unknown was then made. By t h i s means, i t was shown that a l l mannose and glucose residues are of the D s e r i e s , while a l l arabinose residues are of the L s e r i e s . Galactose and xylose were also shown to be of the D s e r i e s , since t h e i r p a r t i a l l y methylated d e r i v a t i v e s gave c.d. curves corresponding to authentic 1,5-di-0-acetyl-2,3»4>6-tetra-0-methyl-D-galactitol and 1) 3>4j5-tetra-O-acetyl-2-0-methyl-D-xylitol, r e s p e c t i v e l y . 4. I s o l a t i o n of Y/estern Red Cedar Hol o c e l l u l o s e Samples of western red cedar were ground to sawdust i n a Wiley m i l l and extracted batchwiae (750 g) with methanol (2 l ) i n a Soxhlet extractor f o r 12 h. Extracted sawdust (225 g» moisture content 5/°) was d e l i g n i f i e d by treatment with sodium c h l o r i t e and a c e t i c a c i d (18) f o r 4 h; 2-octanol was used to suppress foaming. A f t e r a i r drying, the y i e l d of h o l o c e l l u l o s e was 154 g (72$, r e s i d u a l Klason l i g n i n 3$)» 28 5JL ^ctractior. of Me stern Red Cedar Holocellulose with A l k a l i H o l o c e l l u i o s e (140 g) was extracted with potassium hydroxide (24$, 1500 ml) under nitrogen ( 6 ) . The mixture was shaken f o r 6 h, and allowed to stand overnighto F i l t r a t i o n and washing, followed by p r e c i p i t a t i o n i n t o ethanol (6 l ) containing an excess of g l a c i a l a c e t i c a c i d (1 l ) and drying by solvent exchange (ethanol-acetone-petroleum ether ( 5 O - 6 O 0 ) ) y i e l d e d the potassium hydroxide extract (19«7 g» 10*2$ of wood, ash 1 2 . 5 $ ) . Drying by solvent exchange i s c a r r i e d out by s l u r r y i n g the p r e c i p i t a t e with ethanol, c e n t r i f u g i n g , and decanting the ethanol. This i s repeated, with ethanol, then twice with acetone, then twice with petroleum ether (50-60°). G.l.c. gave xylose 75$» mannose 12.8$, arabinose 5«4$» galactose 3«8$> and glucose 3.0$. The s o l i d s remaining a f t e r potassium hydroxide e x t r a c t i o n were s i m i l a r l y extracted with sodium hydroxide (17»5$)containing b o r i c a c i d (4$) (1500 ml). A f t e r p r e c i p i t a t i o n into ethanol-acetic a c i d and drying by solvent exchange, the hemieellulose was obtained (14«1 g> 7*3$ of wood, ash 8.6',J). G.l.c. gave mannose 71«5$> glucose 19$> xylose 4»1$> galactose 2.8;J, and arabinose 2.6$. 6. F r a c t i o n a t i o n of the Potassium Hydroxide Extract Potassium hydroxide extract (18.1 g) was suspended (53) i n sodium c h l o r i t e (100 g / l , 150 ml) buffered with sodium acetate (5 g) and g l a c i a l a c e t i c a c i d (10 ml). A f t e r s t i r r i n g f o r 22 h. the s o l u t i o n was centrifuged to remove a small amount of insoluble material, p r e c i p i t a t e d i n t o 4 volumes of ethanol a c i d i f i e d with g l a c i a l a c e t i c a c i d , recovered and dri e d by solvent exchange (16.3 g). Tne material was then diss o l v e d 29 i n water (150 ml), potassium hydroxide (20$, 150 ml) was added, and barium hydroxide (5$> 1100 ml) was added over 2 h with vigorous s t i r r i n g . The p r e c i p i t a t e was recovered by c e n t r i f u g a t i o n , d i s s o l v e d i n a c e t i c a c i d (50$, 50 ml) and r e p r e c i p i t a t e d into ethanol (200 ml). A f t e r d r y i n g by solvent exchange, polysaccharide GGM-j was obtained (4*4 g> ash 44»2$). The supernatant remaining a f t e r barium hydroxide a d d i t i o n was concentrated to 500 ml then p r e c i p i t a t e d into ethanol (2 l ) containing an excess of g l a c i a l a c e t i c a c i d (200 ml). A f t e r drying, polysaccharide X-] was obtained (11<>2 g, ash 29.2$). 7« Attempted F r a c t i o n a t i o n s Using Ion-Exchange Chromatography A DEAE-cellulose column (3 x 34 cm) was prepared f o l l o w i n g the procedure of Keukom and Kuendig (54). A s l u r r y of DEAE-cellulose i n water was p u r i f i e d by mixing consecutively with hydrochloric a c i d (0.5 M) and sodium hydroxide (0.5 M). Entrapped a i r was removed by sedimentat-i o n of the s l u r r y i n a vacuum desiccator. C e l l u l o s e fragments were removed by decanting. The procedure was repeated three times; the s l u r r y was then c a r e f u l l y poured into a column containing a glass wool plug and a l a y e r of sand 2 cm deep . A f t e r the DEAE-cellulose had s e t t l e d , the column was washed with water u n t i l the e f f l u e n t was n e u t r a l . The DEAE-cellulose was then converted to the carbonate form by washing with ammonium carbonate (0.5 K) as described by Si d d i q u i and Wood (23), then washing with water u n t i l the e f f l u e n t was negative to s i l v e r n i t r a t e . A s o l u t i o n of the potassium hydroxide extract (100 mg) i n water (5 ml) was applied to the top of the column, which was washed with water (200 ml) followed.by a gradient e l u t i o n with ammonium carbonate (0-0.5 M)• 50 A flow rate of 1 ml/ain was used. Fractions were checked f o r carbohydrate material by the Molisch t e s t (55). Two peaks were detected. The f i r s t , appearing at tubes 14-40 (water wash) was shown, a f t e r d i a l y s i n g and freeze drying (34 mg) the combined f r a c t i o n s , to be composed only of glucose. The second peak, a t tubes 104-11° (gradient wash) was shown, a f t e r d i a l y s i n g and freeze drying (63 mg) the combined f r a c t i o n s , to have v i r t u a l l y the same composition as the o r i g i n a l potassium hydroxide e x t r a c t . A s i m i l a r experiment using a mixture of X^ (30 mg) and GGM^ (30 mg) which had been p u r i f i e d by the procedure of Timell (19) gave only one hemieellulose peak, which had the same composition as a non- chromat-ographed mixture of X^ and GGH^» A column ( j x 2 6 cm) of Dowex 1-X2 anion exchange r e s i n was prepared as before, then washed with sodium acetate (0.5 K, 0.5 l ) to convert the r e s i n to the acetate form. A f t e r washing with water u n t i l s i l v e r n i t r a t e gave a negative t e s t , a sample of the potassium extract (150 mg*) was chromatographed. Only one peak was eluted, i n the water wash, and t h i s was shown to have v i r t u a l l y the same composition as the o r i g i n a l potassium hydroxide e x t r a c t . 8. P u r i f i c a t i o n of Arabinoglucuronoxylan A p o r t i o n of X-| (10.1 g) was suspended i n water (200 ml), then sodium hydroxide (17• 5';«) and boric a c i d (4$) (200 ml) were added. A small amount of i n s o l u b l e material was removed by c e n t r i f u g a t i o n . Barium hydroxide (5$, 800 ml) was then added dropwise with s t i r r i n g . The p r e c i p i t a t e formed was recovered by c e n t r i f u g a t i o n to y i e l d Xg 31 TABLE 1. Physical Properties of Hemicellulose Fractions (of7 ~v Equivalent Sugar Ratios by g . l . c . weight ( a l d i t o l acetates) ( t i t r a t i o n ) Gal Glu Kan Ara Jfyl x3 (£ 1«5» water) 16800 775 1.0 1.2 1.7 5.1 9.1 GGM^  -5.8° (c 1,..water ) 8900 - 8.0 16 66 1.6 8.4 GMg -30° (c 0.4, 2N NaOH) — - 1.6 18.5 77 trace 2.9 32 (0.40 g). Paper-chromatography i n solvent A of t h i s f r a c t i o n showed the presence of substantial amounts of hexose, and the f r a c t i o n was not f u r t h e r examined. The supernatant was poured into 3 volumes of ethanol, the p r e c i p i t a t e was recovered by c e n t r i f u g a t i o n , d i s s o l v e d i n i c e - c o l d a c e t i c a c i d (50$, 50 ml), then r e p r e c i p i t a t e d into 2 volumes of ethanol, and recovered by c e n t r i f u g a t i o n to y i e l d (7*05 g> ash 0.4$)• Paper chromatography of X^ hydrolyzate i n d i c a t e d a high pentose to hexose r a t i o . The p h y s i c a l p r o p e r t i e s of X^ are shown i n Table 1. 9. Methylation of Arabinoglucuronoxylan A sample of X^ (1.50 g) a f t e r d i a l y s i s overnight, d e i o n i z a t i o n , freeze drying and drying i n vacuo f o r 24 h at 40°, was methylated i n dry.dimethyl sulfoxide (30 ml) with m e t h y l s u l f i n y l anion (1.5 M, 30 ml) and methyl iodide (6.5 ml). The dimsyl ion was prepared as follows* sodium hydride ( j g, 57$ o i l dispersion) was weighed i n t o a f l a s k f i t t e d with a serum cap. The sodium hydride was washed three times with dry petroleum ether (30-60°), the petroleum ether being drawn o f f by p i p e t t e . DHS0,(30 ml), d i s t i l l e d from calcium hydride and stored over molecular sieves, was transferred into the f l a s k . The mixture was s t i r r e d at 60-70° u n t i l a dark green s o l u t i o n formed (2-3 h), then allowed to cool to room temperature. T i t r a t i o n of t h i s reagent with s u l f u r i c a c i d (N) showed a strength of 2.6 M; however, t i t r a t i o n under dry nitrogen with formanilide u s i n g triphenylmethane as i n d i c a t o r gave a strength of only 1.2 M. The dry polysaccharide was diss o l v e d i n DI-130 (30 ml) i n a f l a s k continuously flushed with dry nitrogen. Dimsyl ion (30 ml) was then introduced with s t i r r i n g into the f l a s k containing the polysaccharide and s t i r r e d overnight. Methyl iodide (6.5 ml) was then 33 added at 10° over 5 min with s t i r r i n g . A f t e r 1-jy h re a c t i o n time, the so l u t i o n was poured into water and the p r e c i p i t a t e formed was removed by spatula, dissolved i n chloroform (50 ml), extracted 3 times with water (25 ml), then evaporated to dryness (1.1 g). F r a c t i o n a t i o n with petroleum ether (30-60°) and chloroform (v/v) gave: f r a c t i o n 1 (90:10), 56 mg; f r a c t i o n 2 (80:20), 316 rag; f r a c t i o n 3 (75*25), 600 mg, r©0 -22o2° (c 1, chloroform), OCII, 38*6$, no i . r . absorption a t ** JD ~ 5 3400 cm""^ . (Values i n brackets are r a t i o s c f petroleum ether to chloroform.) ip._ Characterization of Component Sugars Paper chromatography of the hydrolyzate of f r a c t i o n 3 i n solvents B:.and C showed f i v e components which were t e n t a t i v e l y i d e n t i f i e d by comparison with standards. More p o s i t i v e i d e n t i f i c a t i o n and quantit-a t i v e determination were achieved by g . l . c , i n c l u d i n g co-chromatography with standards, and by mass spectrometry (34»36)« A t y p i c a l g a s - l i q u i d chromatogram and mass spectrum are shown i n Figures 8 and 9» r e s p e c t i v e l y . The r e s u l t s of t h i s a n a l y s i s are given i n Table 2. A p o r t i o n of the hydrolyzate was separated by paper chromatography i n solvent B. The material with corresponding to 2-0- and 3-0-methyl-J>-xylose was eluted, and dissolv e d i n 2 ml of 99 • 8$ BgO. This was freeze d r i e d to e f f e c t deuterium exchange, then.7 mg of the material was diss o l v e d i n 0.4 ml of 99»8$ Bv^ O. A proton magnetic resonance (p.m.r.) spectrum (60 MHz) showed -OCHj si g n a l s at X 6.40 (3-0-methyl-J>-xylose) and t 6.55 (2-0-methyl-D-xylose) i n the approximate proportion of 2:1. A f t e r reduction and a c e t y l a t i o n of the material 34 % SCNSS-M on Chromosorb W ( 8 ' x f ' ) 170° f o r 10 min, then to 220° a t 2°/min Flov/ rate 75 ml/min Figure 8. Gas-Liquid Chromatogram of A l d i t o l Acetates of Methylated Xylan 10CU 80-60 40. 20' 35 200+ m/e 43 58 71 87 101 117 129 145 161 Figure 9» S i m p l i f i e d Mass Spectrum of 2,3,5-Tri-0-methyl-L-arabinose 36 TABLE 2. Methylated sugars from xylan and carboxyl-reduced xylan Component Xylan 1 2 3 4a/4b Compound 2,3,5-Me5-Arat 2,3,4-Me5-Xyl 2,5-Ifej-Xyl 2/3-Me-Xyl as a l d i t o l acetates Retention times Molar.'amounts. (min) based on 2,3-Me2~Xyl 15 20 31 43 0.03 0.055 1.0 0.15 3$ ECSTS3-M, 170° 10 min then to 220° a t 2°/nin Carboxyl-reduced xylan 1 2,3,5-Ma5-Ara 2 3 4 5 2,3,4-I'fe5-Xyl 2,3,4>6-Me^ -Glu 2,3-Me2-Xyl 2/3-Me-Xyl 5$ BD3, 145 to 210° at 2°/rain 12 15 20 23 30 trace 0.07 0.20 1.0 0.31 2,3,5-Me -Ara = 2,3,5-tri-O-methyl-L-arabinose, e t c . 3 — 37 eluted from paper, and collection from g . l . c , a solution of this fraction (1.1 mg) in acetonitrile (0.12 ml) had A G JJHjCN +0.13. The 213 c.d. spectrum of pure 2-0-methyl-D-xylitol tetraacetate gives CH CN 3 +0.37 whereas the 3-0-methyl derivative has A£= 0; hence 213 the mixture contains 35$ of 2-0-methyl-I>-xylitol tetraacetate. 11. Periodate Oxidation of Arabinoglucuronoxylan X (157 ms) w a s reacted with sodium metaperiodate (O .O488 M, 50 ml) 3 at 5° in the dark. The periodate uptake was i n i t i a l l y rapid, becoming essentially constant (1.10 mole/sugar unit) after 1 week. The course of the reaction was followed by ti t r a t i o n of the iodine, produced by reaction of an aliquot with potassium iodide, with sodium thiosulfate. Starch was used as indicator. Iodate and excess periodate were destroyed by the addition of ethylene glycol (0.5 ml); the solution was dialyzed overnight, then reduced with sodium borohydride (130 mg). After neutralization with glacial acetic acid, hydrolysis with trifluoroacetic acid (2N, 10 ml, 6 h) at room temperature, and d i s t i l l a t i o n 4 times with methanol, paper chromatography in solvents A and D showed the presence of a large amount of xylose. After a further reduction with sodium borohyd.ride (20 mg), g . l . c showed the presence of 3 major components, two of which co-chromatographed with glycerol triacetate (16 min) and x y l i t o l pentaacetate ( 4 8 min), an, unidentified component (10 min) and a small peak attributed to ethylene glycol. 12. Isolation and Identification of the Aldotriouronic Acid A sample of X, (0.5 g) was hydrolyzed for 1 h by refluxing in TPA 3 (2 N_). After centrifugation, evaporation and passage through columns 38 of Amberlite I R 1 2 0 (if4") r e s i n and Duolite il-4 (Olf) r e s i n , the a c i d i c f r a c t i o n was eluted with 10$ formic a c i d . Paper chromatography (solvent D) showed the presence of 5 spots with R ^ o s e 1<>17> 0.87, 0 .60, 0.48 and 0 .24. The f r a c t i o n with j^y-iogg O . 4 8 corresponded to that of the genuine a l d o t r i o u r o n i c a c i d 0-(4-0^methyl-o<-r>.glucopyranosyluronic a c i d ) -(l ->2)-0 -p-D-xylopyranosyl-(l->4)-B-xylopyrahose. Preparative paper chromatography i n solvent D was used to i s o l a t e the a c i d ( 6 0 mg). The c r y s t a l l i n e material, which contains 3 moles of water of c r y s t a l l i z a t i o n per mole of a l d o t r i o u r o n i c a c i d , had m.p. 175-177° and C°<3D +50° (c 0.60, water) l i t . ( 5 6 ) m.p. 184°, 0*1 Q +62° (c 1, water)). IT.m.r. (D 2Q, external TMS) of sodium s a l t * r 6.55 (3H s i n g l e t , C-4 OCHj); 4-74 (1H broad s i n g l e t , H-1 of 4-0-methyl-ot-I>-glucopyranosyluronic a c i d ) ; 5«43 and 5*40 (1.5 H doublets, g a n ( * J-] 1 2 1 ? a n < * ® ^"^ ° ^ n o n ~ reducing xylose and H-1 (£$) of reducing xylose); 4.85 (0.5H doublet, 2 = 3 Hz, H-1 (o< ) of reducing xylose). 13. Reduction of xylan A sample of ( 2 0 0 mg). was reduced by the method of Dutton and Kabir ( 4 8 ) using l i t h i u m bbrohydride (LBH). i n r e f l u x i n g THF. To a s o l u t i o n of pyridine (2 ml) and f r e s h l y d i s t i l l e d propionic anhydride (1.5 ml), a s o l u t i o n of the xylan i n formamide (2.5 ml) was added. The r e s u l t i n g s o l u t i o n was l e f t standing overnight, then poured into i c e -col d hydrochloric a c i d ( 2 $ , 75 ml). The p r e c i p i t a t e was c o l l e c t e d by f i l t r a t i o n and dr i e d i n vacuo at 78° overnight (265 mg). The product was then dissolved i n THF (5 ml) and methylated with diazomethane. The diazomethane was prepared from N-rnethyl-N-nitroso-p_-toluenesulfonamide by the method of Vogel (57)• fo a 100 ml long-necked d i s t i l l i n g f l a s k 39 equipped with a downward condenser, potassium hydroxide (3 g) i n water (5 ml), c a r b i t b l (20 ml) and dichloromethane (10 ml) were added. The condenser was connected to 2 f l a s k s i n s e r i e s , set i n i c e - s a l t baths, each f l a s k containing dichloromethane (15 ml). The mixture was brought to r e f l u x , and II-methyl-N-nitroso-p_-toluenesulfonamide (10 g) i n dichloromethane (60 ml) was added. The d i s t i l l a t i o n was continued, with p e r i o d i c shaking of the s t i l l p o t , u n t i l the d i s t i l l a t e was colour-J e s s . The diazomethane was then added to the polysaccharide diss o l v e d i n THF. The methylated polysaccharide was recovered a f t e r standing 1 h by pouring i n t o petroleum ether (30-60°) and c o l l e c t i n g the p r e c i p i t a t e d m a t e r i a l . The d r i e d material (24O mg) was then d i s s o l v e d i n r e f l u x i n g THF (JO ml) and LBH (0.7 g) i n THF (20 ml) was added with s t i r r i n g over 1.5 h. The mixture was refluxed overnight, then excess LBH was destroyed by cautious a d d i t i o n of water. A f t e r d i a l y s i s and freeze drying, the carboxyl-reduced xylan was recovered (131 mg). G.l.c. a n a l y s i s of the hydrolyzate of the carboxyl-reduced xylan gave a xylose to 4-0-methyl-B-glucose r a t i o of 4*5*1. An i . r . spectrum of the material showed no carbonyl absorption. A p o r t i o n (13 mg) of the carboxyl-reduced material, a f t e r • dei o n i z a t i o n , freeze drying and drying i n vacuo at 78° f o r 6 h, was methylated with dimsyl ion and methyl iodide. G.l.c. a n a l y s i s of the hydroljrzate of t h i s material gave the r e s u l t s shown i n Table 2. 14. P u r i f i c a t i o n of Galactoglucomannan Western red cedar GGM-j (18 g) was suspended i n water (150 ml), 40 potassium hydroxide (20"$, 150 ml) added, and s t i r r e d f o r 0.5 h. The inso l u b l e material was removed by ce n t r i f u g a t i o n , dissolved i n a c e t i c a c i d (50;i, 10 ml), p r e c i p i t a t e d into ethanol (50 ml) and dried by solvent exchange to y i e l d GGM^ (1.28 g). This f r a c t i o n , , as ind i c a t e d by g . l . c , was enriched i n pentoses, and was not examined f u r t h e r . To the material soluble i n potassium hydroxide, barium hydroxide (5$, 1 l ) was added dropwise with s t i r r i n g s The p r e c i p i t a t e formed was centrifuged, r e d i s s o l v e d i n a c e t i c a c i d (50$, 50 ml), p r e c i p i t a t e d into ethanol (200 ml) and d r i e d by solvent exchange to y i e l d GGt-L, (5*70 g). G . l . c of t h i s f r a c t i o n showed a hexose to pentose r a t i o of 7*1» To the-barium hydroxide supernatant, ethanol was added to give a f l o c c u l e n t p r e c i p i t a t e (20) which was recovered by c e n t r i f u g a t i o n and dr i e d by solvent exchange to y i e l d C-GI'ij (3*78 EJC^ D-Q - 15«8° (c_ 1.5, water). This f r a c t i o n was shown by g . l . c . to have a pentose to hexose r a t i o of 3.5*1 a n d was not f u r t h e r examined. GGMg (3«5 g) was suspended i n water (125 ml), potassium hydroxide (20$, 125 ml) was added, and barium hydroxide (5$, 500 ml) was added dropwise with s t i r r i n g . The p r e c i p i t a t e was recovered by c e n t r i f u g a t i o n to y i e l d GGM5 (2.50 g, ash 0.75$). The ph y s i c a l p r o p e r t i e s of GGM^  are shown i n Table 1. Further a d d i t i o n of ethanol y i e l d e d a f l o c c u l e n t p r e c i p i t a t e GGMg (1.05 g), which was shown by paper chromatography i n solvent A to be enriched i n pentoses with respect to GGM . This f r a c t i o n 5 was not f u r t h e r examined. Paper chromatography of GGM^  hydrolyzate indi c a t e d a high hexose to pentose r a t i o . 41 15* Hethylation of Galactoglucomannan A sample of GGM^  (0o4 g) a f t e r deionization, freeze drying, and drying i n vacuo at 50° f o r 24 h, was methylated i n dry dimethyl sulfoxide (10 ml) with methylsulfinyl anion (1.5 M, 10 ml) and methyl iodide (5 ml). Af t e r 6 h reaction time, the solution was poured into water, dialyzed f o r 36 h against running tap water, and freeze driedj y i e l d 380 mg. Fractionation, as f o r the methylated xylan gave a product ( f r a c t i o n 3, 80:20, 125 mg) having C^I-Q -4' 3° (° 1» chloroform) and showing no i . r . absorption at 3400 cm . 16. Characterization of Component Sugars Paper chromatography of the hydrolyzate of f r a c t i o n 3 i n solvents B and C showed the presence of s i x major components the molar r a t i o s of which are given i n Table 3« Component 1_ was resolved into 1a and 1b i n the r a t i o 1:2 by chromatography of the aldose acetates at 170° (38)« A l l components co-chromatographed with authentic a l d i t o l acetate samples and had mass spectra consistent with the assigned structures. In addition components 3 and 4 were demethylated and acetylated to give (as collected from g.l.c.) D-mannitol hexaacetate m.p. 115-117° ( l i t . (58) m.p. 123°) and D-glucitol hexaacetate m.p. 95-97° ( l i t . (58) m.p. 99°)• Other minor components, investigated only by mass spectrometry, were tent a t i v e l y i d e n t i f i e d as two 2,6-di-0-methylhexitols and a 2,3,4-t r i - 0 - and 2,3-di-0-methylpentitol, presumably from contaminating xylan. « 17. Periodate Oxidation of Galactoglucomannan Western red cedar GGM^  (148.9 mg) was reacted with sodium metaperiodate (0.0488 M, 50 ml) at 5° i n the dark. The periodate uptake TABLE 3. Methylated Sugars from Galactoglucomannan and Glucomannan. Component Compound Retention times (min) * f Alditol acetates Aldose acetates M o l a r amounts based on GGM AJVL la 2,3,4,6-Me4-Glc tt 27 10,13 0.10 (G:M=1:2) 0.16 (G:M=8:1) lb 2,3,4,6-Me^ -Man 27 20 2 2,3,4,6-Me4-Gal 31 17 0.26 0.03 3 2,3,6-Me3-Man 47 26 2.6 4.2 4 2,3,6-Me3-Glc 49 21,22 1.0 1.0 5 2,3-Me2-Man 59 30 0.34 0.15 6 2,3-Me2-Glc 62 — . 0.23 0.04 t tt 3% ECNSS-M, 170° 10 min then to 220° at 2p/ndn. 3% ECNSS-M, 150°-210° at 2°/min. 2,3,4,6-Me^-Glc = 2,3,4,6-tetra-0-methyl-D-glucose, etc. 43 was i n i t i a l l y very rapid, becoming constant (1.28 moles/sugar u n i t ) a f t e r 1 week. Iodate and excess periodate were destroyed by the a d d i t i o n of ethylene g l y c o l (0.5 -id), the s o l u t i o n was dialyzed overnight, then reduced with sodium borohydride (150 mg). A f t e r n e u t r a l i z a t i o n with g l a c i a l a c e t i c a c i d , d e i o n i z a t i o n , h y d r o l y s i s with t r i f l u o r o a c e t i c a c i d (2 K, 10 ml, 6 h) at room temperature and d i s t i l l a t i o n 4 times with methanol, paper chromatography i n solvents A and D showed the presence of e r y t h r i t o l and g l y c e r o l . G.l.c. showed 2 major peaks, the f a s t e r co-chromatographing with g l y c e r o l t r i a c e t a t e (18 min), the slower with e r y t h r i t o l tetraacetate (-36 min) m.p. 35° ( l i t . (58) m.p. 89°). 18. P u r i f i c a t i o n of Glucomannan Crude sodium hydroxide extract (9*0 g) was suspended i n sodium c h l o r i t e (100 g / l , 75 ml) buffered with sodium acetate (2.5 g) i n g l a c i a l a c e t i c a c i d (5 ml). A f t e r 18 h s t i r r i n g , the mixture was poured into ethanol (500 ml), washed 3 times with water (20 ml), then p a r t i a l l y d r i e d by solvent exchange. While s t i l l moist, the s o l i d was re d i s s o l v e d i n so'dium hydroxide (10$) and b o r i c a c i d (4$) (250 ml) and a small amount of inso l u b l e material was removed by c e n t r i f u g a t i o n . Barium hydroxide (5$, 300 ml) wa3 added dropwise with vigorous s t i r r i n g o Hie p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n , washed twice with sodium hydroxide (5$, 20 ml), redissolved i n i c e - c o l d a c e t i c a c i d (50$, 150 ml), p r e c i p i t a t e d into ethanol (500 ml) and recovered. Repetition of t h i s procedure y i e l d e d GMg (5.80 g, ash 2.2$) (12). The p h y s i c a l data of GI-L, are given i n Table 1. 19. Methviation of Glucomannan P u r i f i e d GM2 0»5 g)» a f t e r d i a l y s i s overnight, freeze drying, 4 4 passage through a 200 mesh screen and drying i n vacuo at 55° f o r 17 h, was dispersed i n dry DM30 (50 ml) under dry nitrogen. Prepared dimsyl ion (25 nil) was then added at room temperature to the polysaccharide suspension. L i t t l e gel formation occurred; the r e a c t i o n mixture was s t i r r e d overnight under nitrogen. Methyl iodide ( 4 ml) was added over 10 min a t 10°, the r e a c t i o n allowed to proceed f o r 3 h, then poured i n t o water, dialyzed f o r 48 h and recovered by freeze drying. The material was i n s o l u b l e i n chloroform and thus a Haworth methylation was c a r r i e d out u s i n g sodium hydroxide (30$, 125 ml) and methyl s u l f a t e (60 ml) with a d d i t i o n of sodium hydroxide (5 g) and methyl s u l f a t e (5 ml) hourly f o r 7 h. A f t e r overnight s t i r r i n g a t 0°, the mixture was n e u t r a l i z e d with s u l f u r i c a c i d (2 K), dialyzed, and the polysaccharide ( 1 . 4 g) recovered by freeze drying. A second Hakomori methylation was then c a r r i e d out to y i e l d f u l l y methylated polysaccharide (1.1 g). F r a c t i o n a t i o n with petroleum ether (3O-6O0) and chloroform (v/v) gavet f r a c t i o n 1 (90i10), 0.128 g, C«0D -17.5°.(c 1, chloroform); f r a c t i o n 2 (80i20), 0.657 g, 0*3 D -23.8°, OGH^ 4 4 . 6 $ ; f r a c t i o n 3 (75:25), 0.344 St C«tfD -23.1°, OCHj 43.0$. F r a c t i o n s 2 and 3 d i d not -1 show any i . r . absorption at 3400 cm . 20. Characterization of gomponent Sugars Paper chromatography i n solvents 3 and C of the hydrolyzate of f r a c t i o n 2 showed the presence of s i x major components the molar r a t i o s of which, as determined by g.l.c, are given i n Table 3 « Component 1_ (50 min) was r e a d i l y shown to be a mixture of the d e r i v a t i v e s of 2,3>4>6-tetra-O-methyl-D-glucose ( l a ) and 2 , 3 , 4 , 6 - t e t r a -45 0-methyl-B-mannose (1b) i n the r a t i o of 8.1:1 by chromatography of the aldose acetates isothermally at 170°. A l l components, as t h e i r a l d i t o l acetates, co-chromatographed with authentic samples and gave mass spectra consistent with t h e i r assigned str u c t u r e . In a d d i t i o n components 5 a n d 4 were deraethylated and ace t y l a t e d to give (as c o l l e c t e d from g.l.c.) D-mannitol hexaacetate m.p. 119-121° and r>-glucitol hexaacetate m.p. 95-96°, r e s p e c t i v e l y . Several very minor components were also detected as noted f o r the methylated GGM. 21. Periodate Oxidation of Glucomannan P u r i f i e d GM^ (149*2 mg) was reacted with sodium metaperiodate (0.0483 M , 100 ml) at 5° i n the dark. The periodate uptake was i n i t i a l l y very rapid, becoming constant (0.81 moles/sugar u n i t ) a f t e r 1 weekt Iodate and excess periodate were destroyed with ethylene g l y c o l (0.5 ml), the s o l u t i o n was d i a l y z e d overnight, and reduced with sodium borohydride (150 mg). A f t e r n e u t r a l i z a t i o n with g l a c i a l a c e t i c a c i d , h y d r o l y s i s with t r i f l u o r o a c e t i c a c i d (2 N, 20 ml, 7 h) at room temperature, d i s t i l l a t i o n 4 times with methanol, and d e i o n i z a t i o n , paper chromatography i n solvents A and B showed the presence of e r y t h r i t o l and f a i n t spots corresponding to mannitol and g l y c e r o l . Periodate oxidation products were acetylated and g . l . c . gave 2 major peaks, which co-chromatographed with g l y c e r o l t r i a c e t a t e (12 min) and e r y t h r i t o l tetraacetate (27 min), m.p. 85°. 4 6 RESULTS MD DISCUSSION Summary of Re sults The xylan had an equivalent weight of 775» suggesting a uronic acid, content of about 27$. This result was substantiated by analysis of both the carboxyl-reduced xylan and the methylated carboxyl-reduced xylan. The uronic acid content appears somewhat higher than that obtained for other softwood xylans which have been studied (4)» The rotation was -36° {c_ 1.5, water), and molecular weight about 16,800, corresponding to a D.P. of about 110. The xylan was obtained in a purity of 96$, based on the ratio of neutral pentose sugars to neutral hexose sugars as determined by g.l.c. of a l d i t o l acetates. On methylation, a value of 38.6$ methoxyl after one Hakomori methylation was obtained. Analysis of this material showed a relatively high amount of trimethylxylose which indicates that there is a moderate degree of branching of the main xylose chain. As expected, periodate oxidation of the xylan, followed by hydrolysis, yielded glycerol and xylose. The l a t t e r compound arises from xylose residues carrying either a 4-0-methyl-o(-D-glucuronic acid moiety on position 2 or an L-arabinose moiety on position 3» while the glycerol arises from unsubstituted xylose residues. The point of attachment of the acid was shown to be through C-2 of xylose by isolation of the crystalline aldotriouronic acid. 4 7 Recently, the c r y s t a l structure of the a l d o t r i o u r o n i c a c i d trihydrate has been determined (59). The GGM had a molecular weight of about 8,900, corresponding to a D.P. of about 55° I t s r o t a t i o n , -3«8° (£ 1, water), i n d i c a t e s a high content of - l i n k e d galactose. The p u r i t y of the material studied, ascertained as f o r the xylan, was 90$. On methylation studies, only a small amount of tetramethyl sugars was found, which i n d i c a t e s the e s s e n t i a l l i n e a r i t y of the polysaccharide. This r e s u l t i s confirmed by periodate oxidation r e s u l t s , which give mainly g l y c e r o l (from the side-chain galactose) and e r y t h r i t o l (from the main chain glucose and mannose). That the p r i n c i p a l dimethyl sugars found were 2,3-di-0-methyl-D-glucose and 2,3-di-0_-methyl-D-mannose i n d i c a t e s that the side chain galactose was l i n k e d through p o s i t i o n 6 of these sugars. The glucomannan, obtained i n 97$ p u r i t y , had a r o t a t i o n of -38° (o_ 0.4» 2 N UaOI-l). A n a l y s i s of both o r i g i n a l and methylated GM showed only a small amount of galactose, which i s l i n k e d through C-6 of e i t h e r mannose or glucose. As f o r the GGM, the r e s u l t s show that l i t t l e branching i s p o s s i b l e . This was substantiated by periodate oxidation, which gave almost e x c l u s i v e l y e r y t h r i t o l . Discussion of Results One of the objects of t h i s study was to see i f the hemicelluloses of western red cedar were s u f f i c i e n t l y d i f f e r e n t from those of the species i n Family Pinaceae previously examined that these d i f f e r e n c e s might act as a chemotaxonomic key. Unfortunately, as seen from the r e s u l t s obtained, the general nature of the hemicelluloses of western 48 red cedar i s s i m i l a r to that of other c o n i f e r s which have been studied. The p r i n c i p a l d i f f e r e n c e s are the r e l a t i v e l y high amount of xylose extracted and the very low per cent of arabinose present. In Family Pinaceae, the r a t i o of arabinose to xylose i s generally 1<7-12 (4)> whereas i n western red cedar i t i s c l o s e r to 1;17-18. This feature may prove to be c h a r a c t e r i s t i c of Family Cupressaceae. The amount of s t r u c t u r a l information which can be gathered from the r e s u l t s i s disappointing. Several f a c t o r s are responsible f o r t h i s , the p r i n c i p a l of these being the random ordering of sugars i n p l a n t hernicelluloses. Experimentally there are problems i n estimating small amounts of dimethyl (monomethyl sugars i n the case of the xylan) sugars i n the presence of large amounts of more hig h l y methylated sugars, and a l s o problems of determining the p u r i t y of hernicelluloses. As demonstrated by Wallenfels (60) a degree of undermethylation of only 0.5$ of the t h e o r e t i c a l maximum of a hexosan w i l l r e s u l t i n an extra 3.9$ dimethylated hexose i n the hydrolysate of the polysaccharide. In a d d i t i o n , i t has been shown (11) that h y d r o l y s i s of polysaccharides under d i f f e r e n t conditions leads to d i f f e r i n g r a t i o s of t e t r a - to dimethyl ( t r i - to monomethyl f o r pentoses) sugars. Thus, the presence of small amounts of d i - (mono-) methyl sugars cannot be used to prove the presence of branching of the polysaccharide. This same f a c t accounts f o r the v a r i a t i o n i n estimates of the degree of branching of the xylan main chain. A n a l y s i s of the methylated a c i d i c xylan gave a value of 5«5 mole per cent 2, 3,4-tri-0^methyl-r>-xylose, corresponding to 1 branch point per 18 xylose residues, while a n a l y s i s of the methylated carboxyl-reduced xylan gave a value of 7 49 mole per cent, corresponding to 1 branch point per 1A xylose residues. Nonetheless i t i s c l e a r that the western red cedar xylan main chain i s moderately branched, u n l i k e that of v i r t u a l l y a l l other softwood hernicelluloses studied (4)» One may a l s o question whether the small amounts of galactose found i n the GM are a r t i f a c t s , since the xylan and GGM are found as contaminants i n each other, and a small amount of xylose i s found i n the GM. While the evidence i s c e r t a i n l y not conclusive, the g r e a t l y d i f f e r e n t s o l u b i l i t y p r o p e r t i e s of GM and GGM point to the f a c t that there are small amounts of galactose inherent i n GM's. To overcome t h i s d i f f i c u l t y , use could be made of electrophoresis combined with g.l.c.-m.s. Using preparative electrophoresis, s u f f i c i e n t q u a n t i t i e s of material could be obtained i n a high degree of p u r i t y to allow a methylation study to be c a r r i e d out, as.the technique of g.l.c.-m.s. of p a r t i a l l y methylated a l d i t o l acetates of the methylated hydrolyzate requires only milligram amounts. While most of the g . l . c . a n a l y s i s of p a r t i a l l y methylated sugars was c a r r i e d out on a BGIT33-M column (a copolymer of ethylene g l y c o l succinate p o l y e s t e r and a n i t r i l e s i l i c o n e polymer), i t became obvious as the study progressed that a butanediol succinate p o l y e s t e r (BDS) column i s superior. This i s a r e s u l t of the higher thermal s t a b i l i t y of the l a t t e r column. Thus baseline d r i f t and column bleeding are- much l e s s with the BBS column than with an EG1IS3-M column. Lower operating temperatures may also be used. Separations of v i r t u a l l y a l l sugar d e r i v a t i v e s encountered are as good as or b e t t e r than on ECNS3-M, with the f o l l o w i n g exception. B - G l u c i t o l hexaacdtate and g a l a c t i t o l 50 hexaacetate separate very poorly on BDS, but well on EC1T33-M. I t is p o s s i b l y f o r t h i s s i n g l e reason that ECNS3-M has become such a popular stationary phase f o r g . l . c . columns, rather than BBS, since these two sugars occur very widely. One of the more i n t e r e s t i n g r e s u l t s to a r i s e from t h i s study was the use of the A G value of the c i r c u l a r dichroism spectrum to determine both configuration of a sugar, and also to a s c e r t a i n the composition of a mixture of two sugar d e r i v a t i v e s , one b e i n g . c h i r a l , the other a c h i r a l . This technique, because of the small sample siz e required, and non-destructive character of the method, holds great promise for. other polysaccharide studies i n which only semimicro q u a n t i t i e s of material are a v a i l a b l e . As noted i n the experimental section, the chemical s h i f t i n p.m.r. spectroscopy i s an a l t e r n a t e method to determine the composition of a mixture of two monomethylated sugars. Thus, f o r the monomethyl xylose f r a c t i o n both methods i n d i c a t e d the presence of 2-0- and 3-0-methyl-D-xylose i n a r a t i o of 1«2. 51 BIBLIOGRAPHY 1. CR. H o l l e r . Chemistry of Organic Compounds. U.3. Saunders Co., Phi l a d e l p h i a , 1965. p. 944. 2. L . E . Wise and E.K. R a t l i f f . Anal. Chem,, Ij? 459 (1947). 5. K. Freudenberg. Science. 148 595 (1965). 4. T . E . T i m e l l , Adv. Carbohydr. Chem. 1£ 247 (1964); 20 409 (1965). 5. R.L. Whistler and E.L. Richards, i n The Carbohydrates. Edited by W. Pieman and D. Korton. 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Verlag von J u l i u s Springer, B e r l i n . 1931. PP«. 490-499. 59. R.A. Moran and G.F. Richards. Carbohydr. Res. 25_ 270 (1972). 60. K. v/allenfels, G. Bechtler, R. Kuhn, H. Trischmann and H. Egge. Angew. Chem. Int. Ed. 2 515 (1963). PART I I THE SYNTHESIS OP 4_0-A-^GALACTOPYRANOSU-L-R?^i'IiNOPYmNOSE 55 INTRODUCTION One of the p r i n c i p a l i n t e r e s t s of t h i s laboratory has been the s t r u c t u r a l e l u c i d a t i o n of polysaccharides. Recently, t h i s i n t e r e s t has centered on the capsular, antigenic slime polysaccharides of b a c t e r i a of the genus K l e b s i e l l a . These s o - c a l l e d K-antigens co n s i s t , i n the K-types studied thus f a r , of a repeating u n i t of from three to six hexose residues. Thus, a polymer of molecular weight of one m i l l i o n may be studied e s s e n t i a l l y as though i t were an oligosaccharide of molecular weight of one thousand or l e s s . In the study of such polysaccharides, i t i s customary to degrade the polymer to a s e r i e s of low molecular weight oligomers, which are e a s i e r to handle experimentally, ''hen an oligomer of unknown structure i s i s o l a t e d , i t i s convenient to have a v a i l a b l e reference compounds of p o s s i b l e structures. These may be obtained by degradation of n a t u r a l l y occurring polysaccharides whose structures and degradation products are known and have been characterized. More commonly, however, these reference compounds must be synthesized. Obviously, the f i r s t step i n the synthesis of a s e r i e s of oligosaccharides i s preparation of a disaccharide from the constituent monosaccharides, and i t i s such a synthesis which i s described i n t h i s p a r t of the t h e s i s . In t h i s laboratory, i n t e r e s t ha3 been placed on synthesis of 56 disaccharid.es containing L-rhamnose as the reducing sugar, f o r two reasons: a) previous to 1972, only one such disaccharide (1,2) had been synthesized and b) L-rhamnose occurs widely i n nature, and more s p e c i f i c a l l y , i t occurs i n the capsular polysaccharide of many s t r a i n s of K l e b s i e l l a ( j ) . The compound 4-0-^-B-galactopyranos3d-L-rhamnopyr-anose, whose synthesis i s herein described, has thus f a r not been i s o l a t e d from a n a t u r a l l y occurring source. However, the corresponding o(-linked disaccharide has recently been reported by Lindberg ( 4 ) to be present i n the capsular polysaccharide of K-type 47 > a ^ d the |J-linked disaccharide w i l l undoubtedly be found eventually. 4-0-^-B-Galactopyranosyl-L-rhaninopyranose was synthesized i n 60 per cent y i e l d by condensation of 2, 3,4>6-tetra-0-aceiryl-<<-T>-galacto-pyranosyl bromide with methyl 2,3-O^isopropylidene-oC-L-rhamnopyranoside usin g mercuric cyanide as a c a t a l y s t i n a c e t o n i t r i l e . This method of preparation i s termed the " H e l f e r i c h r e a c t i o n " (5), and i s a newer development of the o r i g i n a l Koenigs-Knorr disaccharide synthesis (6). This synthesis continues the s e r i e s of L-rhamnose-containing disaccharides thus f a r produced i n t h i s laboratory (7 ,8). While both the disaccharide and the derived a l d i t o l are syrups, the l a t t e r forms a c r y s t a l l i n e peracetate, by means of which the disaccharide was characterized. Methylation and periodate oxidation of the methyl glycoside of the disaccharide provided proof of structure. The structure of 4-0-^-pj-g alactopyranosyl-L-rhaniiaopyrtinose i s shown below, using a Ha.worth s t r u c t u r a l representation. 57 58 DISCUSSION The synthesis of a disaccharide consists of e s s e n t i a l l y f i v e steps, as follo\^st 1) preparation of the reducing sugar intermediate 2) preparation of the non-reducing sugar intermediate 3) condensation of the two intermediates 4) removal of p r o t e c t i n g groups on the disaccharide 5; c h a r a c t e r i z a t i o n and proof of structure 1. Preparation of the reducing sugar intermediate In the synthesis of a disaccharide, one requires that the reducing sugar, the aglycone, have a l l hydroxyl groups but one protected by some bloc k i n g group to ensure that condensation w i l l proceed at only one p o s i t i o n . In the case of 4-0-^-D-galactopyranosyl-L-rhaninopyranose, t h i s was accomplished by forming f i r s t the methyl glycoside of rhamnose (9), then blocking the hydroxyl groups on C-2 and C-3 by means of an isopropylidene group (9)» -^ he c y c l i c isopropylidene k e t a l , which i 3 very a c i d - l a b i l e , can then be removed e a s i l y a f t e r the condensation step. The sequence of reactions i s shown i n Figure 2. The intermediate to be used i n a condensation r e a c t i o n must be pure, as otherwise a large mixture of d i - and t r i s a c c h a r i d e products w i l l r e s u l t . Since the r e a c t i o n of methyl o(-L-rhamnopyranoside with acetone to y i e l d compound 2_ does not go to completion and y i e l d s a syrup, tedious d i s t i l l a t i o n (9) or chromatographic techniques would be required to p u r i f y t h i s intermediate. To overcome t h i s d i f f i c u l t y , 59 Figure 2. Preparation of Reducing Sugar L. 1 Figure 3. Preparation of Non-Reducing Sugar 60 use has been made of the c r y s t a l l i n e 4~ acetate d e r i v a t i v e of compound 2_ (7). Removal of the 4~ acetate group with sodium methoxide (10) i s q u a n t i t a t i v e , and provided the n e u t r a l i z a t i o n of the methoxide ion i s c a r r i e d out at ice-bath temperatures, leads to v i r t u a l l y no l o s s of isopropylidene groups. 2. Preparation of the non-reducing su. ar intermediate The reducing sugar may have any of the s i z hydroxyl. groups ( f o r a hexose) unblocked and thus su i t a b l e f o r condensation. The non-reducing sugar, on the other hand, because of the d e f i n i t i o n ' o f a disaccharide, must be l i n k e d through C-1. The condensation r e a c t i o n , whether i t be SIT-j or o l ^ , i s d e f i n i t e l y a n u c l e o p h i l i c displacement r e a c t i o n . I t involves attack of the oxygen lone p a i r of the free hydroxyl group of the reducing sugar on C-1 of the non-reducing sugar. Therefore, the group b l o c k i n g C-1 of the non-reducing sugar must be a good l e a v i n g group while those groups p r o t e c t i n g C-2 to C-6 of the non-reducing sugar must be stable to the r e a c t i o n conditions. Conventional^/ -, i n the preparation of a ^ - l i n k e d disaccharide, these c r i t e r i a are met by blocking C-1 with bromine (11) and C-2 to C-6 with est e r groups these u s u a l l y being acetates (12). The preparation o f t h i s intermediate, commonly c a l l e d an acetobromo sugar, i s shown i n Figure 3« Since a c i d - l a b i l e groups are often used to p r o t e c t c e r t a i n hydroxyl groups of the reducing sugar, i t i s important that ho traces of a c i d remain from the preparation of the acetobromo sugar. These compounds are often very r e a c t i v e , and may hydrolyze r e a d i l y with evolution o f 61 hydrogen bromide. To overcome t h i s d i f f i c u l t y , f r e s h l y prepared c r y s t a l l i n e acetobromo galactose was used i n the subsequent condensation r e a c t i o n . 3« Condensation of the two intermediates The condensation of two s u i t a b l y blocked intermediates to y i e l d a disaccharide by means of a Koenigs-Knorr type synthesis involves attack of the remaining free hydroxyl group of the reducing sugar on the anomeric carbon of the non-reducing sugar, with expulsion of halide ion and concomitant i n v e r s i o n of conf i g u r a t i o n . The e f f i c i e n c y of the re a c t i o n i s c l e a r l y governed a) by the ease with which the halide ion leaves and b) by the r e a c t i v i t y of the hydroxyl group a t t a c k i n g the anomeric carbon of the non-reducing sugar. Use of s a l t s of heavy metals such as s i l v e r (6) or mercury (13) has been developed to a i d i n the removal of the hali d e i o n . The in t r o d u c t i o n of solvents such as a c e t o n i t r i l e allowed use of soluble mercury s a l t s , thus p r o v i d i n g a homogeneous re a c t i o n medium as opposed to the o r i g i n a l heterogeneous s i l v e r oxide- chloroform mixtures. Combined with modern chromatographic techniques, t h i s has re s u l t e d i n reactions g i v i n g higher y i e l d s . The r e a c t i v i t y of the at t a c k i n g hydroxyl group i s influenced by several things. E i r s t l y , primary hydroxyl groups react more r e a d i l y than secondary. Thus disaccharides l i n k e d through C-6 may be prepared more e a s i l y than others. Secondly, the configuration of a secondary hydroxyl, whether a x i a l or equat o r i a l , w i l l influence i t s r e a c t i v i t y , with the l a t t e r r e a c t i n g more r e a d i l y . F i n a l l y , the presence of very 62 bulky p r o t e c t i n g groups w i l l hinder the a b i l i t y of the f r e e hydroxyl to attack the C-1 p o s i t i o n of the non-reducing sugar. In the present study, use of an isopropylidene k e t a l to block C-2 and C-3 of the rhamnose moiety "locked" the C-4 hydroxyl i n the more rea c t i v e equatorial p o s i t i o n . Use of mercuric cyanide, which forms a complex with hydrogen bromide, aided i n expulsion of the bromide ion and also removed hydrogen bromide from the r e a c t i o n mixture. The e f f e c t i v e n e s s of these procedures was shown by the f a c t that the condensation r e a c t i o n afforded an 8 5 $ y i e l d of c r y s t a l l i n e disaccharide material, based on methyl 2 , 3 - 0-isopropylidene-o(-L-rhamnopyranoside. 4 . Removal of the p r o t e c t i n g groups Since the compound formed i n the condensation r e a c t i o n generally contains d i f f e r e n t types of p r o t e c t i n g groups, which may be e i t h e r a c i d -or b a s e - l a b i l e , i t i s not p o s s i b l e to obtain t h i s compound from, the free disaccharide. Thus one could not i s o l a t e , f o r example, 4-0-fi-D-galactopyranosyl-L-rhamnopyranose from a polysaccharide and characterize i t as compound jS, since the l a t t e r contains both base-l a b i l e groups (acetates) and a c i d - l a b i l e groups (isopropylidene groups). Thus one must remove a l l the p r o t e c t i n g groups, to give the s o - c a l l e d " f r e e " disaccharide, then characterize the material by means of some d e r i v a t i v e . Iii t h i s research, because of an observation of Bebault and Button (7), the isopropylidene group was removed by treatment with t r i f l u o r o -a c e t i c a c i d (14) p r i o r to a c e t o l y s i s . These authors found that i f t h i s was not done, very low y i e l d s were obtained i n subsequent a c e t o l y s i s 63 reactions, due to the formation of a water-soluble compound which was l o s t during washing of the organic solvent l a y e r . A c e t o l y s i s , under conditions developed by A s p i n a l l (15^> followed by deacetylation, afforded 4-0-^-D-galactopyranosyl-L-rhamnopyranose, i n an o v e r a l l y i e l d of 60 per cent, c a l c u l a t e d on methyl 2 ,3-0-isopropylidene-d-L-rhamno-pyranoside. 5. Characterization and proof of structure When a disaccharide i s i s o l a t e d f o l l o w i n g a p a r t i a l h y d r o l y s i s of a polysaccharide, i t may be converted to a c r y s t a l l i n e d e r i v a t i v e i n order to characterize i t . The choice of a c r y s t a l l i n e d e r i v a t i v e of a disaccharide i s l i m i t e d by several f a c t s . I t must be p o s s i b l e to make i t from the f r e e disaccharide. Thus compounds such as 6_ are c r y s t a l l i n e intermediates, and not d e r i v a t i v e s , as explained above. The d e r i v a t i v e should be' e a s i l y prepared i n good y i e l d , '-i-'hese c r i t e r i a are met by use of the peracetylated d e r i v a t i v e of e i t h e r the disaccharide i t s e l f , or i t s derived a l d i t o l . 4-0-^-D^Gc.lactopyranosyl-L-rhamnop;/ranose f a i l e d to c r y s t a l l i z e as i t s peracetate, but the peracetate of 4 - 0 - ^ - 1 } -galacto-pyranosyl-L-rhamnitol r e a d i l y c r y s t a l l i z e d , m.p. 1 1 4 — 1 1 5 ° . The synthesis and c h a r a c t e r i z a t i o n of 4-Pj-p-B-galactopyranosyl-L-rhamnopyranose i s shown i n Figure 4» F i n a l l y , i n the synthesis of a new compound, the actual structure must be shown to be as postulated, i . e . whether the sugar rings are furanose or pyranose, and whether the. linkage i s c< or |3 . The s t r u c t u r a l proof i s c l a s s i c a l l y c a r r i e d out by means of periodate oxidation of e i t h e r the methyl glycoside of the disaccharide or the 64 65 free disaccharide i t s e l f . Since the periodate uptake and products r e s u l t i n g from a subsequent reduction and hy d r o l y s i s w i l l d i f f e r depending on poin t of attachment to the aglycone, t h i s technique serves as proof of the expected structureo Confirmation of t h i s structure i s obtained by a n a l y s i s of the products r e s u l t i n g from h y d r o l y s i s of the pemethylated disaccharide. On periodate oxidation, the methyl glycoside of the disaccharide consumed 2.95 moles of periodate. Subsequent borohydride reduction and methanolysis y i e l d e d g l y c e r o l and 1«rdeoxy-B-erythritol as i d e n t i f i e d by g . l . c . and m 0s. of t h e i r acetate d e r i v a t i v e s . ?he course of the oxidation i s i l l u s t r a t e d i n Figure 5* Methylation of compound _10, followed by h y d r o l y s i s and reduction y i e l d e d 2, 3 , 4,6-tetra-0-methyl-J>-galactitol and 2, J-di-Oj-methyl-L-rhamnitol i n a 1:1 r a t i o , i d e n t i f i e d by g . l . c . and m.s. (16) of t h e i r acetate d e r i v a t i v e s . These r e s u l t s , coupled with n.m.r. data, prove that the linkage of galactose to rhamnose i s through p o s i t i o n 4 of rhamnose, and that both sugars are present as pyranose r i n g s . However, they do not answer the question of whether the linkage i s <* or |3 . This l a s t point i s proven by n.m.r. spectroscopy of the disaccharide a l d i t o l . oince i t has only one anomeric proton, any resonance occurring i n the region X 4*5 to 5»5 must be due to the anomeric proton of the non-reducing sugar. Since the C-2 proton of galactose i s a x i a l when galactose i s i n i t s normal conformation (C1), the coupling constant between the protons of C-1 and C-2 i s expected to be large i f the C-1 proton i s al s o a x i a l . 66 C!I20H + HC0 2 H CH2OH g l y c e r o l >H HQ \ ^ + 2 NCHgOH 1-deoxy-D-e r y t h r i t o l PCS, OCH, CHgOH glycolaldehyde dimethyl a c e t a l Figure 5. Periodate Oxidation of Methyl A-Q-&Galactopyranosyl-et-L-rhamnopyranoside ' ~ H This was found to be the case, with J-j 2 = ^ being found. Therefore the linkage i s ^ (17)» 6. Conclusion The disacciiaride 4-0-^-B-galactopyranosyl-l>rhamnopyranose has been synthesized by a Helferich reaction i n an overall y i e l d of 60 per cent, by condensation of 2,3,4»6-tetra-0-acetyl-«<-12-galactopyranosyl bromide with methyl 2,3-0-isopropylidene-c(-L-rhamnopyranoside. The disaccharide i s readily characterized by i t s a l d i t o l acetate, m.p. 114-115°' Proof of structure was obtained by periodate oxidation and methylation of the disaccharide glycoside. 68 EXPERIMENTAL 1 e General Considerations Me l t i n g points were taken between glass s l i d e s on a Fisher-Johns apparatus and are uncorrected. O p t i c a l r o t a t i o n s were measured with a Perkin-Elmer model 141 polarimeter at 25 + 2°. N.m.r. spectra were recorded on a Varian T-60 instrument or a Varian XL-100 instrument using tetramethylsilane as i n t e r n a l standard except as noted. T . l . c . was c a r r i e d out using solvent systems A and B (see below) on s i l i c a g el G (EM Reagents). The d r i e d p l a t e s were v i s u a l i z e d by spraying with 35/0 ethanolic s u l f u r i c a c i d followed by heating to about 150° f o r 3-5 min. Solutions were concentrated below 45° i n vacuo. G a s - l i q u i d chromatography was performed on an F and M 720 instrument at a gas flow rate of 60 rpj/nin using columns: (a) 2 f t x £ i n 20$ SE 30 (F and M D i v i s i o n , Hewlett Packard, Avondale, Pennsylvania), (b) 4 f t x ^ i n 5$ butanediol succinate on 80-100 mesh Gas Chrom Q. Paper chromato-graphic separations were c a r r i e d out on Whatman No. 1 paper using the upper l a y e r of solvent systems C and D (see below) and were v i s u a l i z e d using s i l v e r n i t r a t e i n acetone f o r non-reducing compounds (18) and £-anisidine spray (19) i n t r i c h l o r o a c e t i c a c i d f o r reducing sugars. Solvent A, ethyl ether-toluene (2:1); B, butanone-water azeotrope; C, ethyl acetate-pyridine-water (4:1:1); D, 1-butanol-ethanol-water (4i1i5). 69 2. Methyl «l-L-Rhamnopyranoside (1_) This compound was prepared e s s e n t i a l l y as described by Levene and Muskat (9)» L-Rhamnose monohydrate (30 g) was disso l v e d i n methanolic hydrogen ch l o r i d e (1.5$, 500 ml). This was prepared by adding 7«8 ml of a c e t y l chloride to 292 ml of methanol which had been d i s t i l l e d over sodium. The s o l u t i o n was refluxed f o r 2 h, allowed to cool and n e u t r a l i z e d with lead carbonate. A f t e r f i l t r a t i o n and concentration, the syrup was disso l v e d i n ethyl acetate (50 ml) and f i l t e r e d to remove any lead carbonate or lead c h l o r i d e . On seeding with a c r y s t a l from a previous synthesis (7), the glycoside c r y s t a l l i z e d overnight; y i e l d 82$. Re c r y s t a l l i z a t i o n from e t h y l acetate gave pure 1_, m.p. 108-109°; 0 3 n -63.2° (c 9.3, water); Rf (solvent B) 0.45; ( l i t . (20,21) m.p. 108-109°} M l j -62.5° (c 9.1, water)); n.m.r. (DO, external TMS)i X 5-35 (1H doublet; J 1 g = 1.5 Hz, H-1), 6.64 (3H s i n g l e t , C-1 OCH^), 8.73 (3H doublet, g = 6 Hz, CH^). 3» Methyl 4-0-Acetyl-2, 3-0-isopropylidene -o(-L-rhamnopyranoside (2) Methyl 2,3-0-isopropylidene-<X-L-rhamnopyranoside (_3_) was prepared e s s e n t i a l l y by the method of Levene and Muskat, with the a d d i t i o n of 2,2-dimethoxypropane (5$ Of the volume of acetone). To a s o l u t i o n of methyl<*-L-rhamnopyranoside (10 g) i n dry acetone (200 ml) containing concentrated s u l f u r i c a c i d (0.4 ml) and 2,2-dimethoxypropane (10 ml), anhydrous copper s u l f a t e (20.g) was added. The mixture was shaken overnight at which time, t . l . c . i n solvent B showed about 90$ formation of 2. A f t e r f i l t r a t i o n , the s o l u t i o n was n e u t r a l i z e d with calcium oxide, f i l t e r e d and concentrated. The syrupy product was acetylated with p y r i d i n e (30 ml) and a c e t i c anhydride (30 ml) e i t h e r f o r 1 h on 70 the steam bath or 6 h at room temperature. The a c e t i c anhydride was then removed by d i s t i l l a t i o n with ethanol; the pyr i d i n e was removed by azeotropic d i s t i l l a t i o n with water. The acetate was c r y s t a l l i z e d from ethanol (50 ml); o v e r a l l y i e l d from 1_ 70$. R e c r y s t a l l i z a t i o n from e.thanol gave pure 2_, m.p. and mixed m.p. 66°, 0*1^  -16.1° (c_ 2.4, chloroform); ( l i t . ( 2 2 ) m.p. 6 1 - 6 3 ° , O Q ^ -14.5° (c 2 . 8 , chloroform)); n.m.r. ( C D C l J i t 5.10 (1H s i n g l e t , E - 1 ) , 6.62 (511 s i n g l e t , C-1 0CH 2), 7.91 (3E s i n g l e t , C-4 OAc), 8.44, 8.66 ( 3 H s i n g l e t s , isopropylidene CH,'s), 8 . 8 3 (5H doublet, J , =6 Hz, CH,). The 60 MHz n.m.r. spectrum 5 5>o 3 i n shown i n Figure 6. An al t e r n a t e synthesis involves use of Amberlite IR120 H r e s i n as the a c i d i c c a t a l y s t as described by Bebault and Button. An important poi n t to note i s that these authors state that the r e s i n was soaked i n acetone overnight p r i o r to use. However, they have now shown i t to be be t t e r to soak the r e s i n i n methanol, as acetone w i l l form polymeric material under the a c i d i c r e s i n conditions. 4. Methyl 2,3-0-Isopropylidene-ot-L-rhamnopyranoside (_3_) Methyl 4-0-acetyl-2, 3-0-isopropylidene-oi-L-rharjnopyranoside (2_) (1.19 g) was di s s o l v e d i n dry methanol (5 ml) and treated with sodium methoxide (0.2 N, 20 Ml) f o r 15 min. Sodium ion was removed from the i c e - c o l d s o l u t i o n with Amberlite IR120 H + r e s i n (presoaked i n methanol); the s o l u t i o n was then also treated with Duolite A4 Oil" r e s i n (presoaked i n methanol) to remove any traces of a c i d . This l a t t e r step was taken as i t was found that concentrated s o l u t i o n s of j) l e f t standing f o r periods of time were degraded to methylc<-L-rhamnopyranoside. The methanol was then evaporated to give a syrupy product j$; y i e l d 1.0 g. 71 C - 2 , 0 - 3 Figure 6 . Methyl 4 - 0-Acetyl - 2 , 3 - 0-isopropylidene-^-L^rhamnopyranoside 72 T . l . c . showed one spot with R f 0.55; -16.8° (c 2.3, acetone); ( l i t . (9,23) 0*1D -15.9° (c 1 .6, acetone); n.m.r. (CDCl^) t 5.14 (1H sing-let, Ii-1), 6.62 (3H s i n g l e t , C-1 OCH^), 8.70 (pH doublet, 5 = 6 Hz, CH^), 8 o 4 8 j 8 0 6 5 (3H s i n g l e t s , endo-, exp- isopropylidene CH^ (24)). The 60 MHz n.m.r. spectrum i s shown i n Figure 7» 5 . 1,2,3 ,4»6-Penta-0-acetyl-^~D-galactopyranose (^) To a suspension of anhydrous sodium acetate (13*2 g) i n a c e t i c anhydride (162 ml) at 100°, anhydrous IVgalactose (30 g) was added with s t i r r i n g over 1 h. The s o l u t i o n was s t i r r e d at 100° f o r 1 h f u r t h e r and allowed to c o o l . Chloroform (100 ml) was added and the s o l u t i o n was poured into i c e water (250 ml), then extracted with chloroform (3 x 50 ml). The chloroform e x t r a c t wa3 washed with sodium bicarbonate (2 x 50 ml) and water (2 x 50 ml). On concentration, the acetate c r y s t a l l i z e d quickly; y i e l d 30.5 g» R e c r y s t a l l i z a t i o n from ethanol gave pure 4, m.p. 142-143°, C04!^ +25.6° (c 2.3, chloroform); ( l i t . (12) m.p. 142°,D*1D +25° ( i n chloroform)); n.m.r. (CDCl^): t: 7 . 8 > 8.00 (15H, OAc's). 6 . 2, 3,4,6-Tetra-0-acetyl-o<-r>-galactopyrano3yl Bromide (j>) Compound 5_ was prepared e s s e n t i a l l y by the method of F l e t c h e r (11) i n about 80$ y i e l d . Compound ^ (15 g) was suspended, i n i c e - c o l d g l a c i a l a c e t i c a c i d (10 ml). G l a c i a l a c e t i c a c i d saturated with hydrogen bromide (30-32$) (25 ml) was added at 0°. The mixture was s t i r r e d a t room temperature f o r 2 h, a t which time a l l the compound 4_ had d i s s o l v e d and t . l . c . i n solvent A showed 1 spot with R^ . 0.55* Chloroform (100 ml) was added and the chloroform s o l u t i o n was poured i n t o i c e (100 g) i n a separatory funnel. The chloroform s o l u t i o n was then washed with 73 C-2, 0-3. isopropylidene 0-6 CH, Eigure 7. Methyl 2,J-O-isopropylidene-^-L-rhamnopyranoside 74 water (50 ml), saturated sodium bicarbonate (3 x 50 ml) and water (2 x 50 nil), and dried over sodium sulfate/calcium s u l f a t e . A f t e r f i l t e r i n g , concentration and s o l u t i o n i n the minimum amount of i c e - c o l d dry ethyl ether, the bromide c r y s t a l l i z e d at 0°. R e c r y s t a l l i z a t i o n from ethyl ether gave pure rp_, m.p. 80-82 °i D*3 +217° (c 1.0, chloroform)} ( l i t . (25) m.p. 83-84°,0*1 +215° (c 1, chloroform)). D Attempts to prepare the bromide by other techniques (26,27) gave much lower y i e l d s . The bromide i s quite unstable, as i t became red-brown, i n colour, due to evolved hydrogen bromide, on storage i n a des i c c a t o r a f t e r 2 days. I t i s thus desirable to use only f r e s h l y prepared bromide f o r the condensation r e a c t i o n . 7. Methyl 4~0-(2, 3,4,6-Tetra-0-.acetyl-|J-12-galactopyranosyl)-2, 3-0-isoprop3'lidene-t<-L-rhamnopyranoside (&) To a s o l u t i o n of methyl 2, 3-0-isopropylidene-o<-L-rhamnopyranoside (j5) (883 mg, 4<>0 mmol) and dry mercuric cyanide (900 mg) i n dry a c e t o n i t r i l e (8.8 ml, d i s t i l l e d over CaHg) was added the bromide 5_ (2.5 g) at once with s t i r r i n g and the s t i r r i n g was continued f o r 1 h a t which time t . l . c . i n solvent Ashowed the r e a c t i o n to be complete. The a c e t o n i t r i l e was evaporated under reduced pressure and the syrup was dissolved i n chloroform (50 ml). The chloroform s o l u t i o n was washed with 1 N potassium bromide (2 x 50 ml), water (50 ml), saturated sodium bicarbonate (3 x 50 ml) and water (2 x 50 ml). The chloroform was evaporated and the syrup, d i s s o l v e d i n hot ethanol (50 ml), c r y s t a l l i z e d on cooling; y i e l d 1.87 g> 3«4 mmol, 85$. R e c r y s t a l l i z a t i o n from ethanol gave pure 6, m.p. 192-193°; l/*! -24.4° (c 2.05, chloroform); R f solvent A) 0.61; n.m.r. (CDCl )i 6.64 (3H s i n g l e t , C-1 OCH,); 7.84-75 8 . 0 2 ( 1 2 H , OAc's), 8.72 (3H doublet, J = 6 Hz, CH ), 3.47, 8.66 5 , b j (3H s i n g l e t s isopropylidene CH^'s). Anal. Calcd. f o r C ^ H ^ O ^ s C, 52.55; H , 6.62. Pound: C, 52.445 H, 6.64. Two points about the condensation re a c t i o n merit a t t e n t i o n . I t was noticed that the isopropylidene group of compound _3_ was hydrolyzed i f care was not taken to remove a l l traces of water and/or a c i d . Also, i f any traces of methanol remained from the preparation of compound 3_» or traces of ethanol present i n chloroform used i n the preparation of compound J5, t h i s would react p r e f e r e n t i a l l y with the bromide. The most e f f e c t i v e means of circumventing these problems was to dry compound j> on the vacuum pump with a heat lamp f o r 1-2 h immediately p r i o r to use i n the condensation reaction, and to use the bromide as a c r y s t a l l i n e material, rather than as a syrup, f o r the r e a c t i o n . 8 . Methyl 4 -0 - (2 , 3 ,4,6-Tetra - 0-acetyl -8-J>galactopyranosyl ) - x3(-L-rhamnopyranoside (j) ~ ~ A s o l u t i o n of methyl 4 -0-(2 , 3,4,6-tetra-0-acetyL-^-3>-galactopyr-anosyl ) - 2 ,3 - 0-isopropylidene-«*-L-rhamnopyranoside (6_) ( 1 .0 g) i n chloroform (45 ml) was treated a t room temperature f o r 1 h with t r i f l u o r o a c e t i c a c i d containing Y/o water (5 ml). The t r i f l u o r o a c e t i c a c i d was removed by d i s t i l l a t i o n with toluene. The remaining syrup showed one spot on t . l . c , (solvent A) 0 . 0 6 and (solvent B) 0 .70; y i e l d 920 mg; n.m.r. ( C B C l j ) : ^ 6.64 (3H s i n g l e t , C-1 OCH^), 7 . 8 4 - 8 . 0 2 ( 1 2 H , OAc's), 8 . 6 8 (3H doublet, J = 6 Hz, CH ). 5,6 3 9. 4 - 0 - ( 2 ,3 , 4 , 6-Tetra - 0-acetyl-S-J>-galactopyranosyl ) - 1 , 2 ,3-tri - 0 -acetyl-«-L-rhamnopyranose ( 8 ) ~ Compound 7_ (900 mg) i n a c e t i c anhydride (5 ml) was shaken with 76 concentrated s u l f u r i c a c i d 2$ (v/v) i n a c e t i c anhydride (10 ml) f o r 3 h (15)<> The re a c t i o n mixture was d i l u t e d with chloroform and the chloroform s o l u t i o n was washed with water (2 z 50 ml), saturated sodium bicarbonate (3 x 50 ml), and again with water (2 x 50 ml). The recovered syrup (825 mg) showed a pajor component on t . l . c . (solvent A) with Bf 0.43, andO*3D -55.4° (c 2.0, chloroform); n.m.r. (CD&j) tf 4.03 (1H doublet, J = 1.5 Hz, H-1), 7.82-8.05 (21H, OAc's), 8.66 (3H doublet, g = 6 Hz, CHj). Attempts to c r y s t a l l i z e t h i s compound a f t e r t . l . C o preparative p l a t e p u r i f i c a t i o n s and s i l i c a gel column chromatography proved unsuccessful. 10. 4-0-^-D-Galactopyranosyl-Lj-rhamnopyranose (j?) Deacetylation of compound ]_ (800 mg) was c a r r i e d out with sodium methoxide (0.2 H, 25 ml) f o r 1 h a t room temperature. Sodium ion was removed with cold Amberlite IR120 If*" r e s i n and the methanol was removed under reduced pressure. The syrup (400 mg) showed one spot on a paper chromatogram run f o r 48 h (solvent C) with R , 0.80; C*3 -8.0° x ' glucose D (c 2.2, water), n.m.r. (DgO, external HIS): X 4.83 (1H doublet, J 1 2 = 1.5 Hz, H-1 of<K-L-form), 5.07 (1H s i n g l e t , H-1 of p-L-form), 5.29 (1H doublet, J 1 t 2 , = 6.8 Hz, H-1»)t 8.60 ( 3 H doublet, J ^ g = 6 Hz, CH^). G.l.c. of the p e r ( t r i m e t h y l s i l y l ) d i s a c c h a r i d e (column a a t 230°) gave two peaks, at 14«4 min (82$) and 17•1 min ( p e r ( t r i m e t h y l s i l y l ) sucrose 22.7 min) (28). 11. Methyl 4-0-^l}-Galactopyranosyl-<*-L-rhaimaopyranoside (lO) Methyl 4-0-(2, 3,4>6-tetra-0^-acetyl-^l>-galactopiyranosyl)-e«-L-77 rhamnopyranoside (690 mg) was deacetylated with sodium methoxide (0.2 H, 30 ml) f o r 1 h at room temperature,, Sodium ion was removed from the i c e - c o l d s o l u t i o n u s i n g Amberlite IR120 H + r e s i n and the methanol was evaporated. T . l . c . (solvent B) showed a major component with Rp 0.11 and a trace of a f a s t e r running compound with R^ . 0.44} y i e l d 400 mg. Ihe syrupy product gave one major spot on a paper chromatogram (solvent C) with R g ] _ u c c s e 2«93;COC3-D -49.1° (c 1.6, methanol)} n.m.r. (DO, external THS)iT5.40 (1H doublet, J = 7.7 Hz, H-1')» . 1 ' j 2' 6.70 (3H s i n g l e t , C-1 OCR,), 8.73 (3H doublet, J = 5.5 Hz, CH..). •> 5,6 3 Reaction of compound 10 (5S.9 rag) with sodium metaperiodate (0.05 M, 100 ml) a t 4° i n the dark showed a r a p i d uptake of 2 mol with the consumption of periodate becoming constant (2.95 mol) i n 24 h (29). Iodate and excess periodate were removed by a d d i t i o n of barium acetate and the r e s i d u a l polyaldehyde was reduced with sodium, borohydride (75 mg). A p o r t i o n of the p o l y a l c o h o l , a f t e r d e i o n i z a t i o n and d i s t i l l a t i o n to dryness with methanol 4 times, was subjected to methanolysis (3/'« HC1 i n methanol, 10 ml, r e f l u x , 6 h) and then n e u t r a l i z e d with Duolite A4 0H~ r e s i n . Glycolaldehyde dimethyl a c e t a l and solvents were removed by evaporation. The syrup showed 2 spots on paper chromatography (solvent D) corresponding to standard 1-deoxy-p-e r y t h r i t o l and g l y c e r o l . G.l.c. of the acetylated material gave (column b at 125°) peaks i d e n t i c a l to authentic standards of the peracetylated d e r i v a t i v e s of 1-deoxy-&-erythritol (10 min) and g l y c e r o l (16 min). The mass spectra of g . l . c . c o l l e c t e d material (30 ) corresponded to those of authentic standards. Methyl glycoside (lp_) (51 • 5 mg) i n dry methyl sulfoxide (1 ml) was 78 methylated (31) by re a c t i o n with methyl s u l f i n y l anion (1.5 M, 1 ml) f o r 4 h and then shaking with methyl iodide (0.5 ml) overnight. The mixture was d i l u t e d with water and extracted with petroleum ether ( 6 5 - 6 8 ° ) . The recovered syrup (11) (46.5 mg) gave one spot on t . l . c . (solvent A) with R f 0.13; -39.0°(c 1.1, chloroform); n.m.r. ( C D C l 5 ) i t : 5.35 (1H doublet, J 1 > 2 = 1.5 Hz, H-1), 5.48 (1H doublet, J = 7.5 Hz), 6.51-6.71 (21H, OCH's), 8.76 (311 doublet, , = 6 Hz, 11,21 2 3,° CHj). Hydrolysis of 1J_ followed by reduction, a c e t y l a t i o n , and i n j e c t i o n onto column c at 225° gave 2 peaks i d e n t i c a l to authentic standards of 1,4,5-tri-0-acetyl-2, 3-di-O-methyl-Ir-rhamnitol (19«7 min) and 1,5-di-C-acetyl-2,3,4,6-tetra-O-methyl-D-galactitol (27.2 min) (16)• The mass spectra corresponded to those of authentic standards. 12. 4-0 -^ - r>-Galactopyranosyl-L-rhanmitol (12) The free disaccharide j3 (150 mg) was reduced with sodium borohydride overnight. A f t e r n e u t r a l i z a t i o n with cation exchange r e s i n , concentration and. d i s t i l l a t i o n with methanol the a l d i t o l J2_ was obtained; R . (solvent C) f o r 48 h 0.53; n.m.r. (D 90, external TKS): *C 5.48 —glucose x '  s <i  1 ^ \ (1H doublet, J 1 l > 2 t = 7»7 Hz, H-1'), 8.71 (3H doublet, g = 6 Hz, CH^). G.l.c. of the p e r ( t r i m e t h y l s i l y l i ) a l d i t o l (column a a t 2 4 0° ) gave one peak at 12.0 min ( p e r ( t r i m e t h y l s i l y l ) s u c r o s e , 16.3 min) (28). The a l d i t o l was acetylated with p y r i d i n e and a c e t i c anhydride to give 4-0-ji-r>galactopyranosyl-L-rhamnitol octaacetate (15) (250 mg, c r y s t a l l i z e d from ethanol (20 ml)). R e c r y s t a l l i z a t i o n from ethanol gave pure 1j), m.p. 114-1150; C«0 -62.8° (c 2.1, chloroform); R ( t . l . c , solvent A) 0.35; n.m.r. (CDCl ) i t 7.84-8.04 (24H, OAc's), 8.67 (JH 79 doublet, J c £ = 6 Hz, CH,). ' Anal. Calcd. f o r C 2 8H 4 00 1 8: C 50.60; H 6.07. Pound: C 50,64; H 6.20. 80 BIBLIOGRAPHY 1. R.V/. Bailey. Oligosaccharides. Perganon Press, London, 19^5• 2. R.J. Beveridge, W.A. Szarek and J.E.I!. Jcnes. Carbohydr. Res. 22. 1°7 (1971). 3. W. ITimmich. 2. Med. Mik r o b i o l . Immunol. 1_5J. 117 (1968); Acta, b i o l . mid. germ. 26 397 (1971). 4. B. Lindberg. unpublished r e s u l t s . 5. B. H e l f e r i c h and J . Z i r n e r . Chem. Per. °J5 2 ( 5 0 4 (1962)-6. V/. Koenigs and E. Knorr. Ber. _3_4 957 (1901). 7. G.M. Bebault and G.G.S. Button. Can. J . Chem. £0 3373.(1973). 8. J . Berry, G.M. Bebault, G.G.S. Dutton and C. Warfield. unpublished r e s u l t s . 9. P.A. Levene and I.E. Muskat. J . B i o l . Chem. J_05_ 431 (1934). 10. R.L. Whistler and M.L. Wolfrorn. Methods Carbohydr. Chem. 2 215 (1963). 11. H.G. Fle t c h e r , J r . Methods Carbohydr. Chem. 2 228 (1963). 12. F.J. Bates ed. Polarimetry, Saccharimetry and the Sugars. U.S. Government P r i n t i n g O f f i c e , Washington. 1942. p. 488. 13. G. Zemplen. Ber. 62 990 (1929). 14. A.J. Acher, Y. Rabinsohn, E.S. Rachaman and D. Shapiro. J . Org. Chrm. -3j5_ 2436 (1970). 15. G.O. A s p i n a l l , R. Kahn and Z. Pawlak. Can. J . Chem. A£ 3000 (1971). 16. K. Bjorndal, C.G. H e l l e r q y i s t , B. Lindberg and S. Svensson. Angew. Chem. Int. Ed. £ 610 (1970). 17. J.N.C. Whyte. Anal. Biochem. 4_2 476 (1971)• 18. W.E. Trevelyan, D.P. Procter and J.S. Harrison. Methods Carbohydr. Chem. 1_ 21 (1962). 19. L. Hough, J.K.N, jones, and W.H. Wadman. J . Chem. Soc. 1702 (1950). 20. E. Fis h e r . Ber. 28 1145 (1895). 21. J . Minsaas. Norska Videnskab. Selskab. Forhandlinger. 6_ 177 (1933)* 81 22. A.C. Richardson and J.Ii. Williams. • Tetrahedron. 2_p_ 1641 (1967). 23. K. Butler, P.F. Lloyd and M. Stacey. J . Chem. Soc. 1531 (1955). 24„ R.D. King and W.G. Overend. Carbohydr. Res. 9_ 423 0 969,). 25. J . Conchie and G.A. Lewy. Methods Carbohydr. Chem. 2 335 (1963). 26. J.K. Dale. J . Am. Chem. Soc. J58 2 1 87 (19 1 6). 27. T. Ishikawa and H.G. Fl e t c h e r , J r . J . Org. Chem. j>4_ 563 (1969). 28. E. P e r c i v a l . Carbohydr. Res. 4 441 (1967)0 29. L. Malaprade. Compt. Rend. 186 382 (1928). 30. G.G.S. Dutton and K.B. Gibney. J . ' Chromatogr. J2 179 (1972). 31. P.A. Sandford and H.E. Conrad. Biochemistry. _5_ 1508 (1966). 

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