Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies on the determination of structural characteristics of three types of hemicelluloses Joseleau, Jean-Paul 1975

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1975_A1 J68_4.pdf [ 6.43MB ]
Metadata
JSON: 831-1.0061081.json
JSON-LD: 831-1.0061081-ld.json
RDF/XML (Pretty): 831-1.0061081-rdf.xml
RDF/JSON: 831-1.0061081-rdf.json
Turtle: 831-1.0061081-turtle.txt
N-Triples: 831-1.0061081-rdf-ntriples.txt
Original Record: 831-1.0061081-source.json
Full Text
831-1.0061081-fulltext.txt
Citation
831-1.0061081.ris

Full Text

STUDIES ON THE DETERMINATION OF STRUCTURAL CHARACTERISTICS OF THREE TYPES OF HEMICELLULOS BY • JEAN-PAUL JOSELEAU Docteur-es-Sciences, University de Grenoble, 1974 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department o f CHEMISTRY We accept t h i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1975 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of <C-U*.t>n.U hry The University of Brit ish Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 - i -A B S T R A C T P a r t I of t h i s t h e s i s d e s c r i b e s the i s o l a t i o n and p u r i f i c a -t i o n of the p r i n c i p a l h e m i c e l l u l o s e from the reed Arundo donax, Mono-cotyledon/ Gramineae. By use of the c l a s s i c a l techniques, a s t r u c t u r a l i n v e s t i g a t i o n demonstrated t h a t t h i s h e m i c e l l u l o s e i s an arabino-4-0-methy1-glucuronoxylan with on average 10 arabino-furanose r e s i d u e s and 5 4-O-methyl-glucopyranuronic a c i d r e s i d u e s o c c u r r i n g as branch p o i n t s w i t h r e s p e c t i v e l y a(l-»-3) and a(l-»-2) l i n k a g e s on the 6(1-^4) l i n k e d backbone of x y l o p y r a n o s y l r e s i d u e s . The r e s u l t s of p e r i o d a t e o x i d a t i o n , m e t h y l a t i o n and osmometry agreed w e l l i n demonstrating an average de-gree of p o l y m e r i z a t i o n of approximately 80. Enzymic h y d r o l y s i s of the p o l y s a c c h a r i d e with an endoxylanase of known s p e c i f i c i t y f o r g(l-»-4) x y l o s i d i c bonds showed t h a t the anomeric nature of the l i n k a g e s between r • the x y l o s e u n i t s was of the 6 c o n f i g u r a t i o n . P.m.r. spectroscopy of the permethylated p o l y s a c c h a r i d e confirmed the above r e s u l t by showing a s t r o n g doublet at p.p.m. 4.24 6 (J 6.5 Hz) corresponding to a B l i n k a g e , and a peak at p.p.m. 5.53 6 corresponding to a anomers. The s t r u c t u r e of the p o l y s a c c h a r i d e was deduced to be of the f o l l o w i n g form : — X y l 4 X y l 1 y 4 X y l — 2 I 3 I | a ja 1UA *Ara In P a r t I I , the i s o l a t i o n of the h e m i c e l l u l o s e s from the wood of Redwood, Sequoia sempervirens, has been c a r r i e d out by the use of d i f f e r e n t methods. T i m e l l ' s method using the s o l u b i l i t y d i f f e r e n c e s - i i -of the hemicelluloses between potassium hydroxide and sodium hydroxi-de allowed the i s o l a t i o n of a glucomannan which was e a s i l y p u r i f i e d by barium hydroxide complexation. For the separation of xylan from galactoglucomannan, a d e l i g n i f i c a t i o n of the polysaccharide mixture was necessary. P u r i f i c a t i o n of the a c i d i c xylan was achieved by com-plexation with cetyltrimethylammonium s a l t , a l s o allowing the recovery of a galactoglucomannan which was further p u r i f i e d by selective p r e c i -p i t a t i o n s with barium hydroxide. The structure of the xylan was deter-mined with emphasis on the methylation procedure. It i s demonstrated that repeated Hakomori methylations on an ac i d i c xylan can be perfor-med without degradation of the main chain. The s t r u c t u r a l features of the xylan consist of a xy l o s y l backbone with an average degree of poly merization of about 5 5 and carrying on average one 4-0_-methyl glucuro-nic acid residue for four xylose residues and one arabinofuranooe r e-sidue every 2 0 xylose residues. A study of the l o c a l i z a t i o n of the uro nic acid substituents was carried out with the use of a s p e c i f i c xyla-nase and was shown not to be randomly distributed on the x y l o s y l main chain. The structures of the glucomannan and galactoglucomannan are also described. Both present the customary structure taht i s a 0 1 1 - ^ 4 ) linked glucomannan with, i n the l a t t e r case, some side chain galactose units ( 1 + 6 ) linked to the main chain. — Man 1 — 4 Glc 1 4 Man 1 4 Man 1 4 Glc 1 6 6 1 1 Gal Gal The results are compared to the data reported i n similar hemicelluloses from coniferous woods and the l i t e r a t u r e for show that i n the pre-- i i i -s ent wood the r a t i o of mannose to glucose i s u n u s u a l l y h i g h . The va-l i d i t y o f the methods of i s o l a t i o n of these p o l y s a c c h a r i d e s i s d i s c u s -sed and i t i s demonstrated t h a t there i s a g r e a t h e t e r o g e n e i t y con-c e r n i n g the composition of the galactoglucomannans p r e s e n t i n t h i s wood. In P a r t I I I , the c e l l w a l l carbohydrate composition of a one-month-old stem from Arundo donax was e s t a b l i s h e d f o r t i s s u e s a t d i f f e -r e n t stages of m a t u r i t y . There i s a r e l a t i o n s h i p between the i n c r e a s e i n x y l o s e and glucose content, the decrease i n arabinose and g a l a c t o s e content, together w i t h the e l o n g a t i o n of the f i b r e s . S i m i l a r changes were a l s o found a t the l e v e l of one e l o n g a t i n g i n t e r n o d e . These va-r i a t i o n s are i n t e r p r e t e d i n terms of p o l y s a c c h a r i d e d e p o s i t i o n i n the c e l l w a l l of the f i b r e s with m a t u r atioa. Each i n t e r n o d e appears as a p h y s i o l o g i c a l u n i t where the p r o g r e s s i v e f i b r o u s c h a r a c t e r of the t i s -sues corresponds to a c h a r a c t e r i s t i c c e l l w a l l p o l y s a c c h a r i d e composi-t i o n . The i s o l a t i o n and p u r i f i c a t i o n of the arabino-4'-CJ-methy 1-glucuro-noxylans,-by stepwise ..alkaline e x t r a c t i o n from t i s s u e s at three d i f -f e r e n t stages of m a t u r i t y , r e v e a l e d t h a t the main change i n the hemi-c e l l u l o s e ' s s t r u c t u r e a c c o r d i n g to growth i s i n the i n c r e a s e of the degree od p o l y m e r i z a t i o n . As the p l a n t matures the average chain l e n g t h of the x y l a n i n c r e a s e s from about 60 i n the youngest internodes to about 150 i n the o l d e r ones. The s t r u c t u r a l f e a t u r e s of the x y l a n which i s a l r e a d y p r e s e n t i n the youngest t i s s u e s are the same r e g a r d -l e s s of the age of the t i s s u e s . More s u b t l e d i f f e r e n c e s , such as the m o d i f i c a t i o n of the s u b s t i t u e n t s o c c u r r i n g on the xylan backbone, were found i n the a c e t y l d i s t r i b u t i o n which i n c r e a s e s , and i n the presence of 4-0-methyl s u b s t i t u e n t s on the g l u c u r o n i c a c i d s which decreases as - i v-the p l a n t ages. In a l l cases examined, the xylose and xylan content of the t i s s u e s reaches a maximum value i n the i n t e r m e d i a t e stage of the maturation and then decreases. The i s o l a t i o n of an exo-xylanase i n the corresponding t i s s u e s suggests t h a t xylans are not only s t r u c t u r a l p o l y s a c c h a r i d e s but a l s o can a c t as reserve elements. - v-TABLE OP CONTENTS Page ABSTRACT - i TABLE OF CONTENTS v LIST OF TABLES i x LIST OF FIGURES x i ACKNOWLEDGMENTS x i l INTRODUCTION 1 HISTORICAL INTRODUCTION 3 I - EXTRACTION AND PURIFICATION 3 1- A l k a l i n e e x t r a c t i o n 4 2- E x t r a c t i o n with dimethyl sulphoxide ..... 4 3- P u r i f i c a t i o n 4 I I - QUALITATIVE AND QUANTITATIVE ANALYSIS 7 1- Chemical h y d r o l y s i s 7 2- Enzymic h y d r o l y s i s 8 3- A n a l y s i s of h y d r o l y s i s products ......... 8 I I I - STRUCTURAL DETERMINATION 9 1- M e t h y l a t i o n a n a l y s i s 9 2- P e r i o d a t e o x i d a t i o n 10 PART I• — THE XYLAN OF ARUNDO DONAX 12 RESULTS a- I s o l a t i o n - P u r i f i c a t i o n 12 - v i -ta- M e t h y l a t i o n study 1 8 c- P e r i o d a t e o x i d a t i o n 3 3 d- N.m.r. spectroscopy 3 3 e- Enzymic h y d r o l y s i s 3 5 DISCUSSION 3 7 EXPERIMENTAL - 4 0 PART I I - HEMICELLULOSES OF REDWOOD 4 7 RESULTS I- ISOLATION OF THE HEMICELLULOSES 4 8 I I T . STRUCTURAL STUDY OF XYLAN S 4 C 2 5 7 a- Carbohydrate a n a l y s i s 5 7 b- M e t h y l a t i o n study 5 7 1 - Hakomori methylations 5 7 2 - M e t h y l a t i o n s by non-degradative methods 6 1 c- P e r i o d a t e o x i d a t i o n 6 2 d- Enzymic h y d r o l y s i s of S ^ C 2 6 7 I I I - STRUCTURAL STUDY OF GLUCOMANNAN . . 6 9 a- Hakomori m e t h y l a t i o n . 6 9 b- S e q u e n t i a l methylations 7 0 c- A n a l y s i s of methylated GM - 7 1 • I V - STRUCTURAL STUDY OF GALACTOGLUCOMANNAN ... 7 4 a- M e t h y l a t i o n of GGM 7 4 b^ A n a l y s i s of methylated GGM 7 6 - v i i -DISCUSSION AND CONCLUSIONS I- ISOLATION AND PURIFICATION OF THE HEMICELLULOSES FROM REDWOOD 79 I I - INTERPRETATION OF STRUCTURE 83 a- Arabino-4-O-methyl—glucuronoxylan 83 b- Mannose-containing h e m i c e l l u l o s e s 85 EXPERIMENTAL PART I I I - CELL WALL CARBOHYDRATES AND STRUCTURAL CHANGES OF XYLANS OF ARUNDO DONAX IN RELATION TO GROWTH 98 RESULTS I- CELL WALL CARBOHYDRATE COMPOSITION AT DIFFERENT STAGES OF MATURITY 100. a- A n a l y t i c a l composition of the 8 i n t e r n o d e s of a 1-month-old reed 100 b- Comparison between the c e l l w a l l carbohydrates o f parenchyma and v a s c u l a r bundles 100 c- B i o m e t r i c study 104 d- V a r i a t i o n s o f carbohydrate c o n s t i t u e n t s i n d e v eloping internodes 106 I I - POLYSACCHARIDES OF ARUNDO DONAX AT THREE STAGES OF MATURITY 108 a- S e l e c t i o n o f the m a t e r i a l 108 b- I s o l a t i o n of the p o l y s a c c h a r i d e s 108 I I I - COMPARATIVE STRUCTURAL STUDY OF XYLANS ORIGINATING FROM TISSUES AT THREE STAGES OF MATURITY 122 IV- ACETYLATED XYLANS 12 7 DISCUSSION AND CONCLUSIONS - v i i i -I - INFLUENCE OF MATURATION OF THE TISSUES ON THE CELL WALL CARBOHYDRATE COMPOSITION 1 2 9 I I - VARIATION OF HEMICELLULOSE CONTENT WITH THE MATURATION OF THE TISSUES 1 3 1 . I I I - COMPARATIVE STRUCTURAL STUDY OF XYLANS AT THREE STAGES OF MATURITY 1 3 4 EXPERIMENTAL • 1 3 8 BIBLIOGRAPHY '. , 1 4 4 - i x -LIST OF TABLES Table Page I. 24% potassium hydroxide e x t r a c t e d - x y l a n a n a l y t i c a l data 15 I I . E x t r a c t i o n of h e m i c e l l u l o s e s from Arundo donax 15 I I I . Composition of f r a c t i o n s B from s e q u e n t i a l e x t r a c t i o n • . 16 I V * F r a c t i o n a t i o n of methylated x y l a n •>..,-,.-.>•...•...19 V. R e l a t i v e i n t e n s i t i e s of the p r i n c i p a l fragments of TMS d e r i v a t i v e s i n mass spectrometry 25 VI. Gas l i q u i d chromatography o f methylated products 28 V I I . P e r i o d a t e o x i d a t i o n o,f xylan from Ar undo 'donax 31 V I I I . H e m i c e l l u l o s l c f r a c t i o n s o b t a i n e d a c c o r d i n g to F i g u r e 9 52 IX. A n a l y t i c a l data of xy l a n ^5 X. F r a c t i o n a l e x t r a c t i o n on methylated S^C^ •• 62 XI.' N e u t r a l sugar a n a l y s i s of the permethy-l a t e d x y l a n S.C^ a/ 62 4 2 X I I . N e u t r a l sugar a n a l y s i s o f the permethy-l a t e d x ylan S 4 C 2 b/ 64 X I I I . P e r i o d a t e o x i d a t i o n on xy l a n S.C_ 65 4 2 XIV. A n a l y s i s o f methylated glucomannan 72 XV. A n a l y s i s 0 f methylated galactoglucomannan .. 75 XVI. Galafitose-glucose-mannose r a t i o i n in t e r m e d i a t e f r a c t i o n s from Redwood ....... 80 XVII. A few examples c f g l u c o s e - d e f i c i e n t galactoglucomannans from gymnosperms 87 - x -XVIII. N e u t r a l sugar composition and l i g n i n content of a l l the internodes from a culm of Arundo donax 101 XIX. Comparative sugar composition of parenchyma c e l l s and v a s c u l a r bundles ... 103 XX. N e u t r a l sugar composition i n three internodes a t d i f f e r e n t l e v e l s of m a t u r i t y 107 XXI. E x t r a c t i o n of h y d r o s o l u b l e p o l y s a c c h a r i d e s 109 XXII. N e u t r a l sugar composition of h y d r o s o l u b l e p o l y s a c c h a r i d e s 110 XXIII to XXX. S e q u e n t i a l e x t r a c t i o n s with a l k a l i n e s o l u t i o n s at d i f f e r e n t c o n c e n t r a t i o n s -A n a l y t i c a l data of the f r a c t i o n s 114 121 XXXI. Sugar composition of p u r i f i e d h e m i c e l l u l o s e s from s e c t i o n s 1 - 3 123 XXXII. M e t h y l a t i o n a n a l y s i s of xylans from 1.0 M KOH e x t r a c t s 124 XXXIII. M e t h y l a t i o n analyses of three xylan f r a c t i o n s i s o l a t e d from s e c t i o n 2 125 XXXIV. A n a l y s i s of DMSO-extracts from s e c t i o n s 1^ -3 128 - x i -LIST OF FIGURES F i g u r e Page 1 - A n a l y s i s of methylated xylan- Gas l i q u i d chromatography of a l d i t o l acetate d e r i v a t i v e s 21 2. A n a l y s i s of methylated x y l a n . Gas l i q u i d chromatography of TMS d e r i v a t i v e s 22 3. Primary fragmentation of a l d i t o l acetate d e r i v a t i v e s of t r i - O - m e t h y l pentoses 24 4. Primary fragments from TMS d e r i v a t i v e s of 2-0- and 2 ,3-di-0-methyl x y l o s e 26 5. P e r i o d a t e consumption by the xy l a n from A rundo donax 30 6. P e r i o d a t e o x i d a t i o n of x y l a n from Arundo donax. Formic a c i d r e l e a s e 32 7. N.M.R. spectrum of methylated x y l a n 34 8. I s o l a t i o n of mannose-containing h e m i c e l l u l o s e s a c c o r d i n g to T i m e l l 49 9. I s o l a t i o n of h e m i c e l l u l o s e s from the wood of Sequoia - Procedure A 50 10. I s o l a t i o n of h e m i c e l l u l o s e s from the wood of Sequoia - Procedure B 54 11. P r i n c i p l e of g - e l i m i n a t i o n on a gl u c u r o n a t e . 60. 12. P e r i o d a t e o x i d a t i o n on S c„ 66 4 2 13. Time course of h y d r o l y s i s of S ^ C 2 b v a xylanase 68 -14. L i g n i n content i n the internodes o f an e l o n g a t i n g stem 102 15. F i b r e l e n g t h d i s t r i b u t i o n i n an e l o n g a t i n g i n t e r n o d e 105 16. E x t r a c t i o n of h e m i c e l l u l o s e s from/tissues a t three stages of ma t u r i t y 112 - x i i -ACKNOWLEDGEMENTS The author wishes to express h i s g r a t i t u d e to h i s re s e a r c h a d v i s o r , P r o f e s s o r G.G.S. Duttoh, f o r h i s i n v a l u a b l e suggestions and encouragement throughout the course of t h i s i n v e s t i g a t i o n . Thanks are a l s o due to P r o f e s s e u r F. Barnoud of the U n i v e r s i t y of Grenoble, f o r h i s help and i n t e r e s t i n the r e s e a r c h d e s c r i b e d i n t h i s t h e s i s . The author a l s o expresses h i s a p p r e c i a t i o n to P r o f e s s o r P. Albersheim, who has k i n d l y agreed to a c t as e x t e r n a l examiner, f o r v a l u a b l e d i s c u s s i o n s d u r i n g the course of t h i s i n v e s t i g a t i o n . He i s l i k e w i s e indebted to Professes. C.A. Mc Dowell f o r h i s hos-p i t a l i t y d u r i n g h i s stay i n the Department of Chemistry and f o r the i n t e r e s t which he showed i n t h i s r e s e a r c h . The author Canada and the support d u r i n g acknowledges with thanks Le C o n s e i l des A r t s du N a t i o n a l Research C o u n c i l o f Canada f o r f i n a n c i a l h i s stay a t the U n i v e r s i t y of B r i t i s h Columbia. -1-I N T R CKD U C T I ON Two d i f f e r e n t types of woody p l a n t m a t e r i a l have been i n v e s t i -gated f o r the n o n - c e l l u l o s i c p o l y s a c c h a r i d e s p r e s e n t i n the c e l l w a l l . The f i r s t i s a gymnosperm t r e e from the American west co a s t , Sequoia sempervirens. The s t r u c t u r a l examination of i t s h e m i c e l -l u l o s e s i s p a r t of a program i n P r o f e s s o r Dutton's l a b o r a t o r i e s i n Vancouver to e s t a b l i s h a chemotaxonomic c l a s s i f i c a t i o n amongst the order C o n i f e r a l e s (1). T h i s p l a n t m a t e r i a l was a l s o chosen because of i t s s u i t a b i l i t y f o r f u t u r e i n v e s t i g a t i o n s i n P r o f e s s o r Barnoud's group i n Grenoble (France) on the b i o s y n t h e s i s of c e l l w a l l po-l y s a c c h a r i d e s using i n v i t r o c u l t u r e s . Such s t u d i e s have alr e a d y been made on l i g n i n b i o s y n t h e s i s (2) The second p l a n t used i n t h i s study c o n s i s t s of a r e e d , Arundo  donax L. c u l t i v a t e d i n the a g r i c u l t u r a l centre of M o n t p e l l i e r (France) and having an economic i n t e r e s t as a source of pulp f o r the paper i n d u s t r y . A c o n s i d e r a b l e amount of work has a l r e a d y been done on the c o n d i t i o n s of growth of t h i s p l a n t as w e l l as on i t s p u l p i n g c h a r a c t e r i s t i c s ( 3 ) . The reed Arundo donax i s p a r t of the Gramineae f a m i l y i n the group of Monocotyledons. In t h i s group the replacement of t r a c h e i d s by v e s s e l s i n d i c a t e s a p h y l o g e n e t i c advance, and on the evidence of t h e i r v e s s e l s t r u c t u r e the Gramineae are considered to be at a very advanced p h y l o g e n e t i c l e v e l (4,5). - 2 -Frcm the p o i n t of view of a study of the e v o l u t i o n of the h e m i c e l l u l o s e s according to growth, Arurido donax represents an i n t e r e s t i n g m a t e r i a l i n that i t has a r e l a t i v e l y s h o r t v e g e t a t i v e c y c l e , c o n s i d e r e d as two ye a r s , and shows an extremely r a p i d r a t e of e l o n g a t i o n during the f i r s t months of i t s growth. Such a p l a n t should p r o v i d e , w i t h i n a p r o p e r l y chosen i n t e r n o d e , a l l the stages of m a t u r i t y . I t was thus a p p r o p r i a t e i n both cases to study the h e m i c e l l u -l o s e s i n a mature p l a n t before s t u d y i n g the c h a r a c t e r i s t i c s of these p o l y s a c c h a r i d e s at an e a r l y stage of m a t u r i t y . -3-H I S T O R I C A L I N T R O D U C T I O N The s t r u c t u r a l i n v e s t i g a t i o n of a p o l y s a c c h a r i d e can be d e p i c -ted i n a few main phases : - e x t r a c t i o n and p u r i f i c a t i o n - q u a l i t a t i v e and q u a n t i t a t i v e a n a l y s i s of the d i f f e r e n t sugar u n i t s c o n s t i t u t i n g the polymer - determination of the nature and p o s i t i o n of the l i n k a g e s b e t -ween the u n i t s - e s t i m a t i o n of the chain l e n g t h of the chain backbone and d e t e r mination of the number o f branch p o i n t s and the nature of b r a n c h i n g s . The p o l y s a c c h a r i d e s i n v e s t i g a t e d i n t h i s work are of the fami-l y of the h e m i c e l l u l o s e s d e f i n e d as"those p l a n t p o l y s a c c h a r i d e s o t h e r than c e l l u l o s e which are unextracted by water or ammonium ox a l a t e s o l u t i o n but which are e x t r a c t e d by aqueous a l k a l i " ( 6 ) . Since numerous authors have reviewed the problems of the s t r u c t u r a l study of p o l y s a c c h a r i d e s (6-10), only a b r i e f survey o f the methods which were used i n the present study w i l l be p r e s e n t e d . I - EXTRACTION AND PURIFICATION These two d i f f e r e n t steps are c l o s e l y r e l a t e d and i n some i n s -tances the mode o f e x t r a c t i o n can be i n i t s e l f a p u r i f i c a t i o n . -4-I - 1 - A l k a l i n e e x t r a c t i o n T h i s e x t r a c t i o n i s u s u a l l y performed by means of a 17.5% sodium hydroxide s o l u t i o n or a 24% potassium hydroxide s o l u -t i o n . When the wood con t a i n s a r a t h e r high percentage of l i g n i n , the a l k a l i n e e x t r a c t i o n cannot be c a r r i e d out d i r e c t l y on the wood-meal and a p r e v i o u s d e l i g n i f i c a t i o n i s necessary. The most u n i v e r -s a l l y used method of d e l i g n i f i c a t i o n i s that suggested by Jayme e t a l . (11) and f u r t h e r developed by Wise et a l . (12) using an aque-ous sodium c h l o r i t e - a c e t i c a c i d s o l u t i o n at 75°. The r e s u l t i n g m a t e r i a l , c a l l e d h o l o c e l l u l o s e , i s t h e o r e t i c a l l y c o n s t i t u t e d of c e l l u l o s e and h e m i c e l l u l o s e s . The h e m i c e l l u l o s e s can then be s o l u -b i l i z e d with a l k a l i n e s o l u t i o n s and the f i n a l r e s i d u e r epresents the g - c e l l u l o s e . I - 2 - E x t r a c t i o n with dimethyl: sulphoxide (DMSCQ During the course o f a l k a l i n e e x t r a c t i o n , the e s t e r i -f i e d c a r b o x y l groups of the h e m i c e l l u l o s e s are s a p o n i f i e d . The use of DMSO, a good p o l y s a c c h a r i d e s o l v e n t , as an agent of e x t r a c -t i o n , was i n t r o d u c e d by Lindberg e t a l . (13) and presents the ad-vantage of p r e s e r v i n g the a c e t y l groups g e n e r a l l y c a r r i e d by the xylans and galactoglucomannans. I - 3 - P u r i f i c a t i o n A l k a l i n e e x t r a c t i o n g i v e s r i s e t o a mixture of p o l y -s a c c h a r i d e s where one s p e c i e s may predominate depending upon the e x t r a c t i n g c o n d i t i o n s . T h i s p u r i f i c a t i o n can be achieved e i t h e r during the course of the e x t r a c t i o n step or a f t e r t h e . e x t r a c t i o n seep. -5-- During the course of the e x t r a c t i o n : Due to t h e i r s o l u b i l i t y d i f f e r e n c e s , c e r t a i n p o l y s a c c h a r i d e s can be s e l e c -t i v e l y e x t r a c t e d by v a r y i n g the c o n c e n t r a t i o n s of a l k a l i i n such a way t h a t a f r a c t i o n a t i o n as w e l l as an exhaustive e x t r a c t i o n are achieved. The p r e f e r e n t i a l s o l u b i l i t y of d i f f e r e n t h e m i c e l l u l o s e s i n sodium or potassium hydroxide s o l u t i o n s has a l s o been used. T i m e l l (14) made use of the p r o p e r t y of xylan and galactoglucomannan being more s o l u b l e i n 24 % potassium hydroxide than glucomannan to separate the l a t t e r with a 17.5 % sodium hydroxide solution with a certair s e l e c t i v i t y . By t h i s means,a f i r s t step of p u r i f i c a t i o n was c a r r i e d out a t the same time as the e x t r a c t i o n . - A f t e r e x t r a c t i o n : P o l y s a c c h a r i d e s are u s u a l l y p r e c i p i t a t e d from the a l k a l i n e e x t r a c t by a c i d i f i e d e t h a n o l . In the case of xylan i t i s p o s s i b l e to f r a c t i o n a t e the h e m i c e l l u l o s e s p r e -sent i n the e x t r a c t by d i r e c t n e u t r a l i z a t i o n with a c e t i c a c i d which y i e l d s the s o - c a l l e d h e m i c e l l u l o s e A c o n s i s t i n g of a high molecular weight f r a c t i o n ; i n a second step the e t h a n o l p r e c i p i t a t i o n g i v e s the h e m i c e l l u l o s e B c o n s i s t i n g of an a c i d i c glucuronoxylan (15). P u r i f i c a t i o n s based on chemical p r o p e r t i e s of the p o l y s a c c h a -r i d e s are numerous and v a r i e d . We w i l l only mention here the most commonly used f o r the s e p a r a t i o n and p u r i f i c a t i o n of x y l a n , g l u c o -mannan and galactoglucomannan. From a crude p r e p a r a t i o n of h e m i c e l l u -l o s e B, W h i s t l e r (16,17) i s o l a t e d three d i f f e r e n t p o l y s a c c h a r i d e s by f r a c t i o n a l p r e c i p i t a t i o n with i n c r e a s i n g c o n c e n t r a t i o n s of etha-n o l . T h i s method sometimes gives good r e s u l t s but the l i m i t s of p r e c i p i t a t i o n are not always c l e a r l y d e f i n e d and c o - p r e c i p i t a t i o n s can occur. A v a r i a t i o n of t h i s procedure i s to use d e r i v a t i v e s of -6-the polymers such as n i t r a t e , acetate o r methyl e t h e r s . Good r e s u l t s have been obtained by f r a c t i o n a l p r e c i p i t a t i o n with o r g a n i c s o l v e n t s i n the presence o f metal ions which form i n s o l u -b l e s a l t s with u r o n i c a c i d — c o n t a i n i n g p o l y s a c c h a r i d e s , or s e l e c t i -ve complexes with c e r t a i n sugars. A good example o f the l a t t e r i s given by p r e c i p i t a t i o n o f mannose-rich p o l y s a c c h a r i d e s by ethanol t i t r a t i o n i n the presence of barium ions (1SJ . Complexation with metals is of wide a p p l i c a t i o n . F e h l i n g ' s s o l u t i o n (19,20) has been employed f o r the formation of copper complexes f o r f r a c t i o n a t i o n between d i f f e r e n t types of p o l y s a c c h a -r i d e s as w e l l as f o r p u r i f i c a t i o n . I n s o l u b l e complexes f o r the s e p a r a t i o n of mannose and g a l a c t o s e - c o n t a i n i n g p o l y s a c c h a r i d e s were i n t r o d u c e d by Meier (21) and Hamilton et a l . (22) and r e p r e s e n t the b e s t method a v a i l a b l e f o r s e p a r a t i o n of galactoglucomannan from xylan and f o r t h e i r p u r i f i c a t i o n ( 1 4 ) . Quaternary ammonium s a l t s form i n s o l u b l e complexes with a c i d i c p o l y s a c c h a r i d e s (23,24) but have a l s o been used f o r the s e p a r a t i o n of an a r a b i n o g a l a c t a n i n t o two f r a c t i o n s of d i f f e r e n t molecular weights ( 2 5 ) . Amongst the p h y s i c a l methods, chromatography on a v a r i e t y of supports has been t r i e d . Poor s e p a r a t i o n s were achieved on d i f f e r e n t types of Sephadex g e l s ( 2 6 ) . The p r i n c i p a l use o f column chromato-graphy was/"for the s e p a r a t i o n of n e u t r a l and a c i d i c polymers on D E A E - c e l l u l o s e (27) . -7-I I - QUALITATIVE AND QUANTITATIVE ANALYSIS There Is only one way of determining the sugar composition o f a p o l y s a c c h a r i d e : h y d r o l y s i s - . The b r e a k i n g down to small u n i t s can be achieved e i t h e r chemically or e n z y m i c a l l y . . I I - 1 - Chemical h y d r o l y s i s A major problem i n a c i d h y d r o l y s i s of a polysaccharide i s the q u a n t i t a t i v e release of the c o n s t i t u t i n g monomers. A l l sugars are more or l e s s degraded by a c i d . Degradations depend on the nature of the sugar, i t s mode of linkage i n the macromolecule and the h y d r o l y t i c c o n d i t i o n s ; temperature, nature of the a c i d , c o n c e n t r a t i o n and length of h y d r o l y s i s . There are no absolute q u a n t i t a t i v e methods. Loss oecuring during h y d r o l y s i s must be evaluated f o r each sugar according to the c o n d i t i o n s used. One of the most widely used methods i s that developed by Saeman and co-workers (28) and r e c e n t l y adapted by R i c h a r d s et ai.(2.9) c o n s i s t i n g of a pretreatment with 72 % s u l p h u r i c a c i d at room temperature and r e f l u x i n 1K: s u l p h u r i c a c i d . H y d r o c h l o r i c a c i d i s mostly used f o r g l y c o p r o t e i n s but causes more degradations. More r e c e n t l y Albersheim et a l . (30) hydrolysed c e l l w a l l p o lysaccharides with 2M t r i f l u o r o a c e t i c a c i d under r e f l u x . This method o f f e r s the advan-tage of avoiding the problems of n e u t r a l i z a t i o n of the excess of t r i f l u o r o a c e t i c a c i d making i t e a s i l y removable. A drawback of-', t h i s method Is that h y d r o l y s i s i s incomplete i n s e v e r a l cases (29, 31). i n our own experience we found s e v e r a l instances where hydro-• 8 -l y s i s by t h i s method was not t o t a l even i n the case of o l i g o -s a c c h a r i d e s of xylose (32). II - 2 - Enzymic h y d r o l y s i s Enzymic h y d r o l y s i s can be considered as a com-plement of the chemical methods. The s e l e c t i v i t y of the enzyme f o r a 4 e f * n i t e t v P e of l i n k a g e and the mode of a c t i o n of the en-zyme l e a d to o l i g o s a c c h a r i d e s which are of great i n t e r e s t f o r the determination of the s t r u c t u r e of a p o l y s a c c h a r i d e . Thus, Bishop and Whitaker (33) c o u l d make a d i s t i n c t i o n between two p o s s i b l e s t r u c -t u r e s of a x y l a n from wheat straw. Another example was given by A s p i n a l l e t a l . (34). II - 3 - A n a l y s i s of the h y d r o l y s i s products A l l k i n d s o f chromatographic methods have been used> but mostly paper and gas l i q u i d chromatography. S o l v e n t systems are very v a r i e d and allow the r e s o l u t i o n of almost any mixture of sugars. But s i n c e 1S58 (35) the technique of gas l i q u i d chromatography of sugars improved c o n s i d e r a b l y and became the main t o o l f o r sugar d e t e r m i n a t i o n . Since carbohydrates are not s u f f i c i e n t l y v o l a t i l e , d e r i v a t i v e s such as methyl e t h e r s , a c e t a t e s t r i f l u o r o a c e t a t e s or t r i m e t h y l s i l y l ethers must be used. An exhaus t i v e review o f t h e i r a p p l i c a t i o n was r e c e n t l y given by Dutton (36) -9-I I I - STRUCTURAL DETERMINATION I I I - 1 ^ Methylation a n a l y s i s . This i s the most general method f o r o b t a i n i n g such inform mation as the p o s i t i o n of the d i f f e r e n t monosaccharides u n i t s i n a polymer, the number and type of branch p o i n t s , the mode of linkage and an e s t i m a t i o n of chain l e n g t h . U n t i l 1964, date of the p u b l i c a t i o n by Hakomori of a new and e f f i c i e n t method of me-t h y l a t i o n , the permethylation of a polysaccharide was a long and d e l i c a t e o p e ration. Several p o s s i b l e methods could be used ; methyl sulphate i n the presence of sodium hydroxide(Haworth (37)) f methyl i o d i d e i n the presence of s i l v e r oxide (Purdie (38)) ; methyl iodj.de and s i l v e r oxide i n the p o l a r solvent N,N-dimethyl f o r -mamide (Kuhn (39)) and s e v e r a l m o d i f i c a t i o n s of these methods (40). The i n s p i r a t i o n of Hakomori's procedure i s the r e a c t i o n described by Corey and co-workers (41) between dimethyl sulphoxide and sodium hydride which gives a methylsulphiny1 carbanion c h a r a c t e r i z e d by a high n u c l e o p h i l i c i t y and a strong deprotonating a c t i v i t y on the hydroxyl groups. The methylating agent i n the method of Hakomori (42) i s methyl iodide i n DMSO. A few r e s t r i c t i o n s have been made pn the u t i l i z a t i o n of t h i s method and w i l l be discussed l a t e r , Though the l a t t e r method brought a considerable improvement, permethyla-t i o n i s very seldom achieved i n one s i n g l e step. One reason f o r t h i s i s the d i f f e r e n c e i n the r e a c t i v i t y of the hydroxyl groups i n a saccharide molecule as demonstrated by Handa and Montgomery (43) . The order of r e a c t i v i t y of the hydroxyl v a r i e s according to -10-the procedure used and to the previous degree of methylation . I t i s thus advisable to vary the types of procedures when dea l i n g with a polysaccharide d i f f i c u l t to methylate. The i d e n t i f i c a t i o n of the numerous p a r t i a l l y methylated d e r i v a t i v e s obtained by h y d r o l y s i s has been considerably improved by the use of gas l i q u i d chromatography and mass spectroscopy. However the separation of a l l the components by gas l i q u i d chromatography i s not always p o s s i b l e . Many compounds which are not separable by gas l i q u i d chromatography of t h e i r p a r t i a l l y methylated a l d i t o l acetates can be separated as t h e i r p a r t i a l l y e t h y l a t e d a l d i t o l acetate d e r i v a t i v e s as shown by Albersheim and h i s colleagues i n a recent pu-b l i c a t i o n (47,48). These authors demonstrated th'@ use of pere thy l a t ion of the polysaccharide p a r a l l e l to the perm'e-t h y l a t i o n using an i d e n t i c a l procedure of e t h e r i f i c a t i o n . I l l - 2 - Periodate o x i d a t i o n Periodate o x i d a t i o n i n v o l v e s the r e a c t i o n of cleavage of the C-C bond between two v i c i n a l hydroxyl groups with formation of two carbonyl fonctions and consum-p t i o n of a molar p r o p o r t i o n of periodate (44) . In the case of a y $ /y— t r i o l s , there i s consumption of two moles of p e r i o -date, formation of two aldehyde groups and release of one mole of formic a c i d . Such a s i t u a t i o n i n a polysaccharide w i l l be the i d e n t i f i c a t i o n of a. terminal non reducing sugar or of (l—*-6) l i n k e d hexopyranose r e s i d u e s . Thus the consumption of p e r i o d a t e , the number of uncleaved residues and the f o r -mic a c i d release give information on the linkages e x i s t i n g - 1 1 -i n a p o l y s a c c h a r i d e ( 4 5 ) . When p e r i o d a t e o x i d a t i o n i s f o l l o w e d by b o r o h y d r i d e r e d u c t i o n of the p o l y a l d e h y d e and by hydro-l y s i s , an e v a l u a t i o n of the c h a i n l e n g t h of the polymer i s g i v e n by e s t i m a t i o n o f the p o l y o l s r e l e a s e d . An i m p o r t a n t m o d i f i c a t i o n o f the p r e c e d i n g sequence o f r e a c t i o n s ^ a n d known as the "Smith d e g r a d a t i o n " (46) tmakes use o f the d i f -f e r e n t s t a b i l i t y t o a c i d h y d r o l y s i s between a c y c l i c a c e t a l and g l y c o s i d i c l i n k a g e s . - 1 2 -PERIODATE OXIDATION AND METHYLATION OF A.XYLAN A r a b i n o - 4 - O - m e t h y 1 - g l u c u r o n o x y l a n -13-P ART 1 Thi s chapter w i l l be concerned with the i s o l a t i o n and s t r u c t u r a l study of the p r i n c i p a l h e m i c e l l u l o s e s p r e s e n t i n the culm of a monocotyledon Gramineae, Arundo donax L. 1 - THE XYLAN 0? ARUNDO DONAX. We used as the s t a r t i n g m a t e r i a l f ield-grown reeds c u l t i -vated at M o n t p e l l i e r . Since t h i s p l a n t has a s h o r t v e g e t a t i v e c y c l e , one-year-old reeds can be considered as mature. RESULTS a - I s o l a t i o n - P u r i f i c a t i o n . Internodes of the culms o f one-year-old reeds were e x t r a c -ted i n two d i f f e r e n t ways to o b t a i n the h e m i c e l l u l o s e s : - 24% potassium hydroxide e x t r a c t i o n . L i p i d s and pigments were e x t r a c t e d from the sawdust by ethanol-benzene (1/1 v/v) i n a sox h i e t apparatus when 6.5 % of the m a t e r i a l was s o l u b i l i z e d . The l i g n i n content, as determined by Klason's procedure, (49) was 19 % and showed t h a t a d e l i g n i f i c a t i o n was necessary p r i o r to -14-h e m i c e l l u l o s e e x t r a c t i o n . D e l i g n i f i c a t i o n was achieved by sodium c h l o r i t e b u f f e r e d with a c e t i c a c i d . The r e s u l t i n g holo-c e l l u l o s e on e x t r a c t i o n with 24 % potassium hydroxide y i e l d e d a mixture of h e m i c e l l u l o s e s which was separated i n t o a hemicel-l u l o s e A , obtained by d i r e c t n e u t r a l i z a t i o n with a c e t i c a c i d and •a h e m i c e l l u l o s e B obtained by ethanol p r e c i p i t a t i o n . The hemicel-l u l o s e A p r e c i p i t a t e d as a l i g h t p r e c i p i t a t e , which could only be c o l l e c t e d by high speed c e n t r i f u g a t i o n (47fOOOg)• These d r a s t i c c o n d i t i o n s of c e n t r i f u g a t i o n could e x p l a i n the unusual y i e l d of h e m i c e l l u l o s e A ( 2 5 %) with respect to the h e m i c e l l u l o s e B ( 8 % ) . Paper chromatography of the a c i d hydrolysate showed i n both f r a c t i o n s xylose as the major sugar, arabinose, and traces of glucose (Tables I and I I I ) , a small amount of a c i d i c sugars was a l s o present. In order to check i f the presence of r e s i d u a l p e c t i n s had i n f l u e n c e d the e x t r a c t i o n of h e m i c e l l u l o s e s , the h o l o c e l l u l o s e was t r e a t e d with hot water ( 9 0 ° ) f o r s i x hours and e x t r a c t e d with 2 4 % potassium hydroxide as before, g i v i n g a h e m i c e l l u l o s e c a l l e d A ( J ^ O ) . i n an other attempt the h o l o c e l l u l o s e was t r e a -ted s u c c e s s i v e l y with hot water ( 9 0 ° ) and 0 . 5 % ammonium oxalate ( 8 0 ° , 6h.) and gave by e x t r a c t i o n with 2 4 % potassium hydroxide the h e m i c e l l u l o s e A (ox.). A n a l y t i c a l data are given i n Table I . - In the second method of e x t r a c t i o n , hydrosoluble polysaccharides were removed p r i o r to d e l i g n i f i c a t i o n , thus a l l o w i n g a b e t t e r p e n e t r a t i o n of the reagents. The sawdust was t r e a t e d s u c c e s s i v e l y - 1 5 -TABLE I - 24% POTASSIUM HYDROXIDE EXTRACTED XYLAN - ANALYTICAL DATA * F r a c t i o n Arabinose Xylose Xylan A 7.2 92.5 Xylan A(1I 20) 6.5 93.0 Xylan A(Ox.) 6.3 . 93.2 & Determined by colorimetry (56) . TABLE I I - EXTRACTION OF HEMICELLULOSES - ARUNDO DONAX •-FROM KOH e x t r a c t i o n F r a c t i o n Y i e l d 20 %* Ash % l a l D («) " 4.3 M . A B 25 8 6.2 - 8 3 0.2 M A 0.9 B 4.8 10.8 - 101 1 .0 M A 0.2 B 11.4 7.5 •90 2.5 M A 0.7 B 8.3 .8.2 90 x on a dry weight b a s i s *s 0.1% s o l u t i o n (ash free) i n 1.0' M NaOH TABLE III COMPOSITIONS OF FRACTIONS B FROM SEQUENTIAL EXTRACTION Arabinose Xylose Uronic acid (moles) (moles) pic ac (g %) Fraction B 10 .89 0.2 M KOH Fraction B 10 89 6.1 1.0 M KOH Fraction B 11 88.5 2.5 M KOH * Values established by gas l i q u i d chromatography of the trimethyl s i l y l derivatives • In anhydro glucuronic acid residues as estimated by decarboxylation - 1 7 -with hot water and 2 % ethylene diamine t e t r a c e t i c a c i d (EDTA) at 70 %, and submitted to' c h l o r i t e d e l i g n i f i c a t i o n . The h o l o c e l -l u l o s e was then extracted with potassium hydroxide s o l u t i o n s of i n c r e a s i n g concentrations : 0.2 M; 1.0 M and 2.5 M. Each e x t r a c t y i e l d e d two h e m i c e l l u l o s e s A and B, obtained as before. In the ~ three cases, f r a c t i o n B was the most abundant (4.8 %, 11.4 % and 8.2 % r e s p e c t i v e l y ) and h e m i c e l l u l o s e A was obtained i n low y i e l d (0.95 %, 0.23 % and 0.70 % r e s p e c t i v e l y ) (Table I I ) . The a n a l y t i c a l data (Tables I - I I I ) show that i n s p i t e of d i f f e r e n t modes of e x t r a c t i o n the monosaccharide composition of the h e m i c e l l u l o s e remained f a i r l y constant. An attempt at f r a c -t i o n a l p u r i f i c a t i o n by ethanol i n the absence and the presence of calcium ions (50), d i d not make any s i g n i f i c a n t d i f f e r e n c e i n the sugar composition of the h e m i c e l l u l o s e . With reference to the d e f i n i t i o n given by Reid and W i l k i e (51) of the term "pure h e m i c e l l u l o s e " , as a " h e m i c e l l u l o s i c m a t e r i a l where p o l y d i v e r s i t y has been avoided", and according to t h e i r a n a l y t i c a l data, the h e m i c e l l u l o s e s extracted by e i t h e r method can be considered as pure. Only f r a c t i o n B e x t r a c t e d with 24 % p o t a s s i u m h y d r o x i d e showed a c e r t a i n d i f f e r e n c e with the other h e m i c e l l u l o s e s B. This d i f f e r e n c e could be explained by the f a c t that the reeds c o n s t i t u t i n g the s t a r t i n g m a t e r i a l had been harvested at d i f f e r e n t times, and i t has been suggested that a seasonal f a c t o r i n f l u e n c e s the composition of h e m i c e l l u l o s e s i n p l a n t s (52). Another l i k e l y e xplanation i s that the monosac-charides from the. 24 % potassium hyaroxide e x t r a c t have been determined c o l o r i m e t r i c a l l y and those from the other e x t r a c t s - 1 8 -have been estimated by gas l i q u i d chromatography of t h e i r t r i m e t h y l s i l y 1 d e r i v a t i v e s ( 5 3 ) . I t was decided that the s t r u c t u r a l study of the xylan from Arundo donax would be c a r r i e d out on the h e m i c e l l u l o s e B from the 1.0 M potassium hydroxide e x t r a c t which showed the greatest p u r i t y and was the most abundant.. This polysaccharide i s a 4-0-methyl-glucurono—arabino-xylan as shown by the presence of 4r0-methyl-p_-glucuronic a c i d ( i d e n t i f i e d by the Rf of the aldobiouronic a c i d on paper chroma-tography) . The uronic a c i d content was estimated by decarboxyla-t i o n followed by conductimetry according to Bylund and Donetzhuber- (54),a m o d i f i c a t i o n of the c l a s s i c a l d e c a r b o x y l a t i o n method which presents the advantage of taking i n t o account the carbon dioxide a r i s i n g from the decarboxylation of n e u t r a l sugars. The uronic a c i d content of the xylan was 6.1 % as expressed i n g % arid 3.75 % as expressed i n moles of 4~0 _methyl g l u c u r o n i c a c i d per anhydroxylose u n i t s . Thus, the molar sugar composition of the xylan i s : xylose : 86.5 - arabinose : 9.7 - 4-0-methyl glucuronic a c i d : 3.6. b - Methy_lation_s tudy^ -The xylan was methylated according to Hakomori (42) using the procedure of Sandford and Conrad (55). since a perjsethylated polysaccharide could not be obtained i n a s i n g l e step, a second -19-TABLE IV - FRACTIONATION OF METHYLATED XYLAN FROM ARUNDO DONAX F r a c t i o n c h l o r o f o r m / y i e l d methoxyl ash p e t r o l e u m e t h e r v/v mg . % % 1 0 : 10 0 2 10 : 90 112-0 3 15 : 75 143.5 0.52 4 * 20 : 80 113.5 32 .9 0.13 5 25 : 75 * 499.5 37.4 0.23 6 30 : 70 341 -0 36.7 0.37 -20-i d e n t i c a l m e t h y l a t i o n was performed, and the i n f r a r e d spectrum of the p r o d u c t showed no h y d r o x y l a b s o r p t i o n i n the 3 450 cm-1 r e g i o n . L i n d b e r g and coworkers have recommended (56) the a v o i -dance of r e p e a t e d t r e a t m e n t s i n the d r a s t i c b a s i c c o n d i t i o n s o f the Hakomori p r o c e d u r e , p a r t i c u l a r l y i n the case o f a p o l y s a c -charide c o n t a i n i n g u r o n i c a c i d r e s i d u e s s u b s t i t u t e d a t the p o s i -t i o n 4 s i n c e a 3 - e l i m i n a t i o n can occur when the p a r t i a l l y methylated polysaccharide i s re-exposed to the methyl s u l p h i n y l anion. We w i l l show i n the next chapter t h a t such a procedure had a c t u a l l y no adverse e f f e c t on the r e s u l t s that we derived from the methylation study (see methylation study of the xylan from Sequoia) . The methylated products were f r a c t i o n a t e d by s e q u e n t i a l e x t r a c t i o n with chloroform- l i g h t petroleum, and the f r a c t i o n 25 : 75 (v/v) gave i n a y i e l d of 20 % a product having a methoxyl content of 37-38 % as determined by the method of Z e i s e l (Table I V ) . This degree of s u b s t i t u t i o n i s lower than the expec-ted t h e o r e t i c a l v alue, but d i d not increase i n s p i t e of a d d i t i o -n a l methylations by Purdie's procedure. This f r a c t i o n was thus considered as permethylated and used f o r subsequent a n a l y s i s . The methylated xylan was hydrolysed and n e u t r a l and a c i d i c sugars separated on i o n exchange columns. The n e u t r a l sugars were examined by paper chromatography and showed four components cor-responding i n Rf values to tri-O-methyl-L-arabinose and t r i - 0 -methy1-D-xylose (Rf 0.91 and 0.87 unresolved s p o t s ) f di-O-methyl-D-xylose (Rf 0.56) and mono-0-methy1-D-xylose (Rf 0.19). FIGURE 2 - ANALYSIS OF METHYLATED XYLAN - GAS LIQUID CHROMATOGRAPHY OF THE TRIMETHYLSILYL ETHER DERIVATIVE a : tri-O-methyl-arabinose + tri-O-rnethyl-xyloae b : di~0_-methy 1-xylose c : 3-O-methyl-xylose d : 2-O-methyl-xylose -23-The fourth spot (Rf 0.46) migrating at a s l i g h t l y lower rate than the di-O-methyl xylose and shov/ing a very weak i n t e n s i t y was not. f u r t h e r c h a r a c t e r i z e d , but could be considered as an a r t e f a c t due to the e p i m e r i z a t i o n o f the di-O-methyl xylose i n t o di-O-methyl lyxose occuring during the n e u t r a l i z a t i o n o f the hydrolysate by barium carbonate. I t has been shown that barium carbonate contains enough free base f o r such an e p i m e r i z a t i o n to take place ( 5 7 ) . A more accurate determination of the nature of the 0-methyl sub-s t i t u t e d monosaccharides was achieved by gas l i q u i d chromatogra-phy of t h e i r a l d i t o l acetate d e r i v a t i v e s (58) ( F i g . l ) ' Retention times corresponded r e s p e c t i v e l y to 1,4-di-0-acety1-2 , 3 , 5 - t r i - 0 - m e t h y l - a r a b i n i t o l ; 1,5-di-0-acetyl- 2,3,4-tri-O-m e t h y l - x y l i t o l ; 1, 4,5-tri-0-acety1- 2 , 3 - d i - 0 - m e t h y l - x y l i t o l ; and an unres olvedmixture of tetra-0_-acetyl-mono-0-methyl-xylitols . I d e n t i f i c a t i o n of the methylated sugars was confirmed by gas l i q u i d chromatography of the t r i m e t h y l s i l y l d e r i v a t i v e s of t h e i r unreduced form. The chromatograph so obtained has the disadvantage of being more complex due to the presence of the anomeric forms, but allows the d i s t i n c t i o n and i d e n t i f i c a t i o n of the unresolved xacnomethyl ethers of xylose as the 2^C_-methyl and 3-0-methyl xylose ( F i g . 2 ) . A more d e f i n i t e c h a r a c t e r i z a t i o n of the methylated sugars was made by mass spectrometry of the d i f f e r e n t d e r i v a t i v e s a f t e r c o l l e c t i o n from the gas chromatograph i n c a p i l l a r y tubes. The i d e n t i t i e s of the a l d i t o l acetates d e r i v a t i v e s were asce r t a i n e d by the presence of c h a r a c t e r i s t i c fragments as•described by -21*-FIGURE 3 - PRIMARY FRAGMENTS GIVEN BY ALDITOL ACETATES OF TRI - 0 - METHYL SUGARS IN A XYLAN 117 "i6 r CH„ - O - C - CH. HC - OCH. CH 30 - CH CH, - C - O - C 161 45 CH 2 - OCH 3 O li CH„ - O - C - CH. HC - OCH. 117 161 1 6 1 C H 3 ° " C H 1 O JL J l l 7 HC - OCH. CH 2 - 0 - C - CH. l,l»-Di-0-acetyl-2,3,5-tri- l,5-Di-0-acetyl-2,3,^-tri-O-aethyl-L-arabinitol O-methyl-D-xylitol TABLE V - A / - R E L A T I V E INTENSITIES OF THE PRINCIPAL FRAGMENTS CF TP. I ME THY LS I L YL DERIVATIVES IN MASS SPECTROMETRY m/e Methylated sugar 73 88 101 115 133 146 159 2 1 7 2 3 3 2 5 9 2 , 3 , 4-Tri-O-Me xylopyranose 80 100 65 20 20 2,3-Di-O-Me xylopyranose 65 78 0 15 25 0 0 2-O-Me xylopyranose 100 0 11 100 5 . 5 2 8 . 3 3-O-Me x ylopyranose 100 ... 0 86 92 60 20 0 . B/-PRINCIPAL FRAGMENTS OF METHYLATED.ALDITOL ACETATES IN MASS SPECTROMETRY - m/e Methylated a l d i t o l 43 45 71 87 101 117 1 2 9 161 1 8 9 2 ,3 ,5-Tri-O-Me Ara + • + + + + + + + 2 ,3 ,4-Tri-O-Me X y l + + + • + 2,3-Di-O-Me X y l + + + + + + - 2 6 -FIGURE 4 - PRIMARY FRAGMENTS FROM TRIMETHYLSILYL ETHER DERIVATIVES OF 2 - 0 -METHYL AMD 2 , 3-DI-O-METHYL. XYLOSE 4 CH ,5 'CH, OSi(CH_) 3 3 (m/e 1 1 3 ) (CH )3SiO-CH=Ssi(CH ) (m/e 191) (CH ) 3 S i O 4 . + 5 CH - CH, OSi(CH3) (m/e 116) •(CH ) 3SiO-CH=OSi (CH ) (m/e 191) 3 +2 CH - CH OCH OSi(CH ) 3 4 3 2 CH = CH - CH +" OCH. OSi(CH 3) 3 " (CH ) 3 S i O OSi(CH ) OCH. OCH OSi(CH ) (m/e 1 4 6 ) (m/e 159) (m/e 83) (m/a 15S) - 2 7 -Lindberg e t al.(56-59) . The most prominent peaks were as f o i l o w s : ( F i g . 3) 2 , 3 , 5 - t r i - 0 _ - m e t h y l - L - - a r a b i n . i t o l d e r i v a t i v e , m/e : 4 3 , 4 5 , 7 1 , 8 7 , 1 0 1 and 1 1 7 ; 2 , 3 , 4 - t r i - 0 - i r . s thy 1-D-xy 1 i to 1 d e r i v a t i v e m/e : 4 3 , 1 0 1 , 1 1 7 and 1 6 1 ; 2 , 3 - d i - 0 - m e t h y 1 - D - x y l i t o l d e r i v a t i v e , m/e : 4 3 , 8 7 , 1 0 1 , 1 1 7 , 1 . 2 9 and 3 89 . The i d e n t i t i e s of the mono-O-methyl d e r i v a t i v e s were c o n f i r -med by the mass s p e c t r a of t h e i r t r i m e t h y i s i l y l d e r i v a t i v e s . The c h a r a c t e r i z a t i o n was made e s s e n t i a l l y on the r e l a t i v e i n t e n -s i t y ( 5 0 ) of the peaks corresponding to the m/e : 1 3 3 , 1 4 6 , 2 1 7 , 2 3 3 and 2 5 9 . (Table V) C o n f i r m a t i o n was a l s o o b tained of the •. i den t i ty of the other methylated d e r i v a t i v e s . As a g e n e r a l r u l e , the mass s p e c t r a of t r i m e t h y i s i l y l d e r i v a -t i v e s are more complex than those of the corresponding a l d i t o l acetate d e r i v a t i v e s ( F i g . 4 ). T h e i r i n t e r p r e t a t i o n r e l i e s upon the s i m i l a r i t y of the fragmentation p a t t e r n of the TMS and per-methylated d e r i v a t i v e s ( 6 0 - 6 2 ) . As a r e s u l t , s u b s t i t u t i o n of a. methyl group f o r a TMS group i n an ion b r i n g s a change of 5 8 u n i t s i n the mass of the fragment . An i l l u s t r a t i o n i s given ( F i g . 4 ) showing the most c h a r a c t e r i s t i c ions i n the fragmenta-t i o n of the t r i m e t h y i s i l y l 2 - 0 - m e t h y 1 - 3 , 4 - d i - 0 - 1 r i m e t h y I s i l y 1 - D -xy l o p y r a n o s i d e and of the trims t h y l a i l y l 2,3,di-0-methy1-4-0-t r i m e t h y l s i l y l - D - x y ' l o p y r a n o s . i d e . Thus a c h a r a c t e r i s t i c m/e w i l l . -28-TABLE VI - GAS LIQUID CHROMATOGRAPHY OF METHYLATED PRODUCTS Methylated sugars Molar r a t i o 5 '? 2 , 3 5 - T r i-O-ne t h y l - L - a r a b i n o s e r 2 ,3 ,4-Tri-O-methyl-D-xylose**. 1 , 0 ~ 74 5 2 ,3-Di-O-methyl-D-xylo'se 3-O-Methyl-D-xylose*** 2 - 4 A^ift' 3 7 2-O-Methyl-D-xylose " ' * r e l a t i v e to 2,3,4-tri-O-methy1-D-xylose *x as t h e i r a l d i t o l a c e t a t e d e r i v a t i v e s i t i as t h e i r t r i m e t h y i s i l y l ether d e r i v a t i v e s appear between the 2-O-methyIxyioside and 3-O-methylxyloside deri-v a t i v e at m/e 159 and m/e 217 r e s p e c t i v e l y corresponding to the formation of the f o l l o w i n g ions : CH =• CH - CH , and , CH = CH - CH i (CH3) S i 6 + O C H 3 (CH3) S i O + 0 S i (CH3) 3 3 3 /e 159 m/e 217 m I t i s worth noting that the. temperature of i n j e c t i o n of the sample must be very accurate when one wants to obtain comparative s p e c t r a , since a d i f f e r e n c e - i n the temperature a f f e c t s the i n t e n s i t y of the fragments. That could e x p l a i n the d i f f i c u l t y i n i d e n t i f y i n g the 2 , 3 , di-O-methyl x y l o s i d e d e r i v a t i v e when compared to the r e -s u l t s of Samuelson and c o l l a b o r a t o r s (58) although there i s no •ambiguity with t h i s compound since i t had already been i d e n t i f i e d by gas l i q u i d chromatography and mass spectroscopy of i t s a l d i t o l acetate d e r i v a t i v e . The q u a n t i t a t i v e e stimation by gas l i q u i d chromatography of the methylated sugars gave the r e s u l t s shown i n Table VI. The a c i d i c f r a c t i o n of the hydrolysate of the methylated xylan was converted to methyl est e r methyl gl y c o s i d e s and reduced with l i t h i u m aluminum hydride i n tetrahydrofuran. H y d r o l y s i s -of the reduced products gave 3-0-me thy 1-D-^xy l o se 2 , 3 , 4 - t r i - 0 - m e t h y l - D - g l u c o s e . This r e s u l t was a confirmation of FIGURE 5 - PERIODATE CONSUMPTION BY THE XYLAN FROM ARUNDO DONAX Hours -31-TABLE VII - PERIODATE OXIDATION OF XYLAN FROM ARUNDO DONAX 1 / PERIODATE CONSUMPTION Time ( h o u r s ) 2 5 18 26 48 69 120 4 C m o l e P e r 0.231 0.326 0.461 0.490 0.545 0.675 0.710 a n h y d r o x y l o s e ) 2 / FORMIC ACID RELEASE Time ( h o u r s ) ,. 48 69 131 152 HCOOH (mole p e r ' a n h y d r o x y l o s e x 10 ) 4.7 5.3 6.2 6.4 FIGURE 6 - PERIODATE OXIDATION OF XYLAN FROM ARUNDO DONAX - FORMIC ACID RE LP] AS E hours -33-the presence of a l d o b i o u r o n i c a c i d . c - P e r i o d a t e o x i d a t i o n A sample of the arabino glucurono xylan was o x i d i z e d with p e r i o d i c a c i d , and a f t e r 120 h. the consumption of p e r i o d a t e was 0.71 mole per anhydroxylose. This f i g u r e , obtained by the sodium a r.seni.te method (63) i w a s confirmed by the U.V. s p e c t r o s -c o p i c method ac c o r d i n g to F e r r i e r and Asp.inail (64) ( F i g . 5 Table V I I ) . The pclyaldehyde obtained by p e r i o d a t e o x i d a t i o n was reduced with sodium borohydride and the r e s u l t i n g p o l y o l was h y d r o l y s e d to give ethylene g l y c o l , g l y c e r o l and.xylose i n the molar propor-t i o n s : 1 : 61 : 13 r e s p e c t i v e l y as determined by gas l i q u i d chromatography of t h e i r t r i m e t h y i s i l y l d e r i v a t i v e s ( 5 3 ) . The r e l e a s e of formic a c i d during p e r i o d a t e o x i d a t i o n was f o l l o w e d by iodometry (65) and a f t e r 120 h. e x t r a p o l a t i o n of the curve ( F i g 6) corresponded to 60 m i l l i m o l e s of formic a c i d per anhydroxylose u n i t (Table V I I ) . d - N . m:_r__g_pe c t r o s copy . Examination of the n.m.r. spectrum of a p o l y s a c c h a r i d e has been shown to give i n f o r m a t i o n on the nature of the anomeric linkages between the d i f f e r e n t sugar u n i t s (66-67) . Because of the problem of s o l u b i l i t y the pennethylated d e r i v a t i v e of the xylan was used f o r recording the n.m.r. spec-trum. The 60 Mhz and 100 Mhz spectra showed i n the anomeric region a peak at p.p.m. 5.53 S corresponding to an a con f i g u r a t i o n "and a doublet at p.p.m. 4.24 5 ( J ^ 2 6.5 Hz) corresponding to a 3 c o n f i g u r a t i o n . The peak of H^3 was much more intense than the H^a s i g n a l but was p a r t l y overlapping with the s i g n a l due to the other protons of the. r i n g , thus not al l o w i n g a q u a n t i t a t i v e estima-t i o n of the r a t i o of c o n f i g u r a t i o n s a to 8 (Fig.7).. However, t h i s i n d i c a t e s that the dominant type of linkage i n the xylan i s of the g c o n f i g u r a t i o n and i s c e r t a i n l y due to the 3 (1—*4) l i n k e d x y l o s y l backbone of the xy l a n . The presence of a linkages i s c e r t a i n l y to be a t t r i b u t e d to arabinos.yl and 4-0_-methyl glucuro-nosyl and 4-p_-methyl glucuronosyl r e s i d u e s , though i t was not p o s s i b l e to d i s t i n g u i s h between these two types of a ancmers e - ^ z y m i c h y d r o l y s i s . \ In a d d i t i o n to the strong negative value of the o p t i c a l r - \ x b r o t a t i o n of the arabino glucurono xylan ( l a - 90°) and to the above n.m.r. data, the use of an enzyme s p e c i f i c f o r the cleavage of 3 (1 —> 4) linkages between x y l o s y l u n i t s provides another proof of the 3 c o n f i g u r a t i o n of the linkages of the xylan chain. When the polysaccharide was incubated with an endo xylanase (68) - 3 6 -th e c l e a v a g e of the 8 (1 —* 4) backbone was i d e n t i f i e d by the e s t i m a t i o n o f the r e d u c i n g c a r b o h y d r a t e s r e l e a s e d and by the presence of x y l o s y l o l i g o c e r s and a c i d i c o l i g o m e r s d e t e c t e d by paper chromatography. D I S C U S S I O N The preceding r e s u l t s allow a deduction of the chemical s t r u c t u r e of the main he m i c e l l u l o s e present i n the stem of the reed Arundo donax. E x t r a c t i o n of the hern i c e l l u l o s e i n a s i n g l e step with 24 % potassium hydroxide (4.3 M) or by i n c r e a s i n g strength of p o t a s s i u hydroxide s o l u t i o n s (0.2 M, 1 .0 M_ and 2.5 M_) gave polysaccharides with very comparable carbohydrate compositions (Table I - I I I ) . The s l i g h t d i f f e r e n c e i n composition of the 24 % potassium hydroxide compared to the others can be accounted f o r by the method of estimation of the sugars ( the 24 % potassium hydroxide has been estimated by a c o l o r i m e t r i c method whereas the other e x t r a c t s have been analysed by gas l i q u i d chromatography). The present h e m i c e l l u l o s e i s then an arabino-4-0-methyl glucurono xylan whose composition i s almost constant independent-l y of the mode of e x t r a c t i o n , thus showing a good chemical p u r i t y . The s t r u c t u r a l study has been c a r r i e d out on the hemicel-l u l o s e B e x t r a c t e d with a i-OM potassium hydroxide s o l u t i o n . This xylan i s c o n s t i t u t e d of arabinose , 4-0=?methyl gluc u r o n i c a c i d and xylose i n the f o l l o w i n g molar proportions ; 1.0 : 0.37 8.9 r e s p e c t i v e l y . H y d r o l y s i s of the f u l l y methylated polysaccharide afforded 2 , 3 , 4-tri-O-mcthyl-D- x y l o s e ; 2 , 3 ,-di-0-me thy .1 -D-xy lose ; 2-0-methyl and 3-O-me thy 1 --D-xy lose ? and 2,3,5 tri-0-roe thy 1 -L arabinose i n the f o l l o w i n g molar proportions ; 1.00 ; 74.5 : 3.7 : 2.4 : 5.2 r e s p e c t i v e l y . I t can be concluded from the presence of 2,3-di-0-methyl xylose as the predominent d e r i v a t i v e that the xylan has a (1 —»• 4) l i n k e d backbone of xylopyranosy 1 u n i t s . The monomethyl xylose residues i n d i c a t e the presence of branch-points i n t h i s backbone e i t h e r on the C-3 or the C-2 p o s i t i o n s . The f a c t that a l l the arabinose was found as the 2,3,5-tri-O-methy1 d e r i v a t i v e i n d i c a t e s that a l l the arabinose residues? were present i n the h e m i c e l l u l o s e as term i n a l non reducing u n i t s and i n t h e i r furanose form. -When the a c i d i c f r a c t i o n of the hydrolyzed methylated xylan was reduced and r e - h y d r o l y s e d , i t afforded 3-0-methy1-D-xylose and 2 , 3 , 4 - tri-0-ine thy 1-D-gl ucose showing that the gl u c u r o n i c a c i d residues were l i n k e d to p o s i t i o n 2 of the x y l o s y l main chain. The molar proportions of the methylated sugars correspond to an average chain length of about 81 anhydroxylose u n i t s per macromole cule . This r e s u l t i s , .in ..good. .agreeaLsnt. w.£th_-the average degree of po l y m e r i s a t i o n of about 75 obtained by periodate o x i -dation (end group a n a l y s i s ) . The consumption of periodate also confirmed the presence of a (1 4) l i n k e d chain. The B c o n f i g u r a t i o n of the above linkage was i n d i c a t e d by the negative value of the o p t i c a l r o t a t i o n ( [a] 2 0 -90°) together D with the presence of a strong d o u b l e t . i n the n.m.r. spectrum -39-of the xylan ( <S 4 .2 4 p.p.ns ; ^ 6-5 Hz) and by the degradative a c t i o n of a fi ( i —* 4) endoxylanase . The i s o l a t i o n by paper chromatography of an a l d o b i o u r o n i c a c i d corresponding i n Rf to an authentic sample '(69) of 2-0_- ( 4-O-me thy 1 -a-D-g lucopy r anosy 1 uro -n i c acid)-D-xylose shows the « c o n f i g u r a t i o n of the (1 —*• 2) g l y c o s i d i c linkage of the 4-0-methyl gluc u r o n i c a c i d side chain attached to the xylose main chain. Prom a l l the above r e s u l t s t h e p r i n c i p a l h emicellulose from Arundo donax appears as an arabino-4-0-methyl glucuronoxy1 an s i -m i l a r to the xylans g e n e r a l l y found i n other Gramineae (70) . - 4 0 -E X P E R I M E N T A L General methods Paper chromatographic separations were c a r r i e d out on Whatman n° 1 paper, or Sc h l e i c h e r and S c h u l l n° 2043 b paper, using the f o l l o w i n g solvent systems : A, e t h y l acetate - a c e t i c a c i d - formic a c i d - water (18 : 3 : 1 : 4) ; B, e t h y l ' a c e t a t e -p y r i d i n e - water ( 4 : 1 : 1 ) ; C, butanone - water- ammonium hydro-xide (100 : 5 : 3) ; D, n-butanol - ethanol - water - ammonium hydro-xide (4 : 1 : 4.9 : 0.1). Separations 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 br a n i l i n e p h t h a i a t e . Gas l i q u i d chromatography was c a r r i e d out using an F <€ M 720 instrument equipped with a thermal c o n d u c t i v i t y d e t e c t o r , or a F€ M 5750 chromatograph f i t t e d with double column and equipped with a flame i o n i z a t i o n d e t ector. Peak areas were determined with an I n f o t r o n i c s CRS-lOO 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 c o n d i t i o n s were used : 8 f t x 1/4 i n . 3 % ECNSS-M on 60-70 mesh Chromosorb' W.For a l d i t o l acetates the program was 165° for 4 minutes then 0.5°/rainute to hold at 185°. For methylated a l d i t o l acetate the program was 130° f o r 4 minutes then l e/minute to hold at 180°. For t r i m e t h y i s i l y l d e r i v a t i v e s a column 8 f t x 1/4 i n . 20 % SF-96 on Diatoport S was used (53), at 2°/minute from 190° to 220°. For periodate o x i d a t i o n products, the co n d i t i o n s were 80° f o r 10 minutes then. 4°/mn to 190°. Uronic a c i d content was estimated by decarboxylation -41 -followed by c o n d u c t i v i t y (54). A l l evaporations were c a r r i e d out under reduced pressure below 40°. Hydrolyses were done with 72 % s u l p h u r i c a c i d fo r 30 minutes at room temperature then d i l u t i o n to 1 molar s o l u -t i o n and held at 100° for 4-6 hours i n sealed tubes, or with t r i f l u o r o a c e t i c a c i d (2 H) at 100° for 2 hours i n sealed tubes. I n f r a r e d spectra were run i n chloroform s o l u t i o n . Mass spectra were obtained e i t h e r by d i r e c t i n t r o d u c t i o n or by gas l i q u i d chromatography coupled to mass spectrometer A.E.I, model MS 9 unless otherwise s t a t e d . N.m.r. spectra were recorded on a Varian A-60 and H A-100 In the mixture benzene d^-chloroform-d (6 : 1 v/v) . I s o l a t i o n of Arundo donax holoce1lulose Dry internodes of Arundo donax were ground to sawdust (317 g) and e x t r a c t e d with ethanol-benzene mixture (1 : 2 v/v) i n a Soxh,,'let e x t r a c t o r f o r 22 hours, g i v i n g 297 g of e x t r a c t i v e - f r e e wood.Several batches were done. E x t r a c t e d sawdust (350 g) was t r e a t e d with b o i l i n g water (3 l i t x e s / 3 x 24 hours) and y i e l d e d 321 g dry weight. An e x t r a c t i o n with ethylene diamine t e t r a c e t i c a c i d (EDTA) f o r 3 hours at 70° (pH 6.4) was then repeated 3 times. The r e s i d u a l f i b r 0 - a s m a t e r i a l 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 f o r 4 hours. A f t e r drying the y i e l d of h o l o c e l l u i o s e was 250 g. -42-E x t r a c t i o n of h o l o c e l l u l o s e w i t h _ a l k a l i H o l o c e l l u l o s e (250 g) was e x t r a c t e d with potassium hydroxide for 24 hours s u c c e s s i v e l y at 0.2 M, 1.0 M and 2.5 M concentrations, under n i t r o g e n , at room temperature. F i 3 t r a t e s were 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 (pH 4.8 at 0°) and the r e s u l t i n g p r e c i p i t a t e was d r i e d by s o l v e n t exchange (ethanol, acetone, e t h y l e t h e r ) , g i v i n g h e m i c e l l u l o s e A. The supernatant was poured i n t o ethanol (4 volumes)and the h e m i c e l l u l o s e B was c o l l e c t e d as above. 5?:£E5E t i o n th _2 4 _% _po tass i um_hy droxide H o l o c e l l u l o s e was e x t r a c t e d as before with a 4.3 M (24 %) s o l u -t i o n of potassium hydroxide f o r 24 hours y i e l d i n g h e m i c e l l u l o s e s A and B. Hemicellulose A was d i f f i c u l t to c o l l e c t and was c e n t r i f u -ged at high speed (47,000 g). The y i e l d s were 25 % and 8 % r e s p e c t i -ve l y with respect to dry t i s s u e s . Carbohydrate_an a l y s i s Samples (about 56 mg) were t r i t u r a t e d i n s u l p h u r i c a c i d (72 %) at room temperature. A f t e r 30 minutes, the s o l u t i o n was brought to normality with water and kept at 100° i n a sealed tube f o r 4-6 hours. Excess a c i d was n e u t r a l i z e d with barium carbonate and the f i l t r a t e passed throuyh Amberlite IR-120 and IR-45 r e s i n s gave the n e u t r a l sugars. A c i d i c sugars were e l u t e d from the IR-45 colman with 10% formic a c i d . Paper chromatography of n e u t r a l and a c i d i c f r a c t i o n s was c a r r i e d out i n solvent systems A and B. N e u t r a l sugars -43-showed xylose as the main component with arabinose, and the presence of traces of glucose and galactose. The a c i d i c f r a c t i o n showed a component corresponding i n Rf with the a l d o b i o u r o n i c a c i d , 2-0- ( 4-0-me thy 1 -D-g 1 ucuror, osy 1) -D-xy 1 ose . Q u a n t i t a t i v e e s t i m a t i o n of n e u t r a l sugars was done by gas l i q u i d chromatography of t h e i r t r i m e t h y i s i l y l ethers d e r i v a t i v e s and uronic a c i d content was determined by decarboxylation (sample of 30 mg). Reduction of a l d o b i o u r o n i c a c i d A p o r t i o n of a l d o b i o u r o n i c was d i s s o l v e d i n methanolic hydro-gen c h l o r i d e (4 %, 10 ml) and heated at 100° f o r 4 hours i n a sea-led tube. A f t e r n e u t r a l i z a t i o n with s i l v e r carbonate, the supernatant was evaporated to dryness and reduced with sodium borohydride. The h y d r o l y s i s products were examined by gas l i q u i d chromatography of t h e i r a l d i t o l acetates and showed x y l i t o l and 4-0-methyl g l u -c i t o l . Me t h y l a t i o n _ o f the xylan_from Arundo_donax Methylation was performed on f r a c t i o n B e x t r a c t e d with l-OM potassium hydroxide. To a sample of xylan (2.5 g) was added f r e s h l y d i s t i l l e d dimethyl sulphoxide (150 ml) under nitrogen. Sodium hydride (50 % o i l suspension, 4.5 g) was washed free of o i l with petroleum ether (three times), then reacted with dry dimethyl sulphoxide (45 ml) -44-for about 3 hours at 60° u n t i l the e v o l u t i o n of hydrogen i n th>i r.cl t i o n ceased. The methylsulphiny1 anion (30 ml) was then auded to the polysaccharide s o l u t i o n and a gel immediately formed. The mixture was s t i r r e d overnight under n i t r o g e n . Methyl iodide (7.5 :.. was added over a p e r i o d of 4 hours, the r e a c t i o n mixture was then poured into water and dialysed for 24 hours. E x t r a c t i o n of the aqueou s o l u t i o n with chloroform i n a l i q u i d e x t r a c t o r f o r 48 hours and evaporation of the e x t r a c t to dryness y i e l d e d 1.51 g of product. The aqueous f r a c t i o n was freeze d r i e d and the two products c o a b i n ^ and remethylated as above. The new chloroform e x t r a c t (1.56 g) showed no absorption at 3400 cm 1 i n the i n f r a r e d . Fractional extract: with chloroform-petroleum ether mixtures gave the r e s u l t s expresse i n Table IV. I d e n t i f i c a t i o n of the methylated sugars was done by paper chromatography i n solvents C and D and by gas l i q u i d chro-matography . Characte rization_of_component_sugars P a r t i a l l y methylated sugars obtained by s u l p h u r i c a c i d hydro-l y s i s (50 mg) were reduced with sodium borohydride. A f t e r n e u t r a l i z a t i o n with 50 % a c e t i c a c i d , d e i o n i z a t i o n with Amberlite IR-120 r e s i n and repeated evaporations from methanol, the methylated a l d i -t o l s were a c e t y l a t e d i n a mixture, of py r i di ne-ace t i c anhydride (1 : 1.5 ml) at 100° f o r 60 minutes. The mixture was cooled and d i l u t e d with water, then evaporated to dryness and the l a s t traces of p y r i d i n e c o - d i s t i l l e d with toluene. Gas l i q u i d chromatography of the mixture of p a r t i a l l y methylated a l d i t o l acetate d e r i v a t i v e s gave the r e s u l t s shown i n Table VI. -45-A second p o r t i o n of the hydrolysate was converted to t r i m e t h y i -s i l y l ether d e r i v a t i v e s . P a r t i a l l y methylated sugars were s o l u b i -l i z e d i n p y r i d i n e (1 ml) and s i l y l a t e d with trime t h y 1 s 1 l y 1 c h l o r i d e (0.3 ml) and hexamethyldisilazane (0.6 ml) . In each case, the peaks corresponding to the d i f f e r e n t compo-nents were c o l l e c t e d i n c a p i l l a r y tubes and examined by mass spec-troscopy by d i r e c t i n t r o d u c t i o n . The most c h a r a c t e r i s t i c peaks are shown i n the chapter "Results". Periodate o x i d a t i o n A sample of xylan (1 g) was s o l u b i l i z e d i n sodium hydroxide (0.2 M, 30 ml) and the s o l u t i o n n e u t r a l i z e d with 50 % a c e t i c a c i d . The volume was adjusted to 100 ml with w a t e r . p e r i o d i c a c i d (0.5 M, 20 ml) was added and the mixture kept at 4° i n the dark. A l i q u o t s (5 ml) were withdrawn at i n t e r v a l s and the consumption of periodate was estimated by the a r s e n i t e method (63). E x t r a p o l a t i o n to zero time showed that a f t e r 5 days of o x i d a t i o n the consumption was 0.71 mole of periodate per anhydroxylose u n i t . Iodate and periodate ions were p r e c i p i t a t e d by a d d i t i o n of barium carbonate and a f t e r c e n t r i f u g a t i o n the supernatant was t r e a t e d with sodium borohyaride (1 g) overnight. The s o l u t i o n was deionized with Amberlite IR-120 r e s i n and a f t e r repeated evaporations with methanol con t a i n i n g 1 % h y d r o c h l o r i c a c i d , the residue was hydrolyzed with 0.5 M s u l -p h u r i c a c i d . A f t e r n e u t r a l i z a t i o n and d e i o n i z a t i o n as usual, the t r i m e t h y i s i l y l d e r i v a t i v e s of the components were chromatographed - 4 6 -on an SF-96 column at 80° f o r 10 minutes, then 4° per minute to h o l d ?t 190° . Ethylene g l y c o l , g l y c e r o l and xylose were found i n the f o l l o w i n g molar p r o p o r t i o n s 1 : 61 : 13 r e s p e c t i v e l y . Formic a c i d r e l e a s e d was estimated by iodometry (65); xylan (113 mg) i n water (120 ml) was o x i d i z e d with sodium metaperiodate (0.2 M , 30 ml) at 4° i n the dark. A l i q u o t s (20 ml) were withdrawn at i n t e r v a l s and the r e a c t i o n was stopped by a d d i t i o n of ethylene g l y c o l (6 d r o p s ) . A f t e r 30 minutes, potassium i o d i d e was added and t i t r a t i o n was performed with sodium t h i o s u l p h a t e (0.01M). - 2 E x t r a p o l a t i o n i n d i c a t e d t hat 6.1 x 10 moles of formic a c i d per anhydroxylan u n i t were r e l e a s e d . - 4 7 -PART II HEMICELLULOSES OF REDWOOD (Sequoia sempervirens) Sequoia sempervirens belongs to the family Taxodiaceae i n . the order of the P i n a l e s and represents one of the most ancient member of the C o n i f e r s . The l i t e r a t u r e covering the studies on he m i c e l l u l o s e s from angiosperms (hardwoods) or gymnosperms (softwoods) has been reviewed by T i m e l l (8,9) . The he m i c e l l u l o s e s of the wood of gymnosperms con-t a i n mainly a glucomannan and an arabino-4-0_-methyl-glucurono -. xyla n . The glucomannan i s very often s u b s t i t u t e d with D-galactose residues g i v i n g then a.galactoglucomannan. The mannose co n t a i n i n g polysaccharides of c o n i f e r s represent the major h e m i c e l l u l o s i c components of these woods. In the Cupressaceae, a family close to the Taxodiaceae, the he m i c e l l u l o s e s of Red Cedar (Thuja p l i c a t a ) h a v e been s t u d i e d (86) and were shown to be s i m i l a r to those of other c o n i f e r s . The only study of the he m i c e l l u l o s e s of Sequoia has been made by Hami1ton, Partlow and Thompson (73) i n 1958 and was concerned with the galac-toglucomannan contained i n a conventional Kraft-wood c e l l u l o s e of Sequoia sempervirens. -48-RESULTS 1 - I s o l a t i o n of the he m i c e l l u l o s e s A l l the e x t r a c t i o n s have been performed on the holoc-elluloso obtained a f t e r ethanol-benzene e x t r a c t i o n followed by sodium c h l o r i t e d e 1 i g n i f i c a t i o n (12) . In a f i r s t experiment the procedure described by T i m e l l (74-75) f o r the i s o l a t i o n and p u r i f i c a t i o n of xyla n s , glucomannans (GM) and galactoglucomannans (GGM) from Softwood was a p p l i e d . This procedure ( F i g . 8) makes use of the d i f f e r e n c e s i n s o l u b i l i t y of the three h e m i c e l l u l o s e s towards sodium hydroxide and potassium hydroxide. When the h o l o c e l l u l o s e of Redwood was s u c c e s s i v e l y e x t r a c t e d with 24 % potassium hydroxide and 17.5 % sodium hydroxide the l a t t e r e x t r a c t gave i n a 5.5 % y i e l d a polysaccharide which contained mannose as the p r i n c i p a l sugar. The carbohydrate composi-t i o n of t h i s f r a c t i o n was analysed by gas l i q u i d chromatography of the t r i m e t h y i s i l y l d e r i v a t i v e s (TMS ethers) and showed galac t o s e , glucose, mannose and xylose i n the f o l l o w i n g molar p r o p o r t i o n s , 0.4 : 1.0 : 4.6 : and 0.2 r e s p e c t i v e l y . Such a composition c o r r e s -ponds to a crude glucomannan. However, a l l attempts to separate as barium hydroxide complexes the polysaccharides contained i n the 24 % potassium hydroxide e x t r a c t f a i l e d to give a sharp separation of the expected xylan and galactog1ucomannan. The a n a l y t i c a l composition of the crude 24 % potassium hydroxide e x t r a c t corresponded to arabinoso, - 4 9 -FIGURE 8 - ISOLATION OF MANNOSE-CONTAINING HEMICELLULOSES ACCORDING TO TIMELL (9) WOOD N a C 1 0 2 HOLOCELLULOSE KOH s o l u b l e i n s o l u b l e H e m i c e l l u l o s e mixture Residue Ba (OH) 2 NaOH,bora te s o l u b l e i n s o l u b l e i n s o l u b l e s o l u b l e Mixture Crude galacto-glucomannan C e l l u l o s e Crude gl u c o -mannan Ba (OH) i ) i i ) s o l u b l e f i n s o l u b l e s o l u b l e ! i n s o l u b l e » KOH Ba(OH) i ) i i ) i n s o l u b l e „ ' „ G a l a c t o g l u c o - . , Galactogluco-F r a c t i o n A — D i s c a r d — — — — — marman I mannan II KOH Ba(OH) s o l u b l e Glucomannan D i s c a r d -50-FIGURE 9 - ISOLATION OF HEMICELLULOSES FROM THE WOOD OF SEQUOIA SEMPERVIRENS - PROCEDURE A EXTRACTIVE-FREE WOOD (210g) NaC10 2 , 70' HOLOCELLULOSE (155g) 24% KOH So l u t i o n Residue Crude S (43g) NaC10 2,25 1 CELLULOSE S x 2 ( 3 4 g ) Ba(OH) S x 3 (18g) KOH(10%) Ba (OH) „ Soluble EtOH j Insoluble(16g) KOH(10%) Ba(OH). GGM (5.5g) GGM J(lg) X(6g) G G M I I I ( 7 - 5 9 > NaOH(17.5%) H 3B0 3(4%) S o l u t i o n EtOH Crude GM NaOH(17;5%) H 3B0 3 (4%) Soluble Ba(OH) GM(11.3g) -51-x y l o s e , g a l a c t o s e , gJucose and mannose in the f o l l o w i n g molar p r o p o r t i o n s , 1.4 : 9.7 : 1.0 : 1.0 : 2.8 r e s p e c t i v e l y . This compo-s i t i o n corresponds to a mixture of xylan and galactoglucomannan i n a 2 : 1 r a t i o . This d i f f i c u l t y i n achieving a good separation between xylan and galactoglucomannan i s l i k e l y due to the presence of some l i g n i n s t i l l present a f t e r c h l o r i t e treatment and which i s s o l u b i l -i s e d i n the 24 % potassium hydroxide e x t r a c t . For t h i s reason, the procedure f o r s e l e c t i v e e x t r a c t i o n of xylans described by Clayton ( 7 6 ) , using a d e l i g n i f i c a t i o n of the h e m i c e l l u l o s e mixture p r e c i p i t a t e d from the 24 % potassium hydroxide e x t r a c t was then a p p l i e d . The d i f f e r e n t , steps corresponding to a combina-t i o n of T i m e l l ' s method and c h l o r i t e d e l i g n i f i c a t i o n of the crude h e m i c e l l u l o s e are presented i n Figure 9. This procedure gave again the same glucomannan and allowed the i s o l a t i o n of a xylan containing only arabinose and xylose as n e u t r a l sugars i n the r e s p e c t i v e mo-l a r p r o p ortions of 1 to 9, but only a very small amount of galactoglucomannan could be obtained i n a reasonably pure s t a t e ( F r a c t i o n GGM 1 - Table V I I I ) . The a n a l y t i c a l data presented i n Table V I I I show th a t the y i e l d s are very low and that an important p a r t of the xylan s t i l l remains i n the f r a c t i o n s GGM I I and GGM I I I . In order to improve the recovery of these two jjolysaccharides from the 24 % potassium hydroxide e x t r a c t , a f r a c t i o n a t i o n was t r i e d as described by Dutton and co-workers ( 7 7 ) . The supernatant of the barium hydroxide p r e c i p i t a t i o n was t i t r a t e d with e t h a n o l , but none of the three -52-TABLE VIII - EEMICELLULOSIC FRACTIONS OBTAINED ACCORDING TO FIGURE 9 F r a c t i o n s Galactose Glucose Mannose Arabinose Xylose Crude GM 4.2 11.7 58.0 3.1 23.0 GM 1.7 13.6 84.0 t t X - t - 5.8 92.4 GGM 19.5 11.0 6.1 2.3 6.5 GGM 19.1 11.3 43.5 2.5 25.8 I I GGMj 5.4 3.9 . 6.8 8.2 76.0 - 5 3 -f r a c t i o n s thus obtained gave a pure galactoglucomannan or a pure xylan . Another method of s e l e c t i v e e x t r a c t i o n of h e m i c e l l u l o s e s from softwood developed by B e e l i k and co-workers (78) was a p p l i e d to the h o l o c e l l u l o s e of Redwood. This method c o n s i s t s of an impre-gnation of the h o l o c e l l u l o s e with 1-2 % barium hydroxide which renders the xylans s o l u b l e i n 10 % potassium hydroxide, but which i n h i b i t s the d i s s o l u t i o n of mannose-containing polymers. Here again, the recovery of the d i f f e r e n t h e m i c e l l u l o s e f r a c t i o n s was very low and no pure polysaccharides were obtained. The best r e s u l t s were obtained when the 24 % potassium hydro-xide e x t r a c t was t r e a t e d with a 2.5 % s o l u t i o n of c e t y 1 t r i m e t h y 1 -ammonium bromide (Cetavlon). This procedure, f i r s t described by Scott (79) and by Bouveng and Lindberg (80)., i s based on the i n s o -l u b l e s a l t formed by Cetavlon and a c i d i c p o l y s a c c h a r i d e s . I t was expected that the uronic a c i d residues of the xylan would form an i n s o l u b l e complex with Cetavlon and leave a s o l u b l e galactoglucoman-nan. Figure 10 shows the d i f f e r e n t steps of the e x t r a c t i o n - p u r i f i -c a t i o n on the h o l o c e l l u l o s e of Sequoia with Cetavlon, and Table IX gives the a n a l y t i c a l data obtained by h y d r o l y s i s of the d i f f e r e n t f r a c t i o n s . By t h i s method of s e p a r a t i o n , the carbohydrate composi-t i o n of the d i f f e r e n t galactoglucomannan f r a c t i o n s i s o l a t e d appears -54-FIGURE 10 - ISOLATION OF HEMICELLULOSES FROM THE WOOD OF SEQUOIA SEMPERVIRENS - PROCEDURE B EXTRACTIVE-FREE WOOD (150g) NaC102,70' HOLOCELLULOSE (1 1 2 g) sol u b l e Ba(OH) H 3B0 3 4% solu b l e KOH(24%) i n s o l u b l e i n s o l u b l e EtOH AcOH Crude S 4X (45g) r Residue S 4GGM I(5.6g) Cetavlon(2.5%) i n s o l u b l e s o l u b l e S 4C 1 (0.28g) EtOH Crude Sc 4 2 i n s o l u b l e r H 20 so l u b l e s4 C 2 ( 4 . 2 g ) KOH (10%) Ba(OH)„ Ba(OH) - H,BO. 2 3 4 EtOH-AcOH S4G G MI I (l - -3g ) S 4C 2 (23.5g) NaoH(17.5%) H 3B0 3(4%) Soluble Ba(OH) S4GM (16g) -55-TABLE I X — ANALYTICAL DATA OF XYLAN S,C Sugar Mole % Arabinose 3.7 Xylose 74.4 Hexoses 3.3 found 4.20 Me thoxy1 % * calculated 4.15 i2Q [cc] (H 20) - 42' * In gram per cent -56-to be r e p r o d u c i b l e and rather constant whatever the degree of p u r i f i c a t i o n . At any step of the p u r i f i c a t i o n the galactoglucomannan composition showed ga l a c t o s e , glucose and mannose i n the molar r a t i o s from 1 : 0.6 ; 3 to 1 : 0.4: 2.2 r e s p e c t i v e l y , the dominant feature of t h i s polysaccharide being the low p r o p o r t i o n of glucose compared with the usual composition of softwood galactoglucomannan. The average r a t i o of mannose to glucose was here 5.5. I t i s of i n t e r e s t to compare t h i s r a t i o with the r a t i o of mannose to glucose of 6 to 6.8 found f o r the glucomannan a f t e r barium hydroxide p u r i -f i c a t i o n ( F i g . IO, Table I X ) . With the use of cetyltrimethylammonium s a l t a f r a c t i o n S4 C2 ( F i g . 10) was obtained i n a 15 % y i e l d , which was c o n s t i t u t e d of arabinose and xylose i n the r e s p e c t i v e molar proportions 1 : 14.2 hexoses were also present (0.9 t o t a l ) . This f r a c t i o n S4 C2 though not obtained as a p r e c i p i t a t e with Cetavlon corresponds i n composition to a xylan. I t i s then c l e a r from the above r e s u l t s that the three types of h e m i c e l l u l o s e s t y p i c a l of the wood of gymnosperms (xylan, gluco-mannan and galactoglucomannan) are present i n the wood of Sequoia sempervirens. However, t h e i r separation was d i f f i c u l t and s e v e r a l procedures had to be used i n order to i s o l a t e enough of each of these polysaccharides with a degree of p u r i t y s u f f i c i e n t to allow f u r t h e r s t r u c t u r a l study. As a r e s u l t , the s t r u c t u r a l i n v e s -t i g a t i o n s were c a r r i e d out r e s p e c t i v e l y on the xylan c a l l e d S4 C2 ( F i g . 10), the galartoglucomannan f r a c t i o n GGM 1 (Fig.10) and the glucomannan GM ( F i g . 9 ) . -57-I I - S t r u c t u r a l study of xylan S4 C2 a - Carbohydrate a n a l y s i s The xylan f r a c t i o n S4 C2 was d i a l y s e d against d i s t i l l e d water and freeze dried,and i t s carbohydrate composition was determined. The n e u t r a l sugars were estimated by gas l i q u i d chromatography of t h e i r a l d i t o l acetate d e r i v a t i v e s and the uronic a c i d content was e s t a b l i s h e d by decarboxylation (54). The a n a l y t i c a l composition i s given i n Table IX. I t i s n o t i c e a b l e that the methoxyl content of 4.2 % reveals that a l l the uronic a c i d u n i t s are methoxylated i n the xylan (18.5 moles % corresponding to 26 % g l u c u r o n i c a c i d by weight). When i t was c a l c u l a t e d as 4-0-methyl g l u c u r o n i c a c i d i t gave a value of 4.15 %. Such a uronic a c i d content can e x p l a i n the f a c t that t h i s xylan i s very e a s i l y s o l u b l e i n water. b - Methylation study b - 1 - Hakomori_methy_lations The procedure of methylation according to Kakomori (42) has now bean recognized as being the most e f f i c i e n t and r a p i d way f o r o b t a i n i n g i n good y i e l d a permethylated p o l y s a c c h a r i d e . This i s the reason why t h i s method was f i r s t a p p l i e d f o r the methylation of the xylan S 4 C2. The f r e e z e - d r i e d xylan was t r e a t e d according to the c o n d i t i o n s of Hakomori methylation although i t was not completely s o l u b l e i n dimethyl sulphoxide (DMSO). At the end of the methylation r e a c t i o n , the mixture was poured, i n t o water but no p r e c i p i t a t e was formed thus i n d i c a t i n g that the degree of e t h e r i f i c a t i o n was s t i l l very low. The aqueous s o l u t i o n was then d i a l y s e d and freeze d r i e d and a second methylation was performed, using s i m i l a r c o n d i t i o n s , followed by a t h i r d treatment with the. same reagents. When poured i n t o water the DMSO-solution of the polysaccharide gave a white p r e c i p i t a t e and a f t e r d i a l y s i s and chloroform e x t r a c t i o n , 1.1 g of methylated product was c o l l e c t e d . A f r a c t i o n a t i o n of the,methylated polysaccharide with petroleum ether-chloroform mixtures gave300 mg f o r the 15 and 20 % chloroform e x t r a c t s with a methoxyl content of 34.6 % and 35.8 % r e s p e c t i v e l y (ash content 0.25 % and 0.23 %) . The combined f r a c t i o n s were then re-methylated according to Purdie's method (38) and a f t e r three such treatments gave 390 mg of methy-l a t e d m a t e r i a l which showed no hydroxyl absorption i n the i n f r a r e d spectrum. A f t e r s u l p h u r i c a c i d h y d r o l y s i s , the n e u t r a l p a r t i a l l y methy-l a t e d sugars were analysed by paper chromatography and by gas l i q u i d chromatography of t h e i r a l d i t o l acetate d e r i v a t i v e s . Further i d e n t i f i c a t i o n of the d i f f e r e n t methylated sugars was obtained by examination of. t h e i r mass s p e c t r a . Q u a n t i t a t i v e r e s u l t s of the a n a l y s i s are shown i n Table XI. Some remarks should be made on the above methylation. The f i r s t i s that the y i e l d of permethylated polysaccharide was very low which v/as p o s s i b l y due to the bad s o l u b i l i t y of S4 C2 i n DMSO. The second remark a p p l i e s to the complications which a r i s e when uronic -59-a c i d residues are present i n a polysaccharide and when an under-methylated polymer i s remethylated i n : t h e presence of the very strong base, me thy1sulphinylmethy 1 anion, used i n Hakomori methylations J4 1 ,56) . In such a s i t u a t i o n the e s t e r i f i e d uronic a c i d residue may react with the s t r o n g l y b a s i c anion e i t h e r to give a me thy1sulphinylmethy1 ketone (A) or may undergo a £- e l i m i n a t i o n ( F i g . 11) which leads to an unsaturated residue (B). 0 0 V II C-CH 2-S-CH 3 :OOCH RO O-Xylan O-Xylan COOH O-Xylan The second r e a c t i o n i s much more important when the methyl uronate i n the p a r t i a l l y methylated polysaccharide i s s u b s t i t u t e d at i t s C-4 p o s i t i o n with a good l e a v i n g group (81-83). In the st r o n g l y b a s i c medium t h i s group i s e l i m i n a t e d and an unsaturated uronate i s formed. The enal ether residue (B) i s very l a b i l e i n a c i d i c medium. Since the xylan S4 C2 contains a high p r o p o r t i o n - 6 0 -FIGURE 11 - PRINCIPLE OF (3 - ELIMINATION ON A GLUCURONATE . By l o s s of two s u b s t i t u e n t s from adjacent atoms, a new double bond i s formed i n the ^  - e l i m i n a t i o n (84) : H — c — c — x —>---• » x = . L Electron-withdrawing groups ( X ) enhance g r e a t l y the a c i d i c character of the' proton ( a c t i v a t e d proton H) which i s cleaved by n u c l e o p h i l i c reagents. This $ - e l i m i n a t i o n i s f a c i l i t a t e d when a good l e a v i n g group ( L ) i s present on the adjacent carbon. In the case of a 4-O-substituted glucuronate; - 6 1 -of uronic a c i d i n the form of 4-0-methyl g l u c u r o n i c a c i d , the successive treatments with Hakomori's reagents might have degraded the o r i g i n a l polysaccharide and might have given erroneous r e s u l t s . In order to i n v e s t i g a t e t h i s p o s s i b i l i t y , a f r a c t i o n of S 4 C2 xylan was methylated by procedures which avoid a re-exposure to the strong base methyl s u l p h i n y l m e t h y l anion. b - 2 - Methylations by_non-degradative_methods In a f i r s t step, 1 gram of the xylan was subjected to a Haworth methylation (37) (sodium hydroxide, dimethyl sulphate) and the r e s u l t i n g undermethylated m a t e r i a l was t r e a t e d twice more using the same c o n d i t i o n s . The p a r t i a l l y methylated product was sol u b l e i n dimethyl sulphoxide and was then remethylated by the method of S r i v a s t a v a (40) (dimethyl sulphoxide, sodium hydroxide and dimethyl s u l p h a t e ) , y i e l d i n g 516 mg of a product which s t i l l showed an hydroxyl band i n the i n f r a r e d . Completion of the methy-l a t i o n was then achieved by remethylating the product by three treatments according to Purdie and gave 405 mg of methylated compound showing no absorption of hydroxyl i n the i n f r a r e d spec-trum . F r a c t i o n a l e x t r a c t i o n of t h i s m a t e r i a l i n mixtures of p e t r o -leum ether/chloroform gave the r e s u l t s presented i n Table X. One p a r t of the methylated polysaccharide ( f r a c t i o n 25 : 75) - - 28° was hydrolysed and the p a r t i a l l y methylated sugars - 6 2 -TAI.LE XI — NEUTRAL SUfiAU ANALYSIS OF THE PKRMETHYLATEI) XYLAN S 4 C 2 Compound Molar r a t i o 2,3,5-Tri--0-- Mc-arabinosc 1. 1 2,3,4-Tri--0-Me- xylose 1. 0 2,3-M-O-Me-xylose 42. 0 2-(3-)Mono-0-Mc-xylose 8. 1 * As their a l d i t o l acetate derivatives ** After four Hakoinori me thy la tions TABLE X — F r a c t i o n a l extraction on methylated S/+ C 2 Cliloroform/petroleiun ether Y i e l d 15 85 87 mg 20 80 12 mg 25 75 140 mg 30 70 165 mg - 6 3 -analysed and c h a r a c t e r i z e d as p r e v i o u s l y described. This showed the presence of the f o l l o w i n g methylated d e r i v a t i v e s : 2 , 3 , 5 - t r i -O-methyl arabinose ; 2,3,4-tri-O-methy 1 xylose ; 2,3-di-O-raethy1 xylose and (2 and 3) mono-O-methy1 xylose i n the respective molar proportions 1.7 : 1.0 : 50.3 6.8. A second p o r t i o n of the methylated xylan was reduced with l i t h i u m aluminum hydride i n tetrahydrofuran g i v i n g a n e u t r a l p o l y -saccharide where a l l the uronic a c i d residues had been c a r b o x y l -reduced . H y d r o l y s i s of t h i s product y i e l d e d 2 , 3 , 5-tri-O-methy1 arabinose ; 2,3,4-tri-O-methyl xylose ; 2 , 3-di-0-methy1 xy l o s e , (2 and 3) mono-O-methy 1 xylose as f o r the f i r s t p a r t but i n a d d i t i o n gave 2 , 3 , 4-tri-O-methyl glucose i n the molar p r o p o r t i o n s , 1.7 : 1.0 : 51.5 : 13.8 and 9.3 r e s p e c t i v e l y (Table XII) , I t can thus be r e a d i l y seen that t h i s second way of methylation gave a methylated polysaccharide with a b e t t e r y i e l d than the f i r s t Hakomori method, and that the h y d r o l y s i s products were obtained i n comparable p r o p o r t i o n s . More comments on these r e s u l t s w i l l be made i n the p a r t "Discussion". c - P e r i o d a t e _ o x i d a t i o n An aqueous s o l u t i o n of S4 C2-xylan was submitted to the a c t i o n of p e r i o d i c a c i d and the consumption of periodate was 0.85 mole per anhydroxylose u n i t a f t e r 120 hours of attack (Table XIII as determined by the a r s e n i t e method ( 6 3 ) . The r e s u l t i n g p o l y -aldehyde was reduced with sodium borohydride and the p o l y o l s r e l e a -TABLE X I I — NEUTRAL SUGAR ANALYSIS OF PERMSTHYLATED XYLAN S 4 C 2* Compound Molar r a t i o Unreduced Carboxyl-reduced 2,3,5-Tri-0_-Me arabinose 1.7 1.7 2,3,4-Tri-O-Me xylose 1.0 1.0 2,3-Di-O-Me xylose 50.3 51.5 Mono-O-Me xylose 6.8 13.8 2,3,4-Tri-O-Me glucose — 9.3 * After sequential methylation -65-TABLE X I I I — PERIODATE OXIDATION ON XYTAN C 2 1/ PERIODATE CONSUMPTION Time (hours) 2 5 18 26 48 69 120 I 0 4 - P e r 465 556 735 782 817 862 910 anhydroxylose unit 10~"^ 2/ FORMIC ACID RELEASED Time (hours) 48 69 131 152 HCOOH per 6 > 3 7 < 1 7 > 6 7 > 6 anhydroxylose unit 9 x 10" 40 26 12 0 -6 7-sed by a c i d h y d r o l y s i s were analysed by gas l i q u i d chromatography of t h e i r t r i m e t h y i s i l y l ether d e r i v a t i v e s (53) ( F i g . 32) . Ethylene g l y c o l , g l y c e r o l and xylose were found i n the f o l l o w i n g molar propor-t i o n , 1.0 : 45.7 : 4.9 r e s p e c t i v e l y . Formic a c i d released during the o x i d a t i o n was estimated by -2 iodimetry and gave by e x t r a p o l a t i o n to zero., time 7.5 x 10 mole of formic ac i d released per anhydroxylose u n i t (Table X I I I ) . d - Enzymic h y d r o l y s i s of S4 C2-xylan When the polysaccharide was submitted to a prolonged attack (48 hours) by an endoxylanase (68), the products of h y d r o l y s i s were d i a l y s e d , and the non-dialysable residue was f r e e z e - d r i e d and submitted to a novel attack by the xylanase. A f t e r three such treatments 30 % of the s t a r t i n g m a t e r i a l remained unattacked by the enzyme ( F i g . 13). Thus, 7 0 % of the (1 4) l i n k e d m a t e r i a l had been cleaved by the enzyme showing the 3 c o n f i g u r a t i o n of the x y l o s y l l i n k a g e s . The uronic a c i d determination performed on the 30 % residue i n d i -cated a r a t i o of uronic a c i d to xylose of about 1 : 2 compared to the r a t i o of 1 : 4 i n the s t a r t i n g m a t e r i a l . -69-I I I - STRUCTURAL STUDY OF GLUCOMANNAN (GM) Thi s p o l y s a c c h a r i d e was obtained with a high y i e l d and seems homogenous i n i t s composition. I t s p u r i f i c a t i o n was r e a d i l y achieved by complexing with barium hydroxide i n the presence of 5 % b o r i c a c i d (see F i g . 9 ) t as shown by the absence of sugars other than g a l a c t o s e , glucose and mannose i n i t s composition. The presence of ga l a c t o s e i n a glucomannan i s a usual phenomenon ( 9 , 8 5 ) . I t must be noted that the p r o p o r t i o n of mannose i s p a r t i c u -l a r l y high i n t h i s h e m i c e l l u l o s e (Table V I I I ) . The s t r u c t u r a l i n v e s t i g a t i o n was c a r r i e d out by met h y l a t i o n and examination of the c o n s t i t u e n t c e t h y l ethers of the sugars a f t e r h y d r o l y s i s . a - Hakomori_methylation Glucomannans being devoid o f u r o n i c a c i d r e s i d u e s , there i s no r i s k of s i d e - r e a c t i o n s by 3 e l i m i n a t i o n , when s e v e r a l methy-l a t i o n s i n the presence of methylsulphiny lme thy .1 anion are per-formed s u c c e s s i v e l y . The glucomannan from Sequoia was methylated a c c o r d i n g to Hakomori, but the product was not completely s o l u b l e i n di m e t h y l -s u l p h o x i d e . Without p r i o r i s o l a t i o n of the products of r e a c t i o n , a new methyla t i o n was performed by d i r e c t a d d i t i o n of the anion i n t o the mixture and then of methyl i o d i d e . A f t e r i s o l a t i o n of the p a r t i a l l y methylated p o l y s a c c h a r i d e , three treatments with -70--Hakomori's r e a g e n t s were r e p e a t e d which gave a p r o d u c t showing no h y d r o x y ! b a n d i n i n f r a r e d s p e c t r o s c o p y . The p o o r s o l u b i l i t y o f the glucomannan i n d i m e t h y l s u l p h o x i d e . i s c e r t a i n l y r e s p o n s i b l e f o r the f a i l u r e o f the Hakomori m e t h y l a t i o n i n one s t e p . T h i s b a d s o l u b i l i t y c o u l d be due t o an i n c o m p l e t e r e m o v a l o f b a r i u m i o n s (86) . A b e t t e r y i e l d was o b t a i n e d when the glucomannan was m e t h y l a -t e d s u c c e s s i v e l y by d i f f e r e n t methods o f m e t h y l a t i o n t h u s making use o f the d i f f e r e n c e i n the o r d e r o f r e a c t i v i t y o f the h y d r o x y l g r o u p a c c o r d i n g t o the p r o c e d u r e used. (43) . b - S e q u e n t i a l me th y l a t i o n s _ b y _Haworth ' s jr__Kuhn 1 s a n d _ P u r d i e ' s p r o c e d u r e s . The glucomannan was f i r s t m e t h y l a t e d by Haworth's p r o c e d u r e (37) (sodium h y d r o x i d e , d i m e t h y l s u l p h a t e ) . The d i a l y z e d t h e n f r e e z e - d r i e d m a t e r i a l was i n t u r n m e t h y l a t e d by the method o f Kuhn (39) ( N N * - d i m e t h y l formamide, m e t h y l i o d i d e and s i l y e r o x i d e , Two s u c h t r e a t m e n t s gave a p r o d u c t w h i c h showed o n l y a weak a b s o r -p t i o n o f the h y d r o x y l band i n the i n f r a r e d s p e c t r u m . A f u l l y methy-l a t e d p o l y s a c c h a r i d e was a c h i e v e d a f t e r f o u r P u r d i e m e t h y l a t i o n s ( m e t h y l i o d i d e , s i l v e r o x i d e ) , and f r a c t i o n a l e x t r a c t i o n w i t h c h l o -r o f o r m - p e t r o l e u m e t h e r gave i n a b o u t 25 % y i e l d i n the 25 : 75 f r a c t i o n a p r o d u c t w i t h no f r e e h y d r o x y l . -71-c - A n a l y s i s o f _ m e t h y l a t e d glucomannan The m e t h y l a t e d h e m i c e l l u l o s e ( f r a c t i o n 25 : 75) was h y d r o -l y s e d w i t h s u l p h u r i c a c i d and the p a r t i a l l y m e t h y l a t e d components were examined by p a p e r c h r o m a t o g r a p h y . C h r o m a t o g r a p h y i n t h e s o l v e n t s y s t e m n_ b u t a n o l :' e t h a n o l : w a t e r : ammonium h y d r o x i d e ( 4 : 1 : 4.9 : 0.1) showed t h r e e main s p o t s w i t h Rg v a l u e s ( r e l a -t i v e t o 2 , 3 , 4 , 6-te.tra-0-me thy 1 g l u c o s e ) o f 1.00, 0.85 and 0.65 c o r r e s p o n d i n g t o 2,3,4,6-tetra-O-methy1 mannose and g l u c o s e , 2,3, 6-tri-O-me t h y l mannose and g l u c o s e and 2,3-di-_0 - m e t h y l mannose r e s p e c t i v e l y (87). C h r o m a t o g r a p h y i n the s o l v e n t s y s t e m m e t h y l e t h y l ketone-water-ammonium h y d r o x i d e (100 : 5 : 3) r e v e a l e d f i v e main s p o t s w i t h Rf v a l u e s o f : 0.80, 0.55, 0.49, O.26 and 0.22 c o r -r e s p o n d i n g t o 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l mannose ; 2 , 3 , 6 - t r i - 0 - m e t h y l g l u c o s e ; 2 , 3 , 6 - t r i - 0 - m e t h y l mannose ; 2 , 3 - d i - 0 - m e t h y l g l u c o s e and 2 , 3 - d i - 0 - m e t h y l mannose r e s p e c t i v e l y (87 ,88) . The above d a t a a r e o n l y t e n t a t i v e r e s u l t s b e c a u s e o f the i m p r e c i s i o n o f the Rf and Rg v a l u e s o b s e r v e d . B e t t e r i d e n t i f i c a -t i o n s were o b t a i n e d by use o f gas l i q u i d c h r o m a t o g r a p h y . P a p e r c h r o m a t o g r a p h y showed the p r e s e n c e o f a t l e a s t f i v e c omponents. Such a number o f d i f f e r e n t p a r t i a l l y m e t h y l a t e d s u g a r s i s d i f f i c u l t t o r e s o l v e i n gas l i q u i d c h r o m a t o g r a p h y as r e d u c i n g s u g a r s o r as t h e i r g l y c o s i d e d e r i v a t i v e s s i n c e t h e two anomers o f the p y r a n o s e and f u r a n o s e forms can be seen f o r each s u g a r , t h u s r e n d e r i n g t h e chromatogram v e r y c o m p l i c a t e d . When U s i n g the r e d u c e d d e r i v a t i v e s a l d i t o l f o r m , o f the s u g a r s , t h e r e i s o n l y one component p e r s u g a r . T h i s i s the r e a s o n why the a l d i t o l TABLE X I V — ANALYSIS OF METHYLATED GLUCOMANNAN * Rt (min.) Compound Molar r a t i o 2,3,4,6-Tetra-O-Me (glucose+mannose) 20.5 1.6 2,3,4,6-Terra-O-Me g a l a c t o s e . 22.5 1.2 2,3,6-Tri-O-Me mannose 28.5 76.2 2,3,6-Tri-O-t-fe glucose 29.7 15.8 2,6-Di-O-Me mannose 32.5 0.9 2 , 3-Di-0-mannose,; 35.8 2.7 2,3-Di-O-Me glucose 37.2 0.5 3t As a l d i t o l acetate d e r i v a t i v e s -73-forms of the methylated carbohydrates were used f o r the gas l i -q uid chromatography a n a l y s i s of the hydr o l y s a t e of the methylated glucomannan. Best r e s u l t s are obtained with the acetate d e r i v a t i v e s of the p a r t i a l l y methylated a l d i t o l s , both i n chromatography and raass-spectroscopy ( 5 6 ) . A n a l y t i c a l data obtained by gas l i q u i d chromatography are expressed i n Table XIV. The products g i v i n g the peaks corresponding to the sugars l i s t e in Table XIV were collected from the chromatograph in capillary tubes and analysed by direct i n t r o d u c t i o n i n mass spectroscopy. The p o s i t i o n s of the O-methyl s u b s t i t u e n t s were determined by using the most prominent peaks (m/e) i n the mass spectra according to B j o r n d a l , Lindberg and Svensson ( 5 8 ) . The 1 , 5 - d i - 0 - a c e t y l - 2,3,4,6-tetra-O-methyl h e x i t o l s were c h a r a c t e r i z e d by t h e i r m/e 45,101,1177129,145,161 and 205. Confirmation of the presence of the two 1 , 4 , 5 - t r i - O - a c e t y l - 2,3, 6-t r i - 0 - m e t h y l h e x i t o l s (mannose and glucose) was obtained by the presence i n t h e i r fragmentation p a t t e r n of the peak of m/e 45,99, 101,113 and 233 as the most c h a r a c t e r i s t i c . The 1 , 3 , 4 , 5 - te t r a-0-. a c e t y l - 2,6-di-O-methy1 h e x i t o i was i d e n t i f i e d by the presence i n i t s mass spectrum of peaks at. m/e 45 ,87 , 117 and 129. Two dimethyl sugars were i d e n t i f i e d as 1 , 4 , 5 , 6 - te tra-0-ace ty 1 - 2 , 3-di -O-rne thy 1 h e x i t o l s by the peaks at m/e 43,101,117 and 261, and by the absence of the peak m/e 45 c h a r a c t e r i s t i c of. those h e x i t o l acetates methy-l a t e d on t h e i r primary a l c o h o l f u n c t i o n . -74f IV - STRUCTURAL STUDY OF GALACTOGLUCOMANNAN (GGM) F r a c t i o n S'4-GGM^  was used f o r the s t r u c t u r a l i n v e s t i g a t i o n . I t i s apparent from Table IX that t h i s f r a c t i o n s t i l l contains about 15 % of a contaminant xylan and that the mannose content i s unusually high for a'galactoglucomannan from a c o n i f e r . Nevertheless t h i s f r a c t i o n was p r e f e r r e d to f r a c t i o n S4-GGM.J.J because of i t s higher y i e l d , being thus more re p r e s e n t a t i v e of the GGM of Red wood. I t was also hoped that a p u r i f i c a t i o n of t h i s p olysaccharide would occur during the course of the methylation steps (89). a - Methylation of galactoglucomannan Although the polysaccharide was not s o l u b l e at f i r s t i n d i -methyl sulphoxide, i t was methylated by the procedure of Iakomori i n the same co n d i t i o n s as p r e v i o u s l y described f o r the glucomannan. A f t e r the second methylation, a l l the product was s o l u b i l i z e d . A t h i r d a d d i t i o n of methylating reagents was a p p l i e d to the s o l u t i o n and the mixture was d i a l y z e d and f r e e z e - d r i e d . The product of reac-t i o n was now r e a d i l y s o l u b l e i n dimethyl sulphoxide. A f t e r one more methylation according to Hakomori and four methylatiohs according to P u r d i e , the methylated product was f r a c t i o n a t e d with chloroform-petroleum ether. The- e x t r a c t i o n s 1 with 15 %; and 20 % chloroform gave i n about 20 per cent y i e l d a product showing no hydroxyl absorption i n the i n f r a r e d spectrum. This sample was used f o r subsequent ana-l y s i s . -75-TABLE X V — A N A L Y S I S OF METHYLATED GALACTOGLUCOMANNAN Rt (min) Molar r a t i o 2,3,5-Tri-O-Me pentose 15.6 3.1 2,3,4,6-Tetra-O-Me (glucose + mannose) 20.8 1.0 2,3,4,6-Tetra-O-Me galactose 23.0 6.1 2,3-Di-O-Me xylose 24.2 18.0 2,3,6-Tri-0-Me mannose 28.8 23.2 2,3,6-Tri-0-Me glucose 30.2 7.3 2j3-Di~()-Me mannose 36.2 4.5 2,3-Di-O-Me glucose 37.8 3.6 ± As a l d i t o l acetate d e r i v a t i v e s Compound - 7 6 -b - A n a l y s i s of methylated galactoglucomannan The p a r t i a l l y methylated sugars released by s u l p h u r i c a c i d h y d r o l y s i s were examined by paper chromatography. E l u t i o n with the solvent system n-butanol ,• ethanol , water , ammonium hydroxide (4 : 1 : 4.9 : 0.1) gave 7 spots with the f o l l o w i n g Rg, ( r e l a t i v e to 2 , 3 , 4,6,tetra-O-methyl glucose) 0.97 : 0.92 : O.05 : 0.82 : 0.7o 0.63 and 0.52 r e s p e c t i v e l y . E l u t i o n with the solvent syst&ra methyl e t h y l ketone, water, ammonium hydroxide (100 : 5 : 3) showed s i x major components. The hydrolysate mixture was reduced with sodium borchydride and the p a r t i a l l y methylated a l d i t o l s were a c e t y l a t e d . The r e s u l t i n g methylated a l d i t o l acetates were examined by gas l i q u i d chromato-graphy and showed 9 peaks, most of which.were i d e n t i f i e d by com-par i s o n with authentic standards, or by comparison of t h e i r reten-t i o n times with the l i t e r a t u r e data. In a l l cases, confirmation of t h e i r i d e n t i t y was obtained by the examination of t h e i r fragmen-t a t i o n p a t t e r n i n mass spectroscopy, (Table XV). Peak ?V 1, Rt 15.8 minutes corresponded to a tri-0_-methyl p e n t i t o l acetate and was concluded to be 1,4,-di-O-acetyl - 2,3, 5- tri-0_-methyl p e n t i t o l by the presence amongst the peaks of i t s mass spectrum of m/e 45,71,87,101,117,129 and 161. The peak at m/e 45 shows that a primary a l c o h o l of the a l d i t o l i s methylated, and the r e t e n t i o n time being much shorter than that of the 2,3,4 6- tetra-<Q-methy 1 h e x i t o l acetates, i n d i c a t e s that the compound i s a pentose d e r i v a t i v e . Peak -fat- 2, Rt 20.8 minutes co-chromatographed with standard samples of 2 , 3 , 4 , 6-te tra-p_-me thy 1-D-glucose and mannose d e r i v a t i v e s . The mass spectrum e x h i b i t e d the c h a r a c t e r i s t i c peaks of 1 , 5 - d i -O - a c e t y l - 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l - h e x i t o l s a t the m/e 4 5 , 145 and 2 0 5 i n a d d i t i o n to 4 3 , 7 1 , 8 7 , 1 0 1 , 1 1 7 , 129 and 1 6 1 . T h i s peak thus, must correspond to e i t h e r t e t r a - m e t h y l glucose or mannose d e r i v a t i v e or to a mixture of both. Peak fcfi 3 gave the same fragmentation In mass spectography as peak j=£ 2 and had a r e t e n t i o n time (Rt 23 minutes) corresponding to 1 , 5 - d i - O - a c e t y l , 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g a l a c t i t o l . Peak 4 was i d e n t i f i e d by i t s r e t e n t i o n time (Rt 2 4 . 2 mi-nutes) as the 2 , 3 - d i - 0 - m e t h y l x y l o s e a l d i t o l a c e t a t e d e r i v a t i v e and showed the expected peaks a t m/e 4 3 , 8 7 , 1 0 1 , 1 1 7 , 129 and 189 as the major peaks of the mass spectrum. Peak j=£ 5 was. the l a r g e s t peak of the chromatogram and as such expected to be the 2 , 3 , 6 - t r i - 0 - m e t h y l mannose d e r i v a t i v e . T h i s was confirmed by the presence i n the mass spectrum of peaks at m/e 4 5 , 113 and 2 3 3 i n a d d i t i o n to 4 3 , 8 7 , 9 9 , 101 and 1 1 7 . Peak 7%^ 6 gave i n mass spectroscopy the same major fragments as peak fi=£ 5 and was thus c o n s i s t e n t with methyl s u b s t i t u e n t s i n p o s i t i o n s 2 , 3 and 6 on an h e x i t o l ( e s s e n t i a l l y peaks at m/e 45 and 2 3 3 ) . The r e t e n t i o n ' t i m e of t h i s compound agreed with 2 , 3 , 6 -tri-O-methy1 g l u c i t o l a c e t a t e . Peaks £4- 7 and fir 8 both gave the same fragmentation i n mass spectroscopy with fragments at m/e 4 3 , 1 0 1 , 117 and the very charac-t e r i s t i c peak at 261 i n d i c a t i v e of the presence of a methyl s u b s t i -tuent on p o s i t i o n 3 and of a c e t y l groups at p o s i t i o n s 4 , 5 and 6 . The corresponding methylated sugars were then i d e n t i f i e d as 2 , 3 -di-O-roethyl mannose and glucose r e s p e c t i v e l y , which were c o n s i s t e n t with the i r retent ion times corresponding to di-methyl hex i to l te t ra -acetates . -79-DISCUSSION AND CONCLUSIONS I - ISOLATION AND PURIFICATION OF THE HEMICELLULOSES FROM RED WOOD I s o l a t i o n o f p o l y m e r s i n good y i e l d s and p r e s e n t i n g the h i g h e s t p u r i t y i s r e q u i r e d f o r the c h e m i c a l s t u d y o f wood h e m i c e l l u l o s e s . As a preliminary step, the h o m o g e n e i t y i n the c a r b o h y d r a t e c o m p o s i t i o n o f a p o l y s a c c h a r i d e f r a c t i o n can be i n d i c a t i v e o f a c e r t a i n d e g r e e o f p u r i t y . C o n i f e r o u s w o o d s have been shown (8,10) t o c o n t a i n t h r e e main h e m i c e l l u l o s e s : x y l a n , glucomannan and g a l a c t o g l u c o m a n n a n , separation of which i s known to be d i f f i c u l t (90) . Our r e s u l t s on the i s o l a t i o n o f t h e h e m i c e l l u l o s e s f r o m the wood o f S e q u o i a s e m p e r v i r e n s show t h a t s e v e r a l methods o f e x t r a c t i o n and p u r i f i c a t i o n have been u s e d . O n l y th e glucomannan c o u l d be e x t r a c t e d r e a d i l y and i n good y i e l d ( a b o u t 8.5 % ) . T h i s p o l y s a c c h a -r i d e showed a r a t h e r c o n s t a n t c o m p o s i t i o n i n g a l a c t o s e , g l u c o s e and mannose i n the m o l a r p r o p o r t i o n 0.1 ; 1 and 6.2 ( o r 6.8). The r a t i o o f mannose t o g l u c o s e seems v e r y h i g h i n t h i s f r a c t i o n when compared t o t h a t u s u a l l y f o u n d i n gymnosperm g e n e r a which r a n g e s from 2.5 t o 4 a c c o r d i n g t o the mode o f i s o l a t i o n and t o t h e s p e c i e s ( 9 1 ) . I t i s n o t i c e a b l e t h a t the c r u d e glucomannan b e f o r e p u r i f i c a t i o n w i t h b a r i u m h y d r o x i d e had a c o m p o s i t i o n i n g a l a c t o s e , g l u c o s e and mannose o f 0.3 : 1 and 4.9 r e s p e c t i v e l y ( F i g . 9, T a b l e V I I I ) . T h i s c o m p o s i t i o n i s c l o s e r t o the n o r m a l c o m p o s i t i o n . - 8 0 -TABLE XVI— GALACTOSE:GLUCOSE :MANNOSE RATIO IN INTERMEDIATE FRACTIONS OF MANNOSE-CONTAINING HEMICELLULOSES FROM RED WOOD n Mode of extraction * Galactose Glucose Mannose Crude GM A ; 0 . 3 1 . 0 4.9 GM A 0 . 1 1 . 0 6 . 2 GGM-1 A. 1 . 0 0 . 6 3 . 1 GGM-II • A 0 . 6 0 . 6 2 . 2 S 4 GGM-I B 1 . 0 0 . 4 2 . 8 S4 GGM-11 B " 1 . 0 0 . 4 2 . 2 Crude GGM . ... . C '"' 1 . 0 2 . 6 5 . 0 ?l GGM c 1 . 0 0 . 7 3 . 0 P 2 GGM c 1 . 0 0 . 7 3 . 1 X A, as in Figure 9 Bj as in Figure 1 0 C, Pj^  and V2 GGM's v/ere obtained by ethanol precipitation from the supernatant of the 24% potassium hydroxide extract after barium hydroxide precipitation which gave the crude GGM. - 8 1 -of a glucomannan from, softwood and agrees more with the composition of the methylated polysaccharide (see below). The separation of xylan from galactoglucomannan .was d i f f i c u l t . However a xylan could be obtained with a comparable composition what ever the mode of i s o l a t i o n and p u r i f i c a t i o n used. As expected f o r a xylan from coniferous wood, the p r o p o r t i o n of 4-O-methyl glucuro-n i c a c i d was high (18.5 %) and the p r o p o r t i o n of arabinose was low (3.7 %) as compared to hardwood xylans. Several d i f f e r e n t methods f o r the separation of the galactoglucomannan have been used but none of them gave a pure polysaccharide f r a c t i o n i n good y i e l d . Moreover, i t seems/from the a n a l y s i s of the intermediate f r a c t i o n s obtained during the course of the d i f f e r e n t p u r i f i c a t i o n s , that there i s a great heterogeneity i n the polysaccharides composed of gal a c t o s e , glucose and mannose. Table XVI gives some of the r e s u l t s obtained at d i f f e r e n t stages of p u r i f i c a t i o n . "The p r o p o r t i o n of mannose to glucose i s always greater than the normal r a t i o of 3 to 1 for softwood (74), and, on the contrary, the p r o p o r t i o n of galactose i s higher than that u s u a l l y present i n c o n i f e r s . Such a low p r o p o r t i o n of glucose, which i s the dominant feature of t h i s p olysaccharide i s d i f f i c u l t to e x p l a i n by a p o s s i b l e degradation, since among gal a c t o s e , glucose and mannose, glucose i s known f o r being the most r e s i s t a n t to h y d r o l y t i c cleavage, and galactose to have a considerable a c i d l a b i l i t y , p a r t i c u l a r l y f o r (.1^ 6:) g a l a c t o s i d i bonds (92). However, the l i t e r a t u r e provides a few examples of g l u c o s e - d e f i c i e n t ga1actoglucomannans(see Table XVII). Rogers and Thomson (92) f o r the GGM of Engelrnann Spruce found a r a t i o of g a l a c t o s e , glucose mannose of 1.7 : 0.7 : 3 and 0.9 : 0.7 : 3 r e s p e c t i v e l y f o r the ascending and descending samples of a GGM - 8 2 -submitted to free, boundary e l e c t r o p h o r e s i s . Meier (93) a l s o found i n isiorvegi an Spruce a ga l a c t o s e - r i ch GGM i n which the r a t i o of mannose to glucose was.7.4 . Our o p i n i o n from the data i n Table XVI i s t h a t galactogl-'uco-mannans and glucomannans are two wide f a m i l i e s of p o l y s a c c h a r i d e s c o n s t i t u t e d of f r a c t i o n s i n which the r a t i o of component sugars can vary g r e a t l y . T h i s suggestion i s s u b s t a n t i a t e d by the r e s u l t s of Dutton and h i s c o l l a b o r a t o r s (94,86) on the h e m i c e l l u l o s e s of Black Spruce and S i t k a Spruce (97) where r a t i o s of mannose to glucose v a r y i n g from 4.5 to 6.7 and 4.0 to 5.2 r e s p e c t i v e l y , were found i n the d i f f e r e n t i n t e r m e d i a t e f r a c t i o n s of p u r i f i c a t i o n . A s i m i l a r s i t u a t i o n i s found i n the mannose c o n t a i n i n g p o l y s a c c h a r i d e s from Western Hemlock (78) with r a t i o s v a r y i n g from 2.1 to 3.6 . Other i n s t a n c e s showing the h e t e r o g e n e i t y of wood p o l y s a c c h a r i d e s have been demonstrated (77) - 8 3 -II - INTERPRETATION OF STRUCTURE a - Arabino-4-O-methy 1 2 ^ u c u r 2 2 2 xylan The s t r u c t u r e of the xylan f r a c t i o n S4-C2 has been e l u c i -dated by the conjunction of the a n a l y t i c a l data obtained by hydro-l y s i s , d ecarboxylation and methoxyl determination (Table IX), along with methylation a n a l y s i s and periodate o x i d a t i o n . Results i n Table IX i n d i c a t e that t h i s xylan c a r r i e s very few arabinose residues but on the other hand has a high content of 4-0-methyl g l u c u r o n i c a c i d . The equ i v a l e n t weight i s 722 correspon-ding to one 4-0-methyl gluc u r o n i c a c i d f o r four xylose residues. The data obtained by methylation are expressed i n Tables:XI and XII. The i n v e s t i g a t i o n by methylation has been c a r r i e d out by using two d i f f e r e n t techniques of methylation. The r e s u l t corresponding to three successive Hakomori treatments (Table XI) compares w e l l with that obtained by Haworth's and Sr i v a s t a v a ' s treatments (Table XII) i n d i c a t i n g an average chain length of 51 and 58 r e s p e c t i v e l y . Such a discrepancy i s not large enough to account f o r a p o s s i b l e degra-dation i n the main chain due to the d r a s t i c c o n d i t i o n s of the f i r s t experiment. When the carboxy1-reduced polysaccharide was analysed (Table XII) an average chain length of 66 was found. This d i f f e r e n c e with the f i r s t r e s u l t s corresponds to the xylose u n i t s l i n k e d to. the g l u c u r o n i c a c i d residues which escape a n a l y s i s when the n e u t r a l sugars are i n v e s t i g a t e d , as the g l y c u r o n i c linkage between a uronic a c i d and a n e u t r a l sugar i s more r e s i s t a n t to a c i d h y d r o l y s i s . - 8 4 -T h i s f a c t appears c l e a r l y i n Table XII with the d i f f e r e n c e i n mono-O-methyl xylose 'between the unreduced and reduced x y l a n . The h i g h e r y i e l d of raono-O-methyl xylose obtained a f t e r three Hakomori methylations i s c e r t a i n l y due to the easy cleavage during a c i d h y d r o l y s i s of the unsaturated uronate c r e a t e d by the $ e l i m i -n a t i o n which took p l a c e i n the s t r o n g l y b a s i c medium (see R e s u l t s ) . From these comparative analyses- i t can be concluded that remethy-l a t i n g an a c i d i c p o l y s a c c h a r i d e with dimethyl sulphoxide can give a b e t t e r r e s u l t as f a r as the n e u t r a l sugars are concerned but does not permit the e s t i m a t i o n of the u r o n i c a c i d r e s i d u e s ( 8 3 ) . The recovery of 2 , 3 , 4-tri-O-methy 1 glucose i n a 13 % r a t i o compared to the 1 8 . 5 % 4 - 0-methyl g l u c u r o n i c a c i d p r e s e n t i n the s t a r t i n g m a t e r i a l (Table IX) shows t h a t only a weak e l i m i n a t i o n took p l a c e during the second procedure of m e t h y l a t i o n , or that the o r i g i n a l m a t e r i a l had been f r a c t i o n a t e d cfuring the methylation p r o c e s s . When chain length was estimated by p e r i o d a t e o x i d a t i o n (Table XII) an average degree of p o l y m e r i z a t i o n of about 52 was o b t a i n e d , and the r e l e a s e of formic a c i d corresponded to about 0 . 7 branch p o i n t s i n the c h a i n . T h i s value of 52 u n i t s i n the chain i s i n good agreement with the r e s u l t of m e t h y l a t i o n . T h i s was c o n f i r -med by a measurement of the number-average molecular weight deter-mined by osmometry on the methylated xylan which gave a Mn value of 8 , 2 3 0 c o r r e s p e n d i n g ' t o a chain length of 5 1 . From the above r e s u l t s and p a r t i c u l a r l y from the m e t h y l a t i o n study, i t can be seen t h a t a l l the arabinose and the 4 - 0-methyl This measurement due to the courtesy of P r o f e s s o r T.E. TIMELL Syracuse, New York -85-g l u c u r o n i c a c i d r e s i d u e s occur as t e r m i n a l non reducing s i d e r e s i d u e s . But no i n f o r m a t i o n can be obtained by the p r e c e d i n g methods of i n v e s t i g a t i o n about the d i s t r i b u t i o n of these s u b s t i t u -ents on the main chain of x y l o s e . On the other hand, an exhaustive a t t a c k of the xylan by an endo-xylanase l e d to only 70 % degraded p o l y s a c c h a r i d e with 30 % m a t e r i a l which was r e s i s t a n t to the a c t i o n of the enzyme. The residue from this attack had a xylose to glucuronic a c i d content of 2 whereas i n the s t a r t i n g m a t e r i a l i t was 4. T h i s r e s u l t which i s an agreement with the mode of a c t i o n of the enzyme (96) i s i n favour of a non random d i s t r i b u t i o n of the 4-0-methyl g l u c u r o n i c a c i d s u b s t i t u e n t s on the x y l o s y l main chain and suggests that these s u b s t i t u e n t s are l o c a l i z e d on p r e f e r e n t i a l areas of the rnacromolecule . A s i m i l a r c o n c l u s i o n has been r e c e n t l y obtained by Rosell- and svensson- on b i r c h xylan (12 0) . However,-another e x p l a n a t i o n of t h i s r e s u l t could be t h a t two d i f f e r e n t l y ^ s u b s t i t u t e d xylans couldhave been p r e s e n t i n the s t a r t i n g m a t e r i a l , one being l e s s s u b s t i t u t e d and thus more s u s c e p t i b l e to enzymic d e g r a d a t i o n . b - Mannose-containing h e m i c e l l u l o s e s The glucomannan and galactoglucomannan f r a c t i o n s were s t u -d i e d e s s e n t i a l l y by m e t h y l a t i o n . Table XIV shows the a n a l y t i c a l data e s t a b l i s h e d f o r the methylated glucomannan. Because of the s m a l l amount of methylated p o l y s a c c h a r i d e i t was not p o s s i b l e to d i s t i n g u i s h between 2,3,4,6-tetra-0-methy1 glucose and mannose. I t i s thus not p o s s i b l e to conclude whether the non-reducing end of the polymer terminates with a glucose or mannose r e s i d u e or i f both -86-a r e p r e s e n t . The predominant, components, 7, .. 3 , 6 - t r i --O-ine t h y 1 man-nose and 2 , 3 , 6 - t r i - O - m e t h y 1 g l u c o s e , i n d i c a t e t h a t t h e main c h a i n i s c o n s t i t u t e d o f (1 — V 4) L i n k e d g l u c o s e and mannose r e s i d u e s . The d i f f e r e n c e between 2 , 3 , 4 , 6 - t e t r a - O - m e t h y 1 g a l a c t o s e and 2 , 3 - d i - 0 -m e t h y l g l u c o s e and mannose may be duo t o some p a r t i a l demethy l a t i o;. of the p o l y s a c c h a r i d e . A l l the g a l a c t o s e i s the n f o u n d as t e r m i n a l p y r a n o s e u n i t s (1 —-*• 6) l i n k e d to mannose or g l u c o s e u n i t s i n t h e c h a i n . Q u a n t i t a t i v e r e s u l t s i n d i c a t e an a v e r a g e c h a i n l e n g t h o f 61. T h i s a v e r a g e d e g r e e o f p o l y m e r i s a t i o n i s i n good agreement w i t h the number-average m o l e c u l a r w e i g h t o f 11,600 d e t e r m i n e d by osmometry on the c o r r e s p o n d i n g m e t h y l a t e d f r a c t i o n , and g i v i n g an a v e r a g e c h a i n l e n g t h o f 57. B u t t h i s r e s u l t can be f o r t u i t o u s s i n c e t h e m e t h y l a t e d f r a c t i o n u n d e r i n v e s t i g a t i o n r e p r e s e n t e d o n l y 25 % of the s t a r t i n g m a t e r i a l and a l s o b e c a u s e the f i n a l r a t i o o f g l u c o s e and mannose i n the m e t h y l a t e d p r o d u c t s d i f f e r s g r e a t l y f r o m the r a t i o p r e s e n t i n the o r i g i n a l p o l y s a c c h a r i d e : 0.07, 1.0, 4.9 v e r s u s 0.12, 1.0 and 6.2 r e s p e c t i v e l y . Such a d i s c r e p a n c y c o u l d be a t t r i b u t e d t o the p r e s e n c e o f d i f f e r e n t glucomannan f r a c t i o n s i n the same wood ; i t seems t h e r e f o r e t h a t o n l y the d o m i n a n t f e a -t u r e o f t h e p o l y s a c c h a r i d e can be e s t a b l i s h e d i n s u c h a s t u d y and t h a t an e x t r e m e l y p u r e m a t e r i a l i s r e q u i r e d i n o r d e r t o g e t a c c u -r a t e c o n c l u s i o n s as a l r e a d y d i s c u s s e d by D u t t o n and Wal k e r (87 ,94) . S i m i l a r r e m a r k s can be made on the r e s u l t s o f t h e g a l a c t o -g l u c o mannan s t u d y . The d o m i n a n t s t r u c t u r a l f e a t u r e s determined by the m e t h y l a t i o n s t u d y ( T a b l e XV) show t h a t t h e p r i n c i p a l c h a i n i s a f l —*• 4) l i n k e d b ackbone o f g l u c o s e and mannose r e s i d u e s an i n d i c a -t e d by t h e p r e d o m i n a n c e of 2 , 3 , 6-tri-0_-methy 1 r e s i d u e s o f mannose and g l u c o f j e . A l l the g a l a c t o s e i s f o u n d as 2 , 3 , 4 , 6 - t e t r a - O - m e t h y l -87-TABLE XVII - A FEW EXAMPLES OF GLUCOSE-DEFICIENT GALACTOGLUCOMANNANS FROM GYMNOSPERMS O r i g i n G a l a c t o s e Glucose Mannose References N o r v e g i a n Spruce 1 . 0 0 . 6 7 4 . 9 0 (93) Engelmann Spruce 1 . 0 0 . 4 1 1 . 7 6 . (92) Ponderosa Pine GGM^ 1 . 0 0 . 8 5 2 . 1 4 (77) Ponderosa Pine GGMg 1 . 0 0 .57 1.35 (77) K r a f t pulp from 1 . 0 0 .83 2 . 5 0 (95) Southern Pine • 8 8 -d e r i v a t i v o , t h u s b e i n g p r e s e n t as s i n g l e r e s i d u e s i n s i d e chains; l i n k e d b y ( l —** 6 ) g l y c o s i d i c bonds t o mannose and g l u c o s e as i n d i c a -t e d by the p r e s e n c e o f 2 , 3 - d i - 0 - m e t h y 1 g l u c o s e and mannose. Here a g a i n t h e r e i s a s l i g h t e x c e s s o f 2 , 3 - d i - 0 - m e t h y 1 s u g ? r compared t o t h e p r o p o r t i o n o f 2 , 3 , 4 , 6 - t e t r a - O - i n e t h y l - g a l a c t o s e . T h i s d i s c r e -p a n c y c a n n o t be a c c o u n t e d f o r t h e p r e s e n c e o f s i d e b r a n c h i n g o f mannose or g l u c o s e s i n c e t h e r e i s o n l y one t e t r a - O - m e t h y l g l u c o s e and mannose f o r the a v e r a g e c h a i n l e n g t h o f a b o u t 4 0 u n i t s , a n d s i n c e t h i s d e g r e e o f p o l y m e r i s a t i o n was c o n f i r m e d by osmometry on the same m e t h y l a t e d p o l y s a c c h a r i d e . The l a t t e r measurement gave a v a l u e o f 8 , 8 0 0 f o r the number-average m o l e c u l a r w e i g h t w h i c h i n f a c t c o r r e s p o n d s t o an a v e r a g e c h a i n o f 4 3 u n i t s . As f o r the glucomannan a c e r t a i n amount o f 2 , 3 - d i - 0 - m e t h y l x y l o s e i s p r e s e n t among the h y d r o l y s i s p r o d u c t o f t h e m e t h y l a t e d p o l y s a c c h a r i d e s . T h i s x y l o s e must c o r r e s p o n d t o t h e c o n t a m i n e n t x y l a n p r e s e n t i n t h e o r i g i n a l p o l y s a c c h a r i d e s ( T a b l e s V I I I and I X ) . The r a t i o s o f g a l a c t o s e , g l u c o s e and mannose i n the methy-l a t e d p o l y m e r compared t o t h e o r i g i n a l f r a c t i o n , h e r e a g a i n , v a r i e d g r e a t l y . The p r o p o r t i o n s o f the t h r e e s u g a r s were l.O : 0 . 4 2 . 8 r e s p e c t i v e l y i n t h e unmethylatedpolysaccharJ.de and a r e 0 . 6 : 1 . 0 2 . 5 i n t h e m e t h y l a t e d f r a c t i o n . T h i s can be c o n s i d e r e d as an a d d i t i o -n a l p r o o f o f t h e h e t e r o g e n e i t y o f t h e g a l a c t o g l u c o m a n n a n p r e s e n t i n S e q u o i a s e m p e r v i r e n s as a l r e a d y n o t i c e d p r e v i o u s l y ( T a b l e XVI) and as seems t o be t h e ca s e f o r o t h e r s p e c i e s ( 7 7 , 8 6 ) . In c o n c l u s i o n on the h e m i c e l l u l o s e s f r o m Redwood, o n l y t h e a r a b i n o 4 - 0 - m e t h y l g l u c u r o n o x y l a n seems t o be homogenous a n " shows a g e n e r a l n a t u r e s i m i l a r t o t h a t o f o t h e r c o n i f e r s . - 8 9 -The p e r c e n t a g e o f u r o n i c a c i d i s r a t h e r h i g h and the x y l a n f r o m S e q u o i a s e m p e r v i r e n s i s v e r y s i m i l a r t o t h a t f o u n d f o r W e s t e r n Res c e d a r (Thuj a p l i c a t a ) . T h i s may be a common c h a r a c t e r of T a x o d i a -ceae and C u p r e s s a c e a e . The s t u d i e s on the g a l a c t o m a n n a n and. g a l a c -t o g l u c o m a n n a n s h o w . t h a t each o f t h e s e has t h e c u s t o m a r y s t r u c t u r e , t h a t i s a p ( l - > 4 ) l i n k e d glucomannan w i t h , i n the. l a t t e r c a s e , some s i d e c h a i n g a l a c t o s e u n i t s . However, the s m a l l amounts i s o l a t e d . o f t h e s e two p o l y s a c c h a r i d e s and t h e i r o b v i o u s h e t e r o g e n e i t y p r e v e n t e d e t a i l e d s t u d i e s f r o m w h i c h more d e f i n i t e s t r u c t u r a l c o n c l u s i o n s c o u l d be drawn. From the r e s u l t s o b t a i n e d f o r glucomannan and g a l a c t o g l u c o m a n n a n we do n o t f e e l e n t i t l e d t o draw c o n c l u s i o n s on t h e c h a r a c t e r i s t i c s o f t h e s e two h e m i c e l l u l o s e s . E X P E R I M E N T A L G e n e r a l methods : As d e s c r i b e d i n P a r t I P r e p a r a t i o n o f H o l o c e l l u l o s e f r o m the wood o f S e q u o i a s e m p e r v i r e n s . The wood o f S e q u o i a s e m p e r v i r e n s was gro u n d i n a m i l l and the s awdust (741 g) e x h a u s t i v e l y e x t r a c t e d f o r t a n i n s , p i g m e n t s and waxes w i t h t h e m i x t u r e e t h a n o l - b e n z e n e (1 : 2 v/v) i n a S p x h l e t a p p a r a t u s , y i e l d i n g an e x t r a c t i v e - f r e e m a t e r i a l (702 g ) . The e x t r a c t i v e - f r e e meal (lOO g) was t r e a t e d w i t h sodium c h l o r i t e (30 g) and a c e t i c a c i d (10 ml a t 7 0 ° f o r 1 h o u r ) . A f t e r four successive treatments, a h o l o c e l l u l o s e was obtained i n 75% y i e l d , with a K l a s o n l i g n i n c o n t e n t o f a b o u t 6 % s t i l l r e m a i n i n g i n t h e h o l o c e l -l u l o s e . I s o l a t i o n o f c r u d e h e m i c e l l u l o s e s . H o l o c e l l u l o s e (112 g) was t r e a t e d w i t h aqueous 24 •% p o t a s -sium h y d r o x i d e ( 1 . 1 1) f o r 24 h o u r s . A f t e r f i l t r a t i o n , t h e s o l u t i o n c o n t a i n e d a c r u d e m i x t u r e o f x y l a n and g a l a c t o g l u c o m a n n a n . The i n s o l u b l e r e s i d u e was e x t r a c t e d f o r 24 h o u r s w i t h 1 7 . 5 % s o l u t i o n o f sodium h y d r o x i d e (1 1) c o n t a i n i n g 4 % b o r i c a c i d . A d d i t i o n o f e t h a n o l (3 volumes) t o the c l e a r e x t r a c t gave a c r u d e g l u comannan. T h i s c r u d e f r a c t i o n was r e - s o l u b i 1 i s e d i n sodium h y d r o x i d e ( 1 7 . 5 % c o n t a i n i n g 4 % b o r i c a c i d ) and d r o p w i s e a d d i t i o n o f an aqueous 5 % b a r i u m h y d r o x i d e s o l u t i o n under v i g o r o u s s t i r r i n g y i e l d e d a + + glucomannan-Ba complex. S o l u b i l i z a t i o n i n 5 0 % a c e t i c a c i d f o l l o w e d by p r e c i p i t a t i o n w i t h e t h a n o l gave glucomannan (GM) (16 g, ash 1 8 . 5 %, y i e l d 8 . 6 . % o f the d r y wood) , s u g a r c o m p o s i t i o n (%) : g a l a c t o s e ( 1 . 8 ) , g l u c o s e ( 1 2 . 2 ) mannose ( 8 4 ) x y l o s e ( 1 . 8 ) , 2 0 [ a ] D - 1 2 ° ( I N NaOH) . I s o l a t i o n o f x y l a n and g a l a c t o g l u c o m a n n a n . . 1 ) Scheme a ( F i g . 9 ) - The 2 4 % p o t a s s i u m h y d r o x i d e e x t r a c t f r o m h o l o c e l l u l o s e ( 1 5 5 g) gave by e t h a n o l p r e c i p i t a t i o n a c r u d e h e m i c e l l u l o s e m i x t u r e (Sx, 4 3 g) w h i c h upon c h l o r i t e d e l i g n i f i c a t i o n a t room t e m p e r a t u r e a c c o r d i n g t o C l a y t o n y i e l d e d S x 2 ( 3 4 g ) . T o a 1 0 % s o l u t i o n o f S x 2 was added d r o p w i s e b a r i u m h y d r o x i d e 5 % ( 1 . 3 1 ) and the r e s u l t i n g 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 g i -v i n g GGMj]- ( 5 . 5 g) . The s u p e r n a t a n t was p r e c i p i t a t e d w i t h e t h a n o l c o n t a i n i n g an e x c e s s o f a c e t i c a c i d and gave S x 3 ( 1 8 g ) . Sx3 was t r e a t e d as S x i and GGM^ . ( 1 g) was o b t a i n e d . To t h e s u p e r n a t a n t o f GGM a d d i t i o n o f 1 5 % e t h a n o l gave GGM .^ ^  ^  ( 7 . 5 g) and f u r t h e r e t h a n o l p r e c i p i t a t i o n i n an e x c e s s o f a c e t i c a c i d y i e l d e d t h e xy-( 2 0 \ l a n X j 6 g, [a] D - 3 1 .6° (H 20) j . A n a l y t i c a l d a t a a r e e x p r e s s e d i n T a b l e V I I I . 2 ) Scheme b ( F i g . 1 0 ) - H o l o c e l l u l o s e ( 1 1 2 g) o b t a i n e d by f o u r s u c c e s s i v e c h l o r i t e t r e a t m e n t s was e x t r a c t e d w i t h p o t a s s i u m h y d r o x i d e ( 2 4 % 1 1 ) f o r 2 4 h o u r s . To the e x t r a c t aqueous 5 % b a r i u m h y d r o x i d e was added d r o p w i s e and t h e p r e c i p i t a t e d b a r i u m -92-coroplex s o l u b i l i z e d . i n 50 % a c e t i c a c i d , y i e l d i n g b'y p r e c i p i t a -t i o n v.'ith e t h a n o l a f r a c t i o n GGM^ (5.6 g, ash 17.7 %, m e t h o x y l . 2 0 . c o n t e n t 2.5 %, [a] ^ '- 0° ( I N NaOH) . A f t e r c e n t r i f u g a t i o n t h e super-n a t a n t w a s P r e c i p i t a t e d w i t h e t h a n o l and gave S i , X (45 g) . A f t e r s o l u b i l i z a t i o n o f t h e l a t t e r i n 10 ?; p o t a s s i u m h y d r o x i d e , t h e n n e u t r a l i z a t i o n w i t h a c e t i c a c i d , a 2.5 % s o l u t i o n o f c e t y l t r i m e t h y l ammonium b r o m i d e ( C e t a v l o n ) was added, g i v i n g by c e n t r i f u g a t i o n a l i g h t p r e c i p i t a t e S 4 C 1 (0.275 g ) . The s u p e r n a t a n t gave by a l c o h o l p r e c i p i t a t i o n a w h i t e p r e c i p i t a t e w h i c h was p a r t i a l l y d i s s o l v e d i n w a t e r , l e a v i n g an i n s o l u b l e , S i, C 3 (4.2 g) . T h i s s o l u b l e p a r t t r e a t e d w i t h b a r i u m h y d r o x i d e - b o r i c a c i d 4 % d i d n o t g i v e any p r e -c i p i t a t i o n , b u t when p o u r e d i n t o an e t h a n o l - a c e t i c a c i d m i x t u r e , a w h i t e p r e c i p i t a t e was o b t a i n e d w h i c h , a f t e r d r y i n g , y i e l d e d St^C? , ' .20 . • » • • C23.5 g, ash 15.1 %, m e t h o x y l 4.2 % [ a j D - 42° ( H 2 0 ) j . The i n s o l u b l e S \ C 3 f r a c t i o n , s o l u b i l i z e d i n 10 % p o t a s s i u m h y d r o x i d e s o l u t i o n and p r e c i p i t a t e d as b e f o r e as i t s b a r i u m complex a f f o r d e d S 4 GGM^ . (1.27 g, ash 22 % ) . M e t h y l a t i o n o f x y l a n S 4 C 2 a) F i r s t e x p e r i m e n t _ ; Hakomori m e t h y l a t i o n s . To x y l a n Si,C'2 (2.1 g) s u s p e n d e d i n d i m e t h y l s u l p h o x i d e (150 ml) was added u n d e r n i t r o g e n t h e d i m s y l a n i o n j s o d i u m h y d r i d e (4.5 g) i n d i m e t h y l s u l -p h o x i d e (50 m l ) j . A f t e r s t i r r i n g o v e r n i g h t a t room t e m p e r a t u r e , m e t h y l i o d i d e (7 ml) was t h e n added d r o p w i s e o v e r a two h o u r s pe-r i o d and the m i x t u r e was p o u r e d i n t o 'Vater 600 ml) and d i a l y z e d . The d i a l y z e d s o l u t i o n was f r e e z e d r i e d and a s e c o n d m e t h y l a t i o n was p e r f o r m e d as above. A f t e r r e c o v e r y o f t h e s e c o n d d i a l y s i s m a t e r i a l by c h l o r o f o r m e x t r a c t i o n , a t h i r d t r e a t m e was p e r f o r m e d on t h e d r y c h l o r o f o r m e x t r a c t s , w h i c h a f f o r d e d 1,1 g o f m e t h y l a t e d p r o d u c t . F r a c t i o n a t i o n i n c h l o r o f or in-pp.tr oleum e t h e r gave i n t e r a l i a 300 mg i n t h e ]5 % c h l o r o f o r m e x t r a c t and 200 mg i n the 25 % e x t r a c t , w i t h a m e t h o x y l c o n t e n t o f 34.6 and 35.8 % r e s p e c t i v e l y (ash c o n t e n t 0.25 and 0.23 % ) . These two f r a c t i o n s were j o i n e d and s u b m i t t e d t o a s e r i e s o f t h r e e P u r d i e m e t h y l a t i o n s when no h y d r o x y l band a b s o r p t i o n was s e e n i n t h e i n f r a r e d s p e c t r u m . b) S e c o n d e x p e r i m e n t : Haworth, S r i v a s t a v a , P u r d i e m e t h y l a t i o n s . S 4 C 2 x y l a n (1 g) was s o l u b i l i z e d i n sodium h y d r o x i d e (8 %, 20 ml) and s o l i d s o d i u m h y d r o x i d e (3 g) was added. The m i x t u r e was s t i r r e d a t 50° and d i m e t h y l s u l p h a t e (30 ml) and sodium h y d r o x i d e (30 % 80 ml) were added s l o w l y o v e r a two h o u r p e r i o d . The m i x t u r e was t h e n h e a t e d a t 100° t o decompose the e x c e s s d i m e t h y l s u l p h a t e . A f t e r n e u t r a l i z a t i o n a t 0° w i t h 5 N s u l p h u r i c a c i d , t h e s o l u t i o n was d i a l y s e d . T h i s p r o c e d u r e was r e p e a t e d t h r e e t i m e s and t h e f i n a l s o l u t i o n was e x t r a c t e d f o r 16 h o u r s w i t h c h l o r o f o r m . The r e m a i n i n g aqueous f r a c t i o n was d i a l y z e d and f r e e z e d r i e d . The two f r a c t i o n s were combined and r e m e t h y l a t e d a c c o r d i n g t o S r i v a s t a v a (40) . The p a r t i a l l y m e t h y l a t e d p r o d u c t was s u s p e n d e d i n d i m e t h y l -s u l p h o x i d e and s o d i u m h y d r o x i d e (25 g) and d i m e t h y l s u l p h a t e (25 roi) were added u n d e r s t i r r i n g o v e r 8 h o u r s . S t i r r i n g was m a i n t a i n e d o v e r n i g h t . The exce.~s d i m e t h y l s u l p h a t e was decomposed by h e a t i n g the m i x t u r e a t 100° f o r 1 h o u r , and a f t e r a d d i t i o n o f w a t e r (100 ml) tne s o l u t i o n was n e u t r a l i z e d n e a r 0° w i t h 10 N s u l p h u r i c a c i d . C h l o r o f o r m e x t r a c t i o n o f the d i a l y s e d m a t e r i a l a f f o r d e d 5 16 mg o f a p r o d u c t w h i c h s t i l l showed a s l i g h t a b s o r p t i o n a t 3400 r n " 1 i n t h e -94-i n f r a r e d . Two a d d i t i o n a l P u r d i e m e t h y l a t i o n s gave 405 mg o f methy-l a t e d x y l a n which was f r a c t i o n a t e d a c c o r d i n g t o T a b l e X. R e d u c t i o n o f m e t h y l a t e d x y l a n . A p o r t i o n o f the m e t h y l a t e d x y l a n was d i s s o l v e d i n f r e s h l y d i s t i l l e d t e t r a h y d r o f u r a n and l i t h i u m aluminum h y d r i d e was added. The m i x t u r e was s t i r r e d o v e r n i g h t a t room t e m p e r a t u r e i n t h e d a r k and t h e e x c e s s h y d r i d e was d e s t r o y e d by a d d i t i o n o f e t h y l a c e t a t e . A f t e r d i l u t i o n w i t h w a t e r and f i l t r a t i o n on s i n t e r e d • g l a s s , t h e s o l u t i o n was e v a p o r a t e d t o d r y n e s s . Water was added and t h e s o l u -t i o n d e i o n i z e d on A m b e r l i t e r e s i n s IR-120 and IR-45. The r e s u l t i n g n e u t r a l m e t h y l a t e d p o l y s a c c h a r i d e was t h e n h y d r p l y s e d i n the u s u a l manner. P e r i o d a t e o x i d a t i o n o f X y l a n S i t C 2 • The p o l y s a c c h a r i d e (1.00 g) was s o l u b i l i z e d i n w a t e r and t h e f i n a l volume a d j u s t e d t o 100 ml. To t h e s o l u t i o n , 20 ml o f aqueous 0.5 M p e r i o d i c a c i d was added. The s o l u t i o n was k e p t a t 4° C i n t h e d a r k and 5 m i l l i t e r s a l i q u o t s were removed a t i n t e r v a l s and t i t r a t e d by t h e a r s e n i t e method (see T a b l e X I I I ) . The i n i t i a l -l y r a p i d p e r i o d a t e u p t a k e became c o n s t a n t a f t e r 120 h o u r s (0.85 rool./anhydro p e n t o s e u n i t ) . i o d a t e and p e r i o d a t e i o n s were p r e c i p i t a t e d w i t h b a r i u m c a r -b o n a t e and, a f t e r c e n t r i f ugation, the supernatant was t r e a t e d w i t h s o -dium b o r o h y d r i d e . The s o l u t i o n was deionized w i t h A m b e r l i t e IR-120 r e s i n and a f t e r r e p e a t e d . d i s t i l l a t i o n w i t h m e t h a n o l , the r e s i d u e was h y d r o l y s e d i n 0.5 H s u l p h u r i c a c i d . The h y d r o l y s a t e was n e u t r a -l i z e d i n t h e u s u a l way and the n e u t r a l s u g a r s examined by gas l i q u i d c h r o m a t o g r a p h y as t h e i r t r i m e t h y l s i l y l d e r i v a t i v e s . Column SF.96 (4 m X 0.3 cm) a t 80° f o r 10 m i n u t e s , t h e n programmed a t 4°/ininute t o h o l d a t 1 9 0 ° . E t h y l e n e g l y c o l , g l y c e r o l and x y l o s e were f o u n d i n the r a t i o s 1 : 45.7 : 4.9 r e s p e c t i v e l y . F o r the e s t i m a t i o n o f f o r m i c a c i d r e l e a s e , t h e x y l a n (98 mg) was d i s s o l v e d i n w a t e r (120 m l ) , a s o l u t i o n o f p e r i o d a t e (0.2 M, 30 ml) was added and t h e m i x t u r e w a s kept i n the dark at 4°. A l i q u o t s (20 ml) , were w i t h d r a w n a t i n t e r v a l s and the r e a c t i o n s t o p p e d by t h e a d d i t i o n o f e t h y l e n e g l y c o l (6 d r o p s ) . i o d o m e t r i c t i t r a t i o n w i t h O.Ol M sodium t h i o s u l p h a t e gave t h e r e s u l t s i n T a b l e X T I I . E n z y m i c d e g r a d a t i o n . A r a b i n o - 4 - 0 - m e t h y 1 g l u c u r o n o x y l a n S 1 4 C 2 (500 mg) s o l u b i l i z e d i n a c e t a t e b u f f e r ( O . l M, pH 5, 50 ml) c o n t a i n i n g sodium a z i d e _ 3 ( f i n a l c o n c e n t r a t i o n 5.1 '6 M) , was i n c u b a t e d w i t h t h e x y l a n a se a t 40° w i t h s h a k i n g . I n c u b a t i o n was m a i n t a i n e d f o r 48 h o u r s and t h e m i x t u r e v/as d i a l y s e d f o r . 24 h o u r s a g a i n s t d i s t i l l e d w a t e r a t 40° . The non d i a l y s a b l e m a t e r i a l was f r e e z e d r i e d and w e i g h e d and s u b -m i t t e d t o a renewed attack o f 48 h o u r s under the above c o n d i t i o n s . A t t h e end o f a t h i r d a t t a c k , 144 mg o f non d i a l y s a b l e p o l y s a c c h a r i d e was o b t a i n e d (29 % o f s t a r t i n g m a t e r i a l ) . A n a l y s i s o f t h i s r e s i d u e showed a r a b i n o s e , x y l o s e , h e x o s e s and 4 - 0 - m e t h y l g l u c u r o n i c a c i d i n the f o l l o w i n g p r o p o r t i o n s , 3.1 : 5 7 . 8 : 10.2 : 28.9 r e s p e c t i v e l y . M e t h y l a t i o n o f glucomannan. The p o l y s a c c h a r i d e (2 g) was s o l u b i l i z e d i n sodium h y d r o x i d e (8 %, 40 ml) and s o l i d sodium h y d r o x i d e (5 g) was added. A c e t o n e (100 ml) was added t o p r e v e n t foam f o r m a t i o n . T h e m i x t u r e was s t i r -r e d a t room t e m p e r a t u r e and s i m u l t a n e o u s l y d i m e t h y l s u l p h a t e (100 ml) and sodium h y d r o x i d e (30 %, 200 ml) were added e v e r y 30 m i n u t e s i n p o r t i o n s o f l O ml and 20 ml r e s p e c t i v e l y , w i t h v i g o r o u s s t i r r i n g . The t e m p e r a t u r e was t h e n r a i s e d t o 85°V The c l o u d y s o l u t i o n was d i a l y z e d f o r 48 h o u r s a g a i n s t r u n i n g t a p w a t e r and f r e e z e d r i e d . The w h i t e m a t e r i a l was d i s s o l v e d i n NN_'-dimethyl formamide (50 ml) and m e t h y l i o d i d e (12 ml) and s i l v e r o x i d e (6 g) were added i n p o r -t i o n s o v e r one h o u r . a t room t e m p e r a t u r e and under s t i r r i n g . S t i r r i n g was m a i n t a i n e d f o r 24 h o u r s i n t h e d a r k . The m i x t u r e was c e n t r i f u g e d and t h e p a l e y e l l o w s o l u t i o n and w a s h i n g s ( d i m e t h y l formamide) were r e m e t h y l a t e d w i t h the same c o n d i t i o n s f o r 24 h o u r s . A f t e r c e n t r i f u g a t i o n , the s i l v e r s a l t s were, p r e c i p i t a t e d by a d d i t i o n o f w a t e r (120 m l ) . The m i x t u r e was t h e n e x t r a c t e d w i t h s e v e r a l p o r -t i o n s o f c h l o r o f o r m t o g i v e a f t e r e v a p o r a t i o n t o d r y n e s s a y e l l o w s y r u p (661 mg). T h e ' i n f r a r e d s p e c t r u m showed o n l y a weak a b s o r p t i o n due t o h y d r o x y l g r o u p s . P u r d i e m e # t h y l a t i o n s were n e c e s s a r y t o a c h i e v e a c o m p l e t e m e t h y l a t i o n . The p a r t i a l l y m e t h y l a t e d p r o d u c t was d i s s o l v e d i n me-t h y l i o d i d e and s i l v e r o x i d e was added i n p o r t i o n s i n t o the g e n t l y b o i l i n g s o l u t i o n u n d e r r e f l u x . A f t e r c e n t r i f u g a t i o n and e x t r a c t i o n o f t h e s o l u t i o n w i t h warm c h l o r o f o r m , t h r e e a d d i t i o n n a l t r e a t m e n t s were p e r f o r m e d as above. F r a c t i o n a l e x t r a c t i o n w i t h c h l o r o f o r m - p e t r o --9 7-leutn e t h e r m i x t u r e s 7- *-r f o r t h e 2b % c h l o r o f o r m e x t r a c t . 5bO mg o f a p r o d u c t w h i c h d i d not show any a b s o r p t i o n a t 3 400 cm 1 i n t h e i n f r a r e d . M e t h y l a t i o n o f G a l a c t o g l u c o m a n n a n . To the g a l a c t o g l u c o m a n n a n f r a c t i o n GGM^ (1.8 g) i n s u s -p e n s i o n i n d i m e t h y l s u l p h o x i d e (150 ml) was added under n i t r o g e n a s o l u t i o n o f d i m e t h y l - s u l p h i n y l a n i o n (4 g) i n d i m e t h y l s u l p h o x i d e (50 m l ) . The m i x t u r e , p l a c e d i n a r o u n d b o t t o m f a s k f i t t e d w i t h a r u b b e r cap, was s t i r r e d o v e r n i g h t a t room t e m p e r a t u r e . M e t h y l i o -d i d e (8 ml) was added a t i n t e r v a l s w i t h a syringe t h r o u g h t h e r u b b e r cap. W i t h o u t i s o l a t i o n o f t h e p r o d u c t s , a s e c o n d a d d i t i o n o f r e a g e n t s l e a d t o a c l e a r s o l u t i o n . A t h i r d - t r e a t m e n t w i t h t h e m e t h y l a t r n g a g e n t s was p e r f o r m e d and t h e m i x t u r e was p o u r e d i n t o w a t e r (500 ml) and d i a l y s e d , t h e n f r e e z e d r i e d . The f r e e z e d r i e d m a t e r i a l , n o w r e a -d i l y s o l u b l e i n d i m e t h y l s u l p h o x i d e fwas r a m e t h y l a t e d as b e f o r e and t h e n was s u b m i t t e d t o 4 P u r d i e m e t h y l a t i o n s u s i n g the c o n d i t i o n s a l -r e a d y d e s c r i b e d . F r a c t i o n a l e x t r a c t i o n w i t h c h l o r o f o r m - p e t r o l e u m e t h e r m i x t u r e s gave f o r t h e 15 % and 20 % e x t r a c t s 340 mg o f a p r o d u c t w h i c h showed no a b s o r p t i o n o f h y d r o x y l band i n t h e i n f r a r e d . -98-PART I I I CELL WALL CARBOHYD RATS S AND STRUCTURAL CHANGES OF XYLANS OF ARUNDO DONAX IN RELATION TO GROWTH P o l y s a c c h a r i d e s and p r o t e i n s a r e f u n d a m e n t a l c o n s t i t u e n t s o f p l a n t c e l l w a l l s . I t has been d e m o n s t r a t e d t h a t t h e p o l y s a c c h a -r i d e c e l l w a l l c o m p o s i t i o n i s c o n t r o l e d and changes d u r i n g d e v e l o p -ment (97) as i s shown by t h e d i f f e r e n t n a t u r e o f the p o l y s a c c h a -r i d e s s y n t h e t i z e d i n p r i m a r y and s e c o n d a r y . w a l I s • ( 9 8 ) . F u r t h e r m o r e , t h e c e l l w a l l o f v a r i o u s m o r p h o l o g i c a l p a r t s ( r o o t s , h y p o c o t y l s , f i r s t i n t e r n o d e s and p r i m a r y l e a v e s ) o f bean p l a n t s were each f o u n d t o have a c h a r a c t e r i s t i c s u g a r c o m p o s i t i o n (.99) . I t i s n o t i c e a b l e t h a t few workers; have t r i e d t o c o r r e l a t e t h e c a r b o h y d r a t e c o m p o s i t i o n and t h e r a t e o f e l o n g a t i o n o f t h e p l a n t c e l l w a l l s . However, Monro and h i s c o l l a b o r a t o r s (100) were u n a b l e t o f i n d a d i f f e r e n c e i n the s u g a r c o m p o s i t i o n o f t h r e e zones o f t h e h y p o c o t y l o f l u p i n . In a s t u d y o f the m e t a b o l i s m o f c e l l w a l l p o l y -s a c c h a r i d e s f r o m P h a s e o l u s a u r e u s , F r a n z (101) compared the p o l y s a c -c h a r i d e d i s t r i b u t i o n o f 2 t o 4-day-o.ld c e l l s w i t h t h a t o f 4-week-o l d c e l l s . The f o r m e r showed 30 % o f a - c e l l u l o s e , 50 % o f h e m i c e l -l u l o s e s and 20 % o f p e c t i n s whereas the l a t t e r were c o n s t i t u t e d o f 60 % a c e l l u l o s e , 30 % h e m i c e l l u l o s e s and 10 % p e c t i n s . He e s t a b l i s -h ed a l s o t h a t changes were t a k i n g p l a c e e s s e n t i a l l y a t t h e l e v e l o f h e m i c e l l u l o s e s and p e c t i n s i n r e l a t i o n t o g r o w t h . Glucose-'''c i n 4 - d a y - o i d bean h y p o c o t y l s was i n c o r p o r a t e d more r a p i d l y i n h e m i c e l - . l u l o s i c f r a c t i o n s t h a n i n a - c e l l u l o s e . These i m p o r t a n t d a t a s u g g e s t a t u r n o v e r o f t h e p o l y s a c c h a r i d e s i n t h e g r o w i n g w a l l s . -99- . D e t a i l e d s t u d i e s have been r e p o r t e d by R e i d and W i l k i e on young p l a n t s o f Avena s a t i v a ( 1 0 2 ) . They d e m o n s t r a t e d t h a t n o t o n l y v a r i a t i o n s o c c u r r e d between the d i f f e r e n t p a r t s o f t h e p l a n t b u t a l s o w i t h i n one p a r t as a f u n c t i o n o f g r o w t h . The i n c r e a s e o f m a t u r i t y was a c c o m p a n i e d by an i n c r e a s e i n the x y l o s e c o n t e n t and a d e c r e a s e i n t h e g l u c o s e c o n t e n t . S i m i l a r r e s u l t s were o b t a i n e d by B u c h a l a and W i l k i e (103) on t h e t o t a l h e m i c e l l u l o s e c o n t e n t i n wheat. T h i s p a r t o f our r e s e a r c h w i l l be c o n c e r n e d w i t h t h e c h a n -ges i n t h e c a r b o h y d r a t e c o m p o s i t i o n o f t h e c e l l w a l l s o f A r u n d o  donax a t d i f f e r e n t s t a g e s o f m a t u r i t y . The r a p i d i t y o f growth o f t h i s p l a n t t o g e t h e r w i t h i t s m o r p h o l o g i c a l s t r u c t u r e i n w e l l d e f i n e d • i n -t e r n o d e s p r o v i d e s , a t a g i v e n t i m e o f g r o w t h , s e v e r a l s t a g e s o f d e v e l o p m e n t o f the c e l l w a l l s . The o v e r a l l growth o f t h e stem c o r -r e s p o n d s t o t h e c o m b i n a t i o n o f t h e p r o g r e s s i v e e l o n g a t i o n i n t i m e "and s p a c e o f t h e i n t e r n o d e s . F u r t h e r m o r e , a t the b a s i s o f each i n -t e r n o d e , a n i n t e r c a l a r y m e r i s t e m a t i c zone r e m a i n s a c t i v e u n t i l the f i n a l s t a g e o f e l o n g a t i o n i s r e a c h e d ( a b o u t 21 cm). A s t u d y o f t h e i n f l u e n c e o f t h i s m e r i s t e m on t h e c a r b o h y d r a t e c o m p o s i t i o n o f d i f -f e r e n t z o n e s o f the d e v e l o p i n g i n t e r n o d e s r e l a t i v e t o t h e i r d i s t a n c e t o t h e i n t e r c a l a r y m e r i s t e m w i l l be p r e s e n t e d ( 1 0 4 ) . The s t r u c t u r e o f the r e e d Arundo donax was s u i t a b l e f o r t h e i d e n t i f i c a t i o n and t h e s e l e c t i o n o f zones o f a c t i v e i n c o r p o r a t i o n o f t h e c e l l w a l l p o l y s a c c h a r i d e s i n the s c o p e o f a s t u d y o f t h e s t r u c -t u r a l e v o l u t i o n o f i t s main h e m i c e l l u l o s e ( 1 0 5 ) , t h e a r a b i n o g l u c u -r o n o x y l a n . - 1 0 0 -RESULTS I - CELL WAL'' CARBOHYDRATE COMPOSITION i^I DIFFERENT STAGES OF MATURITY a - A n a l y t i c a l c o m p o s i t i o n o f t h e 8 c o n s t i t u t i v e i n t o r n o d e s o f a o n e - m o n t h - o l d r e e d . O n e - m o n t h - o l d r e e d s had an a v e r a g e h e i g h t o f 1 . 2 meter, and c o n s i s t e d o f e i g h t d i s t i n c t i n t e r n o d e s from 0 . 5 t o 21 c e n t i m e t r e s i n l e n g t h betwen the apex and t h e b a s e o f t h e c u l m . A l l t h e 8 i n -t e r n o d e s were c u t - o f f and i n d i v i d u a l l y e x t r a c t e d w i t h e t h a n o l - b e n -zene t o remove p i g m e n t s and l i p i d s , t h e n w i t h b o i l i n g w a t e r t o r e -move t h e s o l u b l e c a r b o h y d r a t e s . On t h e d r y t i s s u e s a t o t a l h y d r o l y -s i s a c c o r d i n g t o K l a s o n ( 4 9 ) was p e r f o r m e d . T h i s t y p e o f h y d r o l y s i s a l l o w s i n t h e same e x p e r i m e n t the e s t i m a t i o n o f t h e t o t a l c e l l w a l l c a r b o h y d r a t e s and o f the l i g n i n c o n t e n t ( T a b l e X V I I I , F i g . 1 4 ) . b - C o m p a r i s o n between the c e l l w a l l c a r b o h y d r a t e s o f parenchyma and v a s c u l a r b u n d l e s t i s s u e s a t two s t a g e s o f _ m a t u r i t y . The t i s s u e s o f t h e stem o f A r u n d o donax c o n s i s t o f numerous v a s c u l a r b u n d l e s ( 1 0 6 ) j o i n e d by a f u n d a m e n t a l p arenchyma. The r e l a -t i v e p r o p o r t i o n o f t h e s e two t y p e s o f t i s s u e s v a r i e s w i t h t h e d e g r e e o f m a t u r i t y . V a s c u l a r b u n d l e s and parenchyma wore s e p a r a t e d under a b i n o c u l a r l e n s and the two t y p e s o f c e l l s were a n a l y s e d f o r t h e i r c a r b o h y d r a t e c o m p o s i t i o n . The e x p e r i m e n t was c a r r i e d o u t on two -101-TABLli X V I I I - NEUTRAL SUGAR COMPOSITION A N I) LTGMIN CONTENT OF ALL THE INTERNODES CONSTITUTING THE CULM OF A 1 -MONTH -OLD IXP.ZD (Arunc'o d'jp.ax; Internode Length Ligm'.n Moles % cm % Axabinose Xylose Galactose Glucose 1 0.5 a 11.6 34.2 3.8 50.0 2 1.5 2.9 10.0 48.6 3.3 38.1 3 3 . 5.4 4.8 53.5 0.8 40.8 4 9 7.2 3.7 56.2 0.8 39.1 5 15 9.1 3.2 50.5 0.6 45.8 6 21 10.7 3.3 52.2 0.5 44.0 7 21 11.7 3.2 51.5 0.7 44.5 8 21 12.8 3.9 45.7 1.3 49.0 a not determined * 1 i ifiiiiiniiiiiiii *——: 1—^ |J—i—mnn f in • I I I I . I IH I I I I ' I •i i i i i ini.i i i i i i i i i i i ii ^ ^^.^a,^, " number of i n t e r n o d e 1 2 3 4 5 6 7 8 F I G U R E 14 - L I G N I N C O N T E N T I N T H E I N T E R N O D E S OF A N E L O N G A T I N G S T E M -103-TABLE XIX COMPARATIVE MOLAR PERCENTAGES OF SUGARS IN HYDROLYSATES OF PARENCHYMA CELLS' AND VASCULAR BUNDLES Internode Type of c e l l Arabinose Xylo: 9 cm Parenchyma Bundles 7.3 15.9 Galactose 24.3 31.7 9.0 10.3 Glucose 53.1 38.5 !1 cm Parenchyma Bundles 2.8 . 2.6 51.2 45.7 0.9 2.4 44.9 49.2 - 1 0 4-s amples r e p r e s e n t i n g two d i f f e r e n t p h y s i o l o g i c a l l e v e l s , i n t e r n o d e fe£ 4 (9' cm l o n g ) and i n t e r n o d e fcf 6 (21 cm l o n g ) . The median zone o f t h e two i n t e r n o d e s was t r e a t e d as b e f o r e , and the r e s u l t s a r e shown i n T a b l e XIX. c -. B i . o r a e t r i c s t u d y . An e v a l u a t i o n o f t h e h i s t o l o g i c a l changes occurring w i t h i n one e l o n g a t i n g i n t e r n o d e was p r o v i d e d by a b i o m e t r i c s t u d y o f the f i b r e s l e n g t h s on a f a s t g r o w i n g zone ( i n t e r n o d e 4 ) . T h r e e zones were e x c i s e d : zone 1 a d j a c e n t t o t h e i n t e r c a l a r y m e r i s t e m a t i e t i s s u e s ; zone 2 c o r r e s p o n d i n g t o the t i s s u e s s i t u a t e d between the t h i r d and f i f t h c e n t i m e t r e ; and zone 3 c o r r e s p o n d i n g t o the t i s s u e s s i t u a t e d above t h e s i x t h c e n t i m e t r e , a t t h e t o p o f t h e i n t e r n o d e . On the t h r e e s a m p l e s , s e p a r a t i o n o f t h e f i b r e s was p e r f o r m e d by an a l k a l i n e c o o k i n g under p r e s s u r e a t 1 7 0 ° . The d r a s t i c c o n d i t i o n s r e q u i r e d f o r a c o m p l e t e s e p a r a t i o n o f the f i b r e s were t o o v i o l e n t f o r zone 1 and the r e s u l t i n g p a r t i a l d e s t r u c t i o n o f t h e f i b r e s w h i c h a r e v e r y f r a g i l e - a t t h i s s t a g e o f m a t u r a t i o n d i d n o t a l l o w s t a t i s t i c a l measurements on t h i s s a m p l e . However, the r e s u l t s r e l a -t i v e t o zones 2 and 3 a r e g i v e n i n the b i o g r a m o f F i g u r e 15. The f i b r e s l e n g t h s d i s t r i b u t i o n was measured on an a v e r a g e o f 400 i s o l a t e f i b r e s . L i k e w i s e , the e s t i m a t i o n o f t h e a v e r a g e d i a m e t e r s measured i n t h e m i d d l e p a r t o f t h e f i b r e s were 10-12 m i c r o n s f o r zone 2 and 20-22 m i c r o n s f o r zone 3 r e s p e c t i v e l y . The above r e s u l t s show c l e a r l y the i m p o r t a n t e l o n g a t i o n o f t h e f i b r e s w h i c h t a k e s p l a c e over a v e r y s h o r t d i s t a n c e w i t h i n a zone A 2 ' zone A^" Minimum l e n g t h 0 . 3 mm 0 . 6mm Maximum l e n g t h 3 . 3mm 5 . 5 mm A v e r a g e l e n g t h 1 . Omm .1 . 9mm -r mm 20 10 H 1 2 3 4 mm A v e r a g e d i a m e t e r 10 -12 li 20 -2 2 u * e s t i m a t e d on more t h a n 400 f i b r e s z o n e s A and A^ e x c i s e d f r o m a 10 cm l o n g i n t e r n o d e A 3 FIGURE 15 FIBRE LENGTH DISTRIBUTION IN AN ELONGATING INTERNODE - 1 0 6 -de ve 1 o p i n g i n te r. node . Gonsequen t l y s i g n i f i c a n t d i f f e r e n c e s \ie r e t o be e x p e c t e d a c c o u n t i n g f o r t h i s r a p i d b i o c h e m i c a l - , e v o l u t i o n o f the c e l l w a l l and w h i c h would, c o r r e s p o n d t o an i m p o r t a n t d e p o s i t i o n o f t h e c o n s t i t u t i n g w a l l p o l y s a c c h a r i d e s . d -' V a r i a t i o n _ o f the c a r b o h y d r a t e : c o n s t i t u e n t s i n ' d e v e l o p i n g i n t e r n o d e s . T h r e e i n t e r n o d e s were s e l e c t e d i n a young d e v e l o p i n g r e e d . I n t e r n o d e A, 9 cm l o n g ; i n t e r n o d e B , the f i r s t i n t e r n o d e h a v i n g r e a c h e d the f i n a l l e n g t h o f 2 1 cm ; and i n t e r n o d e C the l a s t i n t e r -node a t the b a s e o f t h e stem. T h r e e z o n e s were e x c i s e d on each i n t e r n o d e a c c o r d i n g t o t h e i r d i s t a n c e from t h e b a s e , as i n d i c a t e d i n T a b l e XX. The c a r b o h y d r a t e c o m p o s i t i o n o f t h e n i n e z o n e s so o b t a i n e d i s g i v e n i n T a b l e XX, and t h e changes i n c o m p o s i t i o n a r e c o n s i d e r e d i n terms o f m a t u r a t i o n o f t h e t i s s u e s ( c f . " d i s c u s s i o n " ) . -107-TABLE- X X NEUTRAL SUGAR COMPOSITION IN THREE INTERNODES AT DIFFERENT LEVELS OF MATURITY Anatomical p o s i t i o n Distance froni the of the t i s s u e s base of the i n -ternode Moles % Internode 9 cm 0.5 - 2.5 cm 3.5 - 5.0 cm 6.5 - 9.0 cm Arabinose Xylose Galactose Glucose 21.5 9.0 3.2 32.0 41.2 52.5 12 .0 3.6 0.8 30.0 45.7 43 .5 F i r s t Internode 21 cn 0.5 - 2.5 cm 3.5 - 5.0 cm B 3 10.0 - 18.0 cm 5.5 3.0 2.7 47.5 52 .0 52.5 2.1 1.5 0.6 44.5 43.0 44-0 Lower Internode 21 cm 0.5 - 2.5 cm 3.5 - 5.0 cm 10.0 - 18.0 cm 2.3 2.2 2.1 51.5 52 .5 50-0 1.4 0.8 0.5 44-0 44.7 47 .0 -108-I I - POLYSACCHARIDES OF ARUNDO DONAX  AT THREE STAGES OF MATURITY a - S e l e c t i o n _ o f the m a t e r i a l . On t h e b a s i s o f t h e p r e c e d i n g r e s u l t s and o b s e r v a t i o n s , t h r e e s e c t i o n s were s e l e c t e d f o r a c o m p a r a t i v e s t u d y o f the x y l a n s p r e s e n t i n the c e l l w a l l s a t t h r e e s t a g e s o f m a t u r i t y i n t h e deve-l o p i n g stem o f o n e - m o n t h - o l d r e e d s . e S e c t i o n 1 c o r r e s p o n d e d t o t h e f i r s t i n t e r n o d e s s i t u a t e d u n d e r n e a t h the apex and r a n g e d from 0.5 t o 3 cm i n l e n g t h ; , S e c t i o n 2 c o r r e s p o n d e d t o t h e 4th and 5 t h i n t e r n o d e s h a v i n g an a v e r a g e l e n g t h o f 9 and 15 cm. « S e c t i o n 3 c o r r e s p o n d e d t o t h e 7th and 8th i n t e r n o d e s , b o t h m e a s u r i n g 2 1 cm i n l e n g t h . b - I s o l a t i o n _ o f _ t h e _ p o l y s a c c h a r i d e s . F r e s h t i s s u e s were g r o u n d a t -10° and b o i l e d i n e t h a n o l t o i n a c t i v a t e e n z y m a t i c s y s t e m s . A l l t h e y i e l d s e x p r e s s e d h e r e a f t e r w i l l be r e l a t i v e t o t h e d r y w e i g h t a f t e r a l c o h o l t r e a t m e n t . -109-TA15LE XXI EXTINCTION OF 1IYD110S0EUKLE 1'01.YSACCHA111i.• ES * Section of the stem Mode of extraction Cold water Hot water Oxalate Total7> 1.5 0.7 1.6 3.8 0.5 0.5 0.7 1.7 1.5 0.3 0.2 2.0 Section 1 Section 2 Section 3 * In percent of dry weight after 80 % ethanol treatment -110-TABLE XXII NEUTRAL SUGAR COMPOSITION OF HYDR0S0L11BLE POLYSACCHARIDES F r a c t i o n Sugars ( moles % ) Galactose Glucose Mannose Arabinose xylose R bam nose if jcose So (cold) 29.0 33.0 0.9 29.6 5.1 1 . 8 0.8 2 1I20 I I 35.6 12.1 1.7 38.8 8.8 2.3 0.7 3 H 20 I I 41.0 16.0 1.0 22.0 13.0 6.6 0.7 ln2° (bot) 3 . 5 61.0 1.7 18.0 13.0 1.4 0.6 2H 20 it 30.8 10.0 2.1 35.0 19.0 3.0 0.6 3 H 20 I I 13.0 • 16.0 1.0 1.0 62 . 0 5.3 0.8 XH 20 (Ox.) 26 . 5 26.5 - 34.0 9 . 0 2.0 0.6 2 II 20 I I 26 . 5 17.2 - 29.0 19.0 5.0 1.0 3„ . 16.5 16.0 _ 28.0 3 1.0 6.0 1.0 H 20 -111-b - 1 - E l i m i n a t i o n o f _ h y d r o s o l u b l e p o l y s a c c h a r i d e s . Aqueous e x t r a c t i o n s were c a r r i e d o u t f i r s t a t room t e m p e r a -t u r e , t h e n a t 8 0 ° . The' t i s s u e s were d r i e d and s u b m i t t e d t o e t h a n o l -b e n z e n e e x t r a c t i o n , and a f i n a l e x t r a c t i o n o f t h e h y d r o s o l u b l e po-l y s a c c h a r i d e s was a c h i e v e d w i t h a 0.5 % ammonium o x a l a t e s o l u t i o n a t 9 0 ° . A l l f r a c t i o n s were d i a l y z e d and f r e e z e d r i e d ( T a b l e s XXI, XXII) . b - 2 - S e q u e n t i a l a l k a l i n e e x t r a c t i o n s _ o f h e m i c e l l u l o s e s . D e l i g n i f i c a t i o n o f the t i s s u e s p r i o r t o a l k a l i n e e x t r a c t i o n was p e r f o r m e d i n c o n d i t i o n s a d j u s t e d a c c o r d i n g t o the d i f f e r e n t l i g n i n c o n t e n t o f the s a m p l e s . The f i r s t a l k a l i n e e x t r a c t i o n was a c c o m p l i s h e d by u s i n g p o t a s s i u m b o r o h y d r i d e . The p u r p o s e o f t h i s t r e a t m e n t was t o e f f e c t i n s i t u r e d u c t i o n o f t h e p o l y s a c c h a r i d e s t o p r e v e n t the p o l y m e r from a p o s s i b l e " p e e l i n g " t a k i n g p l a c e d u r i n g a l k a l i n e e x t r a c t i o n . B u t the a l k a l i n i t y o f t h e b o r o h y d r i d e s o l u t i o n (pH a b o u t 10) i s s u f f i c i e n t t o a c h i e v e an e x t r a c t i o n o f c e r t a i n p o l y s a c c h a r i d e s , (107) and c o m p l e t e t h e r e c o v e r y o f h y d r o s o l u b l e m a t e r i a l . On each sample, n e u t r a l i z a t i o n o f t h e e x t r a c t by a c e t i c a c i d gave the f r a c -t i o n s P - KBH^ and f u r t h e r p r e c i p i t a t i o n w i t h a l c o h o l gave t h e f r a c t i o n s P g - KBH^, whose a n a l y t i c a l d a t a a r e shown i n T a b l e s X X I I I , XXIV. -112-FIGURE 16 - EXTRACTION OF HEMICELLULOSES ON TISSUES AT THREE STAGES OF MATURITY HOLOCELLULOSE KBH 4 1% (24 h) f i l t r a t e residue AcOH ce n t r i f u g . supernatant EtOH P -KBH . s 4 P -KBH. A 4 1 f i l t r a t e AcOH 0.2M KOH + KBH4 1% (24 h) residxie I supernatant EtOH P -0.2M S centr. 1.0M KOH + P -0.2M A = i f i l t r a t e residue AcOH centr. supernata nt EtOH' / Ps-M p -M A = 2.5M KOH + KBH 4 0.5% (24 h) f i l t r a t e residue AcOH r— . supernatamt EtOH Pg-2.5M centr. P,-2.5M A = 1 f i l t r a t e 4.3M KOH c e l l u l o s e AcOH T supernatant EtOH P -4.3M. centr. P,-4.3M A = S e q u e n t i a l e x t r a c t i o n s w i t h p o t a s s i u m h y d r o x i d e s o l u t i o n s of. i n c r e a s i n g c o n c e n t r a t i o n s have been c a r r i e d o u t as i n d i c a t e d i F i g u r e 16. F o u r c o n c e n t r a t i o n s were used, i n t h e p r e s e n c e o f p o t a s i u m b o r o h y d r i d e : 0.2 M p o t a s s i u m h y d r o x i d e c o n t a i n i n g 1 % b o r o -h y d r i d e ; 1.0:M c o n t a i n i n g 0.5 % b o r o h y d r i d e ;" 2.5 M c o n t a i n i n g 0.5 % b o r o h y d r i d e ; and 4.3 M p o t a s s i u m h y d r o x i d e . In most cases,, e a c h o f t h e above e x t r a c t i o n s gave r i s e t o two f r a c t i o n s , P o b t a i n e d by d i r e c t n e u t r a l i z a t i o n and P „ , o b t a i n e d by e t h a n o l p r e c i p i t a t i o n . The a n a l y t i c a l r e s u l t s c o r r e s -p o n d i n g t o a l l t h e s e p o l y s a c c h a r i d i c e x t r a c t s a r e shown i n T a b l e s XXV t o XXX. -114-TABLE XXIII - EXTRACTION I.'ITU 1 t POTASSIUM KOKOliiTJiM v>Y. l r A - K B H 4 2 . 0 3 . 4 11.6 ? P A - K T , 1 I 4 1.1 3 . 0 7 . 9 3 P A - K B 1 1 4 0-9 4 . 0 1 P S - K B H 4 0.5 4.1 4 . 3 2 P S - K B I I 4 4 . 3 1.8 0.3 • 3 P S - K B I 1 4 1-3 2.5 -Sfc As i n Tabic XXX TABLE XXIV ~ 1% POTASSIUM EXTRACTS - ANALYTICAL DATA Fractious G alactose'-'Glucose Mannose Arabinose Xylose U r o n i c a c i d % V K B H 4 7 - 5 , 3 - 8 3 ' 5 • N ' ° 4 4 ' ° 1 - 2 2V A_KBH 4 t t - 7.7 92.0 2.0 3p A-KBH 4 1.2 0.5 - 5.5 92.3 3.1 lp s-KBH 4 8.9 16.0 3.0 10.0 43.0 4.5 2P S-KBH 4 6-4 3.3 - 13.0 77.0 5.0 3P S-KBH 4 1-5 1.0 - 92.0 0.6 t = traces - 1 1 6 -TABLE XXV — EXTRACTION WITH 0.2M KOH-KBH4 1 % Fractions Yield' Ash Nitrogen fa] (* lp A_ 0.2M. 3.4 2.5 10.3 lp s_ 0.2M 4 - 6 1-5 2..-5 -64 2 P o.2M. 6 - 7 1-° 0.2 -77 3P S-0.2M 4 - 2 1-1 0.25 -59 -117 T A B L E XXVI - 0.2' M . KOH E X T R A C T S — ' A N A L Y T I C A L D A T A Fractions Galactose Gluco.se Mannose Arabinose xylose, uronic acid /-1P A-0.2M 12.0 15.0 3.5 10.5 - 59.Q 1.6 lPc;-0.2M 3.6 4.9 t. 22.0 70.0 6.0 2Ps-0.2M 1.8. 2.2 - 11.5 85.0 2.5 3P S-0.2M 1.8 - 10.1 86.0 2.7 - 11 8 -TABLE XXVII - EXTRACTION WITH 1.OM KOH - KBH 0.5 % 'P -M A = l p A ~ i ' i ' 1 3.9 4.9 2 - 6 . 7.8 0.1 3 PA~= 4.1 4.2 0.1 F r a c t i o n s Y i e l d % Ash N i t r o g e n [a] . ( ° ) l p S ^ 1 2 - ° 3.8 0.3. - 52 2pS~l± 1 7 - 5 2.7 0.1 - 70 3 ps - M 8.5 0.9 0.1 - 72 -119-TABLE X X V I I I - 1 .011 KOH EXTRACTS' - ANALYTICAL DATA F r a c t i o n s G a l a c t o s e G l u c o s e Mannose A r a b i n o s c X y l o s e U r o n i c a c i d l pA"M 1.5 1 - 5.7 92 2 PA_ M t t - 4.5 94 3 P f i - M 0.9 0.5 - 5.1 93 l ps -i^ 3 - 6 1^-5 • - 12.0 70 4.5 2 PS~= 1 * 1 2 10 - 6.0 91 2.1 3 P S ~ I 1 - 4 3.3 - 5.7 9o 2.1 - 1 2 0 -TABLE XXIX - EXTRACTION WITH 2.5M KOH - KBH O 5 % 4 P -2.5M A = ANALYTICAL DATA F r a c t i o n s Y i e l d % Ash N i t r o g e n £«J (°) 1P s-2.5M 9.3 0.8 - -42 2 P g - 2 . 5 M 1 2 . 5 4 . 5 - - 5 4 3 P G - 2 . 5 M . 6 .0 1.5 . - - 6 6 F r a c t i o n s G a l a c t o s e G l u c o s e Mannose A r a b i n o s e X y l o s e U r o n i c a c i d % 1P -2.5M 1.9 9.3 - 5 . 3 8 3 . 5 4.2 2P g-2.5M 1.2 5.6 - 4.9 88.3 1.3 3P -2.5M 4.1 32.5 - 12.5 50.9 2.0 •121-TABLE XXX - EXTRACTION WITH 4.3 M KOH F r a c t i o n s . Y i e l d Ash l pS ~ 4 ' 3 - 2.3 3.8 2. ' P S-4.3M 1.1 5.1 3 P S-4.3M 0.4 10.8 ANALYTICAL DATA F r a c t i o n s G a l a c t o s e G l u c o s e Mannose A r a b i n o s e X y l o s e U r o n i c a c i d % 1 P g - 4 . 3 M 5.2 37 0.7 15.0 41.2 4.9 2 P g-4.3M 1.3 18.4 0.5 4.5 71.5 2.3 3 P s-4.3M 1.4 15.6 0.6 5 . 4 7 7 > 0 * X Not d o t e rm i n e d -122-I I I - COMPARATIVE STRUCTURAL STUDY OF XYLANS ORIGINATING FROM TISSUES AT THREE STAGES OF MATURATION The h i g h e s t y i e l d s o f p o l y s a c c h a r i d e s were o b t a i n e d i n the 1.0 m o l a r p o t a s s i u m h y d r o x i d e e x t r a c t s , and r e p r e s e n t e d a b o u t 50 % o f t h e t o t a l r e c o v e r y o f h e m i c e l l u l o s e s . A l t h o u g h t h e a n a l y s i s o f the c a r b o h y d r a t e c o m p o s i t i o n o f t h e s e p o l y m e r s r e v e a l e d t h e h i g h l y d o m i n a n t p r o p o r t i o n o f x y l o s e , p u r i f i c a t i o n o f t h e f r a c t i o n s was n e c e s s a r y b e f o r e a t t e m p t i n g a s t r u c t u r a l s t u d y . Due t o t h e i r g r e a t e r abundance the i n v e s t i g a t i o n was c a r r i e d o u t on the f r a c t i o n s P -H c o r r e s p o n d i n g t o s e c t i o n s 1,2 and 3. P u r i f i c a t i o n ; was a c h i e v e d on a DEAE-Sephadex A-25 column and the x y l a n s r e c o v e r e d i n minimum y i e l d o f 70 % were t r u e a r a b i n o g l u -c u r o n o x y l a n s . T h e i r h o m o g e n e i t y was c h e c k e d by g e l f i l t r a t i o n o f t h e complex f o r m e d w i t h Red p r o c i o n dye (108) on B i o g e l and A g a r o s e A-0.5 m. The complex gave e v e n t u a l l y a s i n g l e b and on e l e c t r o p h o r e s i s on c e l l u l o s e a c e t a t e s t r i p s . The r e m a r k a b l e c o n s -t a n t c o m p o s i t i o n o f t h e x y l a n s i n d e p e n d e n t l y o f t h e p h y s i o l o g i c a l d e v e l o p m e n t o f t h e t i s s u e s examined i s shown i n T a b l e XXXI. The o n l y d i f f e r e n c e b e a r s upon t h e u r o n i c a c i d c o n t e n t w h i c h a p p a r e n t l y d o u b l e d f r o m t h e y o u n g e r t i s s u e s t o t h e more mature one s . S t r u c t u r a l i n v e s t i g a t i o n o f t h e a r a b i n o g l u c u r o n o x y l a n s from s e c t i o n s 1 t o 3 v/as c a r r i e d o u t m a i n l y by t h e m e t h y l a t i o n t e c h n i q u e . P e r m e t h y l a t e d p o l y s a c c h a r i d e s were al w a y s o b t a i n e d a f t e r one t r e a t -ment a c c o r d i n g t o the p r o c e d u r e o f H a k o m o r i , f o l l o w e d by two t r e a t -ments w i t h t h e r e a g e n t s o f P u r d i e . F o r a l l t h e t h r e e x y l a n s t h e - 1 2 3 -TAB LK XXXI - SUGAR COMPOS ITION OF PURIFIED H y MI CE L I, U LO I? F. S FROM SECTIONS 1 - 3 (1.0M KOH e x t r a c t s ) H e m i c e l l u l o s i c f r a c t i o n ' S e c t i o n 1-A S e c t i o n 2-A S e c t i o n 3-A A r a b i n o s e 5 . 7 4 . 5 5 . 1 M o l e s % X y l o s e U r o n i c a c i d 92 a 94 a 93 a S e c t i o n 1-S S e c t i o n 2-S S e c t i o n 3-S 6.5 5.2 5 . 7 92 90 90 4 . 5 2 . 1 2 . 1 F r a c t i o n s A were o b t a i n e d by d i r e c t a c e t i c a c i d a c i d i f i c a t i o n o: KOH e x t r a c t F r a c t i o n s G : e t h a n o l p r e c i p i t a t i o n on t h e s u p e r n a t a n t f r o m f r a c t i o n s A i n w e i g h t % - e s t i m a t e d by d e c a r b o x y l a t i o n (a) n o t d e t e r m i n e d -12 4-TABLE .'• XXXII - METHYLATION ANALYSIS OF XYLANS FROM 1.0 M KOH EXTRACTS M e t h y l a t e d s u g a r s * S e c t i o n 1 S e c t i o n 2 S e c t i o n 3 2 , 3 , 5 - T r i - O - M e - a r a b i n o s e 2, 3 , 4 - T r i - O - M e - x y l o s e 2 , 3 - D i - O - M e - x y l o s e (2-0 + 3 - Q ) M e - x y l o s e 2 , 3 , 4 , 6 - T e t r a - O - M e - g l u c o s e D P n 1.9 3.5 5.2 1.0 1.0 1.0 58.5 84.0 140.5 3.3 6.0 10.0 - 0.8 6 3.7 9 1 .0 151.5 * i d e n t i f i e d by GLC-MS o f t h e i r a l d i t o l a c e t a t e d e r i v a t i v e s TABLE X X X I I I , - METHYLATION ANALYSIS OP THREE XYLAN FRACTIONS ISOLATED FROM SECTION 2 M e t h y l a t e d s u g a r s 2,3,5-Tri-O-Me-arab xnose 2 , 3 , 4 - T r i - O - M e - x y l o s e 2,3-Di-0-Me-xylos< (2-0 + 3 - 0 ) M e - x y l o s e 0.2M KOH e x t r a c t 7 . 4 1 .0 1 . OM KOH i . OM. KOH f r a c t i o n A * f r a c t i o n S 13.3 4 . 1 1 . 0 82 .5 5.9 3.5 1 . 0 84.0 6.0 DP 97.8 88 . 4 91.0 * f r a c t i o n s A and S as, i n T a b l e XXXI -126-m e t h y l a t e d p r o d u c t s o b t a i n e d a f t e r h y d r o l y s i s c o r r e s p o n d e d t o . 2 , 3 , 5 - t r i - O - m e t h y l - L - a r a b i n o s e ; 2 , 3 , 4 - t r i - O - m e t h y 1 - D - x y l o s e ; 2 , 3 - d i - O - m e t h y l - D - x y l o s e and a m i x t u r e o f 2 and 3 mono-O-methyl-D-x y l o s e . In t h e h y d r o l y s a t e o f the x y l a n from s e c t i o n 3, a s m a l l amount o f 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g l u c o s e was a l s o f o u n d t o be p r e -s e n t (0.55 % ) . A l l s u g a r d e r i v a t i v e s were i d e n t i f i e d by t h e c o n j u n c -t i o n o f p a p e r c h r o m a t o g r a p h y , gas l i q u i d c h r o m a t o g r a p h y and mass s p e c t o g r a p h y . The q u a n t i t a t i v e r e s u l t s o f the a n a l y s i s a r e g i v e n i n T a b l e X X X I I . The e s t i m a t i o n o f the c h a i n l e n g t h cideduced from t h e s e r e s u l t s was 64, 91 and 151 f o r s e c t i o n 1, s e c t i o n 2 and s e c -t i o n 3 r e s p e c t i v e l y . A l t h o u g h t h e above s t u d y has been c a r r i e d o u t on t h e most ab u n d a n t f r a c t i o n s o f t h e H e m i c e l l u l o s e , i t was i m p o r t a n t t o v e r i f y the h o m o g e n e i t y o f the x y l a n s i n d e p e n d e n t l y o f t h e mode o f e x t r a c t i o n T h a t was done by s u b m i t t i n g two o t h e r f r a c t i o n s o f t h e x y l a n from s e c t i o n 2, namely, the f r a c t i o n o b t a i n e d by e t h a n o l p r e c i p i t a t i o n o n the 0.2 M p o t a s s i u m h y d r o x i d e (2 P -M) and t h e f r a c t i o n o b t a i n e d by d i r e c t n e u t r a l i z a t i o n o f the j,. O H p o t a s s i u m h y d r o x i d e e x t r a c t . A s i m i l a r s t r u c t u r a l i n v e s t i g a t i o n as above was c a r r i e d o u t on t h e s e two f r a c t i o n s and t h e r e s u l t s o f t h e m e t h y l a t i o n s t u d y a r e p r e s e n t e d i n T a b l e X X X I I I . I t can be se e n t h a t the a v e r a g e d e g r e e o f p o l y m e r i z a t i o n i s q u i t e s t a b l e w h a t e v e r t h e mode o f e x t r a c t i o n was . -12 7-I V ~ ACETYLATED XYLANS - COMPARISON AT THREE STAGES OF.MATURITY OF THE PLANT. I t i s now w e l l e s t a b l i s h e d t h a t n a t i v e x y l a n s f r o m hardwoods a r e a c e t y l a t e d . D i m e t h y l s u l p h o x i d e w h i c h i s a good s o l v e n t f o r . p o l y s a c c h a r i d e has been shown t o e x t r a c t a c e t y l a t e d p o l y s a c c h a r i d e s w i t h p r e s e r v a t i o n o f the a c e t a t e s . U s i n g t h e p r o c e d u r e d e s c r i b e d by H a g g l u n d and c o w o r k e r s ( 1 3 ) , a c e t y l a t e d p o l y s a c c h a r i d e s from s e c t i o n s 1,2 and 3 were i s o l a t e d . The y i e l d s were 1.7 %, 5.2 % and 1.9 % o f t h e d r y t i s s u e s r e s p e c t i v e l y . The e s t i m a t i o n o f t h e a c e t y l g r o u p s v/as done by gas l i q u i d c h r o m a t o g r a p h y o f t h e m e t h y l a c e t a t e r e l e a s e d a f t e r m e t h a n o l y s i s o f t h e a c e t y l a t e d p o l y s a c c h a r i d e s , u s i n g t h e method d e s c r i b e d by M e i e r (109) w i t h a s l i g h t m o d i f i c a -t i o n ( T a b l e X X X I V ). On t h e same s a m p l e s , th e u r o n i c a c i d c o n t e n t was e s t i m a t e d and gave v e r y c l o s e r e s u l t s f o r t h e t h r e e l e v e l s . On t h e o t h e r hand, the r e l a t i v e p r o p o r t i o n s o f 4-0-methyl g l u c u r o n i c a c i d t o g l u c u r o n i c a c i d , d e t e r m i n e d by the amounts o f 4-0-methyl g l u c o s e and g l u c o s e o b t a i n e d a f t e r c a r b o x y l - r e d u c t i o n o f t h e a l d o b i o u r o n i e a c i d s r e l e a s e d a f t e r h y d r o l y s i s , were g r e a t l y d i f f e r e n t - I t i s a p p a r e n t from T a b l e XXXIV t h a t t h e r e i s a s t r o n g i n c r e a s e i n t h e p r o p o r t i o n o f g l u c u r o n i c a c i d as t h e p l a n t m a t u r e s . -128-TABLE- XXXIV - ANALYSIS OF DMSO-EXTRACTS FROM SECTIONS i - 3 S e c t i o i i S arc X y l G a l G l c A c e t y l * * U r o n i c R a t i o . , 4 - O - r - t e G l c a c a d _ i . < - u j . u / G l c A S e c t i o n 1 17.0 37.8 8.8 36.2 . 4.7 5.2 15.0 (0.5-3cm) S e c t i o n 2 4.0 90.0 0.5 5.4 10.7 5.9 2.5 (9-15cm) S e c t i o n 3 3.5 92.0 t 3.0 8.0 5.7 1.5 (2 1 cm) * i n wei g h t %- c a l c u l a t e d on a x y l a n b a s i s e s t i m a t e d from x y l o s e con ten t - 1 2 9 -DISCUSSION I - INFLUENCE OF MATURATION OF THE TISSUES ON THE CELL WALL CARBOHYDRATE COMPOSITION OF THE STEM OF ARUNDO DONAX The m o r p h o l o g i c a l s t r u c t u r e o f t h e r e e d i n a s u c c e s s i o n o f i n t e r n o d e s o f i n c r e a s i n g l e n g t h from t h e a p i c a l e x t r e m i t y t o t h e b a s e i s s u c h t h a t t h e y o u n g e s t i n t e r n o d e s a r e a t t h e t o p o f t h e stem. N e v e r t h e l e s s , t h i s s i t u a t i o n i s c o m p l i c a t e d by t h e p r e s e n c e a t t h e b a s e o f e a c h i n t e r n o d e o f an i n t e r c a l a r y m e r i s t e m where m i t o t i c a c t i v i t y r e m a i n s f o r a i o n g t i m e . I t r e s u l t s f r o m t h i s zone o f a c t i v e c e l l u l a r d i v i s i o n t h a t w i t h i n one e l o n g a t i n g i n t e r n o d e a l l the s t a g e s o f m a t u r a t i o n a r e e n c o u n t e r e d between the l o w e r and t h e u p p e r p a r t s o f the i n t e r n o d e . However, i t can be c o n s i d e r e d t h a t t h e a v e r a g e age o f t h e c e l l s i n c r e a s e s as t h e p o s i t i o n o f t h e i n t e r n o d e i s more d i s t a n t from th e apex. I t can t h u s be s e e n from the d a t a i n T a b l e X V I I I t h a t m a t u r a t i o n o f the t i s s u e s r e s u l t s i n a r a p i d i n c r e a s e i n x y l o s e and g l u c o s e and a d e c r e a s e i n t h e p r o p o r -t i o n o f a r a b i n o s e and g a l a c t o s e . T h i s i s an o v e r a l l i n d i c a t i o n o f the r a p i d s i t e o f s y n t h e s i s o f x y l o s e - c o n t a i n i n g p o l y m e r s a t an e a r l y s t a g e o f the d e v e l o p m e n t o f the p l a n t ( v e r y s h o r t i n t e r n o d e s ) , and o f a s i m u l t a n e i t y i n t h e b i o s y n t h e s i s o f x y l a n s and c e l l u l o s e as shown by the a l m o s t p a r a l l e l b e h a v i o u r o f x y l o s e and g l u c o s e . The e v o l u t i o n o f t h e s e c a r o o h y d r a t e s i s i n o p p o s i t i o n w i t h t h e phenomenon o f l i g n i f i c a t i o n w h i c h i s a slow p r o c e s s ( T a b l e X V I I I ) -130-and whose maximum i s r e a c h e d a t the end o f t h e . v e g e t a t i v e c y c l e , i n c r e a s i n g up t o 19 % i n the f . u l l y mature t i s s u e s . When the c a r b o h y d r a t e c o m p o s i t i o n o f v a s c u l a r . bundles' .and p arenchyma c e l l s was compared a t two d i f f e r e n t p h y s i o l o g i c a l l e -v e l s , i t was shown ( T a b l e XIX) t h a t x y l a n s were s y n t h e s i z e d f a s t e r in. the v a s c u l a r b u n d l e s t h a n i n the. c o r r e s p o n d i n g parenchyma c e l l s . The presence of a greater proportion of parenchyma i n the youngest internodes c o u l d a c t as a d i l u t i o n f a c t o r f o r the a p p a r e n t l o w e r c o n t e n t o f x y l a n i n t h e s e t i s s u e s . However, a s i d e from i n t e r n o d e 7%^ 1 , the r e l a t i v e p r o p o r t i o n s o f x y l o s e t o g l u c o s e i n t h e v a s c u l a r b u n d l e s a g r e e w i t h t h a t f o u n d i n t h e c o r r e s p o n d i n g r e s p e c t i v e i n t e r n o d e s ( T a b l e X V I I I ) . These r e s u l t s can be r e l a t e d t o the b i o c h e m i c a l mo-d i f i c a t i o n s o c c u r r i n g as t h e f i b r o u s c h a r a c t e r and w a l l m a t u r a t i o n i n c r e a s e . A b i o m e t r i c s t u d y p r o v i d e d an e v a l u a t i o n o f the f i b r e l e n g t h w i t h i n the f a s t g r o w i n g i n t e r n o d e /=^4.The d a t a i n F i g u r e -15 show t h a t o v e r s h o r t d i s t a n c e i n a d e v e l o p i n g i n t e r n o d e t h e a v e r a g e l e n g t and d i a m e t e r o f a f i b r e a b o u t d o u b l e . Such an e l o n g a t i o n o f about 85 % r e p r e s e n t s an i m p o r t a n t d e p o s i t i o n o f t h e c e l l w a l l c o n s t i t u e n t and t h u s a s i g n i f i c a n t d i f f e r e n c e must be f o u n d a c c o u n t i n g f o r t h i s r a p i d b i o c h e m i c a l a c t i v i t y , a t the l e v e l o f t h e w a l l p o l y s a c c h a r i d e s As e x p e c t e d when the c a r b o h y d r a t e c o m p o s i t i o n s o f t h r e e zones e x c i s e d between the b a s e and the t o p o f t h r e e i n t e r n o d e s a t t h r e e d i f f e r e n t s t a g e s o f m a t u r a t i o n were compared, the r e l a t i v e p r o p o r -t i o n s o f t h e d i f f e r e n t s u g a r s were f o u n d t o be c h a r a c t e r i s t i c o f the d e g r e e o f m a t u r a t i o n o f t h e t i s s u e s ( T a b l e XX) . The i n c r e a s e i n x y l o s e between the m e r i s t e m a t i c zone and t h e upper s e c t i o n i s from 32 t o 52 %. G l u c o s e undergoes, a p a r a l l e l i n c r e a s e whereas t h e r a t i o o f x y l o s e t o g l u c o s e r e m a i n s a l m o s t c o n s t a n t t h r o u g h o u t the m a t u r i n g i n t e r n o d e s . I n i n t e r n o d e s B and C h a v i n g r e a c h e d t h e i r d e f i n i t i v e l e n g t h , the i n t e r c a l a r y m e r i s t e m a t i c zone becomes p r o g r e s s i v e l y . i n o p e r a t i v e and a l l the d i f f e r e n t s e c t i o n s show a r e m a r k a b l y s i m i l a r c o m p o s i t i o n w i t h x y l o s e r e p r e s e n t i n g 50 t o 52 % and g l u c o s e 43 t o 45 % o f t h e t o t a l c a r b o h y d r a t e . A l l t h e above d a t a d e m o n s t r a t e t h a t ' t h e r e I s a c l o s e r e l a t i o n s h i p between m a t u r i t y and c a r b o h y d r a t e c o m p o s i t i o n o f the f i b r o u s t i s s u e s . W i t h i n a d e v e l o p i n g i n t e r n o d e , a l l the s t a g e s o f m a t u r i t y a r e r e p r e s e n t e d p o i n t i n g o u t t h a t t h e i n t e r n o d e i s a. p h y s i o l o g i c a l u n i t i n the stem o f the r e e d . I I - VARIATION,OF HEMICELLULOSE CONTENT.WITH  THE MATURATION OF THE TISSUES. S i n c e t h e aim o f t h i s r e s e a r c h was a c o m p a r i s o n o f the hemi-c e l l u l o s e s according to the growth cy c l e , i t was important to protect the p o l y s a c c h a r i d e s from a p o s s i b l e d e g r a d a t i o n d u r i n g t h e i r i s o l a t i o n . T h i s i s the r e a s o n why the a l k a l i n e e x t r a c t i o n s have been c a r r i e d o u t i n t h e p r e s e n c e o f b o r o h y d r i d e w h i c h p r e v e n t s t h e r e a c t i o n o f p e e l i n g by 3 - e l i m i n a t i o n ( 1 1 0 ) . A l t h o u g h R e i d and W i l k i e c o n s i d e r e d t h a t " t o t a l h e m i c e l l u -l o s e s " (51) would be more r e p r e s e n t a t i v e o f the r e l a t i o n s h i p between p o l y s a c c h a r i d e s and g r o w t h , we p r e f e r r e d a s t e p w i s e e x t r a c t i o n o f the h e m i c e l l u l o s e s w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f a l k a l i w h i c h a l l o w s a b e t t e r a c c e s s i b i l i t y t o the p o l y s a c c h a r i d e s and an e x h a u s --132- .. r t i v e p e n e t r a t i o n o f the s o l v e n t s . The t o t a l h e m i c e l l u l o s e c o n t e n t a s-deduced, from T a b l e s XXIV t o XXXI v/as 3 4 % f o r t h e y o u n g e s t t i s -s u e s , was as much as 4 4 % f o r s e c t i o n 2 b u t was o n l y 25 % i n the more mature t i s s u e s . The t o t a l x y l o s e p r e s e n t i n t h e s e p o l y s a c c h a r i -des i n c r e a s e d from 6 8 %. ( s e c t i o n 1 ) t o 8 9 % ( s e c t i o n 2) and t h e n d e c r e a s e d t o 8 1 % ( s e c t i o n 3 ) , whereas the p e r c e n t a g e o f a r a b i n o s e d e c r e a s e d w i t h i n c r e a s i n g m a t u r i t y . T h i s phenomenon can be e v i d e n c e o f s e c o n d a r y t h i c k e n i n g o f the c e l l w a l l s s i n c e s e c o n d a r y w a l l s i n v a r i a b l y c o n t a i n x y l a n s ( 1 1 1 ) The p r e s e n c e o f a maximum y i e l d o f x y l o s e , t h u s o f x y l a n , i n s e c t i o n 2 c o r r e s p o n d i n g t o the most a c t i v e s i t e o f e l o n g a t i o n o f the f i b r e s , i s s i m i l a r t o the s i t u a t i o n d e s c r i b e d by M e i e r ( 1 1 2 ) f o r the d eve-l o p i n g w a l l s o f f i b r e s and t r a c h e i d s i n B i r c h wood and P i n e wood. T h i s a u t h o r d e m o n s t r a t e d i n d e e d t h a t the c o n t e n t o f x y l a n f i r s t r e a c h e d a maximum and t h e n d e c r e a s e d as s e c o n d a r i z a t i o n o f the c e l l w a l l p r o c e e d s . S i m i l a r l y , R e i d and W i l k i e (102) n o t e d w i t h young l e a v e s o f Avena a t o t a l h e m i c e l l u l o s e c o n t e n t o f 16 % f o r t h e y o u n g e s t l e a v e s o f 28 % f o r the a d u l t p l a n t , and o f as much as 36 % f o r an i n t e r m e -d i a t e m a t u r i t y . T h i s was accompanied-j-,y a d i m i n u t i o n o f t h e x y l o s e c o n t e n t i n the o l d e r t i s s u e s . The a u t h o r s e x p l a i n e d t h e s e v a r i a t i o n s as the r e s u l t o f a d i l u t i o n o f x y l a n s by o t h e r p o l y s a c c h a r i d e s . T h i s i n t e r p r e t a t i o n has a l s o been t h a t o f A l b e r s h e i m and h i s c o l l a b o r a -t o r s (113) f o r young P h a s e o l u s and f o r Zea mays.But t h e s e a u t h o r s s u g g e s t t h a t d i l u t i o n c o u l d n o t be the o n l y e x p l a n a t i o n o f s u c h v a r i a t i o n s , and t h a t i n c o r p o r a t i o n o r .'.disappearance o f c e r t a i n p o l y -s a c c h a r i d e c o n s t i t u e n t s w o u l d a l s o t a k e p l a c e w i t h d i f f e r e n t r a t e s . A n o t h e r i n t e r p r e t a t i o n c o u l d be s u g g e s t e d by a n a l o g y w i t h the r e -s u i t s o f F r a n z (101) on c e l l w a l l s o f P h a s e d u s a u r e u s , who o b s e r -ved t h a t the h e m i c e l l u l o s e c o n t e n t d e c r e a s e d f r o m 50 % t o 30 % d u r i . - i 1 4 the g r o w t h . He showed by g l u c o s e -' C i n c o r p o r a t i o n t h a t most o f the a c t i v i t y was f o u n d f i r s t i n the h e m i c e l l u l o s e f r a c t i o n and t h e n was t r a n s f e r r e d to the a - c e l l u l o s e . as the p l a n t aged. T h i s s u g g e s t e d a t u r n o v e r o f the p o l y s a c c h a r i d e s d u r i n g p i a n t g r o w t h . I t can t h e n be assumed t h a t i n Arundo donax, an a c t i v e phase o f s y n t h e s i s o f x y l a n s would t a k e p l a c e , i n the r a p i d l y e l o n g a t i n g i n t e r n o d e s and t h a t t h i s b i o s y n t h e t i c a c t i v i t y w o u l d slow down a t th e end o f t h e p e r i o d o f e l o n g a t i o n when o t h e r p o l y s a c c h a r i d e s w o u l d be s y n t h e s i z e d more a c t i v e l y o r when a p o s s i b l e m e t a b o l i z a t i o n of some x y l a n s would s t a r t . I t i s t e m p t i n g t o s u b s c r i b e t o the p o s s i b i l i t y o f a t u r n o v e r s i n c e we r e c e n t l y f o u n d a x y l a n a s e a c t i v i t y o f the exo-enzyme t y p e i n d e v e l o p i n g i n t e r n o d e s o f Arundo donax. But t h i s r e s u l t has t o be c o n f i r m e d by further experiments b e f o r e a n t i c i p a t i n g any c o n c l u s i o n . - 1 3 4 -I I I - COMPARATIVE s ^ R " C T U R A L _ S ^ j ^ ^ F _ 2 L ^ N « AT THREE STAGES OF M A T U R I T Y . The arabino glucurono xylans from s e c t i o n s 1,2, and 3 p r e -sented a remarkably s i m i l a r composition c o n s i s t i n g of about 6 % 1 arabinose and 90 % xylose (Table X X X I ). The u r o n i c a c i d content was apparently 4.5. %. i n S e c t i o n 1 and 2.1 % i n S e c t i o n s 2 and 3. But the d i f f e r e n c e i n the u r o n i c a c i d content might be only apparent since an i n s i t u r e d u c t i o n with borohydride was done be f o r e e x t r a c -t i o n of the h e m i c e l l u l o s e s . As a matter of f a c t , we could observe t h a t the borohydride r e d u c t i o n of the wood of Aspen leads. to the i s o l a t i o n of a 4-0-methyl glucuronoxylan i n which a few u r o n i c a c i d r e s i d u e s had been reduced i n s i t u (114). T h i s f i n d i n g would fa v o r the p o s s i b i l i t y of the presence i n v i y o of e s t e r or l a c t o n e forms of the u r o n i c a c i d r e s i d u e s which would then become s u s c e p t i b l e to r e d u c t i o n by b o r o h y d r i d e . E s t e r l i n k a g e s with l i g n i n have been . suggested (115,116).. In such a s i t u a t i o n , i n s i t u r e d u c t i o n of a l i g n i f i e d m a t e r i a l would lead to erroneous e s t i m a t i o n of the u r o n i c a c i d content. N e v e r t h e l e s s the apparent decrease of u r o n i c a c i d as the p l a n t ages agrees with e a r l i e r r e s u l t s ' (117,118). The i n v e s t i g a t i o n of s t r u c t u r e of the arabinoglucuronoxylans from S e c t i o n s 1 to 3 was c a r r i e d out mainly by the study of the h y d r o l y s i s products of the permethylated p o l y s a c c h a r i d e . For a l l the three S e c t i o n s (Table X X X I I ) the methylated sugars found were i d e n t i c a l . 2,3,5 - x ri-O-methy1-L-arabinose was the only arabinose d e r i v a t i v e found and i n d i c a t e d that a l l the arabinose r e s i d u e s were -135-p r e s e n t as t e r m i n a l n o n - r e d u c i n g u n i t s i n t h e i r f u r a n o s e form ; 2 , 3 , 4-tri-0_-methy 1 - D - x y l o s e c o r r e s p o n d e d t o the t e r m i n a l non r e d u -c i n g end o f the (1 •? '4) l i n k e d x y l o s e backbone as shown by the p r e d o m i n a n c e o f 2 , 3 - d i - 0 - m e t h y i - D - x y l o s e . B r a n c h p o i n t s c o u l d o c c u r a t the 2- or 3- p o s i t i o n c f c e r t a i n x y l o s e u n i t s as i n d i c a t e d by the p r e s e n c e o f some r e s i d u e s o f mono-O-methy1-D-xylose. I t can be a l s o n o t e d t h a t t h e r e was a s m a l l - a m o u n t o f 2 , 3 , 4 , 6 - t e t r a - O - m e t h y l - D -g l u c o s e amongst the h y d r o l y s i s p r o d u c t s o f the x y l a n f r o m S e c t i o n 3-As d e v e l o p e d above, t h i s g l u c o s e c o u l d o r i g i n a t e from the i n s i t u r e d u c t i o n o f some u r o n i c a c i d r e s i d u e s . The q u a n t i t a t i v e r e s u l t s i n T a b l e XXXII c o n f i r m t h e - i d e n t i t y o f s t r u c t u r e o f t h e x y l a n t h r o u g h o u t th e d e v e l o p m e n t o f t h e p l a n t t i s s u e s and b r i n g a b o u t t h e , c o n s i d e r a b l e i n c r e a s e o f t h e a v e r a g e c h a i n l e n g t h w h i c h goes from 63 f r o m the y o u n g e s t i n t e r n o d e s t o 91 f o r the i n t e r n o d e s h a v i n g r e a c h e d h a l f t h e d e f i n i t i v e h e i g h t and t o 150 f o r t h e f u l l - g r o w n i n t e r n o d e s . The h o m o g e n e i t y o f t h e x y l a n s under s t u d y was n o t due t o t h e mode o f e x t r a c t i o n as shown by t h e g r e a t s t a b i l i t y i n s t r u c t u r e and c h a i n l e n g t h e s t i m a t e d on the d i f f e r e n t f r a c t i o n s e x t r a c t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f a l k a l i i n S e c t i o n 2 ( T a b l e X X X I I I ) . T h i s p o i n t s o u t t h a t the a v e r a g e d e g r e e o f p o l y m e r i s a t i o n o f t h e h e m i c e l l u l o s e i s r e l a t e d t o t h e d e g r e e o f m a t u r a t i o n o f the c e l l w a l l s components, and i t i s i n t e r e s t i n g t o n o t e t h a t a t t h e e a r l i e s t s t a g e o f d i f f e r e n c i a t i o n o f the f i b r o u s t i s s u e s a x y l a n s i m i l a r i n c o m p o s i t i o n and s t r u c t u r e t o the d e f i n i t i v e one was a l r e a d y p r e s e n t , showing" 5 t h u s the v e r y r a p i d b i o s y n t h e t i c a c t i v i t y o f the c e l l w a l l . -136-A l l t h e p r e c e d i n g d a t a o b t a i n e d by c l a s s i c a l methods g i v e o n l y average, r e s u l t s on t h e d o m i n a n t f e a t u r e s o f the p o l y s a c c h a r i -d e s . I t seems t h a t when l o o k i n g a t b i o c h e m i c a l e v o l u t i o n i n a mate-r i a l o f such an a n a t o m i c a l c o m p l e x i t y and where p o l y d i s p e r s i t y o f t h p o l y s a c c h a r i d e s c e r t a i n l y o c c u r s ( 5 1 ) , more c h a r a c t e r i s t i c v a r i a t i o n i n t h e c o m p o s i t i o n and s t r u c t u r e o f t h e p o l y s a c c h a r i d e s i n r e l a t i o n , t o growth c o u l d be due t o 'minor d i f f e r e n c e s w h i c h have n o t o f t e been b r o u g h t i n t o a t t e n t i o n i n t h e p a s t . In o r d e r t o i l l u s t r a t e t h i s p o i n t , the v a r i a t i o n i n the d i s t r i b u t i o n o f a c e t y l g r o u p s i n the x y l a n c h a i n has been i n v e s t i g a t e d on the d i m e t h y 1 s u l p h o x i d e e x t r a c t e d h e m i c e l l u l o s e s . The d a t a i n T a b l e XXXIV show t h a t e x c e p t f o r S e c t i o n 1 where a m i x t u r e o f p o l y s a c c h a r i d e s has been s o l u b i l i -z e d w i t h d i m e t h y l s u l p h o x i d e , t h e c a r b o h y d r a t e c o m p o s i t i o n o f the f r a c t i o n s o b t a i n e d i s i d e n t i c a l w i t h t h a t o f the a l k a l i - e x t r a c t e d x y l a n s . A l l the a c e t y l has been c a l c u l a t e d r e l a t i v e t o the c o m p o s i -t i o n o f p u r e a r a b i n o g l u c u r o n o x y l a n s and i t i s a p p a r e n t t h a t t h e a c e t y l c o n t e n t i s t h e l o w e s t i n the y o u n g e s t t i s s u e s ( 4 . 7 %) and i n -c r e a s e s t o 1 0 . 7 % and 8 . 5 % as t h e p l a n t a g e s . As f o r t h e r a t i o o f 4 - O - m e t h y l g l u c u r o n i c , a c i d to g l u c u r o n i c a c i d , t h e r e i s a s t r o n g i n -c r e a s e i n t h e p r o p o r t i o n o f g l u c u r o n i c a c i d w i t h the m a t u r a t i o n o f the t i s s u e s . T h i s l a s t r e s u l t i s i n o p p o s i t i o n t o t h e f i n d i n g s o f Kauss and H a s s i d ( 1 1 9 ) on the b i o s y n t h e s i s o f u r o n i c a c i d s and t h e o b s e r v a t i o n o f B u c h a l a and W i l k i e ( 1 1 8 ) on o a t who f o u n d an i n c r e a s e i n t h e p r o p o r t i o n o f 4 - O - m e t h y l g l u c u r o n i c a c i d r e l a t i v e to g l u c u r o -n i c a c i d i n t h e l e a f t i s s u e s as the p l a n t m a t u r e s . I t i s s t i l l t o o e a r l y to draw any conclusions from our l a s t r e s u l t s on the v a r i a t i o n s of a c e t y l g r o u p s and 4 - 0 - m e t h y l s u b s t i t u t i o n o f g l u c u r o n i c a c i d , b u t we b e l i e v e •137-t h a t t h e s e v a r i a t i o n s must c e r t a i n l y p l a y a r o l e a t the l e v e l o f the b i o s y n t h e t i c i n c o r p o r a t i o n o f p o l y s a c c h a r i d e s i n the p l a n t c e l l w a l l i E X P E R I M E N T A L G e n e r a l methods - -As d e s c r i b e d i n P a r t i . . P r e p a r a t i o n o f t h e m a t e r i a 1. The 1-month-old r e e d s were h a r v e s t e d a t the end of'May,-- and i m m e d i a t e l y f r o z e n i n l i q u i d n i t r o g e n p r i o r t o s t o r a g e a t -25°in s e a l e d p l a s t i c b a g s . B e f o r e u s e , the l e a f s h e a t h s were removed and t h e i n t e r n o d e s were c u t i n t o s m a l l p i e c e s and e x t r a c t e d w i t h 80 % b o i l i n g e t h a n o l f o r 15 m i n u t e s , t h e n w i t h a m i x t u r e o f m e t h a n o l -c h l o r o f o r m ( 1 : 1 ) f o r 16 h o u r s a t room t e m p e r a t u r e . P r e p a r a t i o n o f t h e f i b r e s f o r b i o m e t r i c s t u d i e s . F r a g m e n t s o f i n t e r n o d e s were s u b m i t t e d t o t h e a c t i o n o f a 20 % sodium h y d r o x i d e s o l u t i o n f o r 150 m i n u t e s a t 170° i n a t i g h t l y c l o s e d s t e e l c o n t a i n e r . The t i s s u e s were washed w i t h w a t e r and t r e a -t e d w i t h s o d i um c h l o r i t e a c c o r d i n g t o Wise and c o w o r k e r s f o r 15 m i n u t e s a t 7 0 ° . The d e l i g n i f i e d f i b r o u s - m a t e r i a l was d i s p e r s e d i n w a t e r and used' f o r b i o m e t r i c measurements. T o t a l h y d r o l y s i s . F i n e l y g r o u n d t i s s u e s were e x t r a c t e d w i t h b o i l i n g w a t e r f o r 15 m i n u t e s and d r i e d , w i t h e t h a n o l . T h e d r i e d t i s s u e s were h y d r o l y s e d a t room t e m p e r a t u r e f o r t h r e e h o u r s w i t h 72 % s u l p h u r i c a c i d , w h i c h was t h e n d i l u t e d to n o r m a l and h e a t e d a t 100° i n a s e a l e d tube f o r 5 h o u r s . A f t e r n e u t r a l i s a t i o n w i t h b a r i u m c a r b o n a t e and p a s s a g e t h r o u g h Amber l i t e IR-120 ( H -i-} a n d I R - 4 5 ( a c e t a t e form) , the n e u t r a l c a r b o h y d r a t e s were c o n c e n t r a t e d t o a s m a l l volume. The n e u t r a l s u g a r s were e s t i m a t e d by gas l i q u i d c h r o m a t o g r a p h y o f t h e i r a l d i t o l a c e t a t e d e r i v a t i v e s . L i g n i n e s t i m a t i o n . Samples (1.0 g) were e x t r a c t e d w i t h b o i l i n g w a t e r and t h e n w i t h a b e n z e n e - e t h a n o l m i x t u r e (1 : 1) i n a S o x h l e t a p p a r a t u s 'for 24 h o u r s . The f i n e l y g r o u n d t i s s u e s were t r i t u r a t e d f o r 3 h o u r s i n 72 % H2SO4 (15 ml) u n t i l c o m p l e t e l y d i s s o l v e d , t h e n w a t e r (560 ml) was added and the m i x t u r e r e f l u x e d f o r 5 h o u r s . The i n s o l u b l e l i g n i was c o l l e c t e d by f i l t r a t i o n and d r i e d t o c o n s t a n t w e i g h t . I s o l a t i o n o f h e m i c e l l u l o s e s . H o l o c e l l u l o s e was s u c c e s s i v e l y e x t r a c t e d f o r p e r i o d s o f 24 h o u r s w i t h 1 % aqueous p o t a s s i u m b o r o h y d r i d e , and t h e n w i t h p o t a sium h y d r o x i d e s o l u t i o n s o f i n c r e a s i n g c o n c e n t r a t i o n s : 0.2 M c o n t a i n i n g 1 % p o t a s s i u m b o r o h y d r i d e ; 1.0 M - 0.5 % b o r o h y d r i d e ,-2.5 M - 0.5 % b o r o h y d r i d e and f i n a l l y 4.3 M p o t a s s i u m h y d r o x i d e . N e u t r a l i z a t i o n o f the e x t r a c t s below 5° w i t h a c e t i c a c i d gave p r e c i p i t a t e s c o l l e c t e d by centra, f u g a t i o n . The s u p e r n a t a n t was d i a l y s e c o n c e n t r a t e d to a b o u t 100 ml and p o u r e d i n t o 3 volumes o f e t h a n o l t o g i v e p r e c i p i t a t e s P . A l l the p r e c i p i t a t e s were washed w i t h -14 0-e t h a n o l , a c e t o n e and e t h y l e t h e r and a i r d r i e d . P u r i t y t e s t on f r a c t i o n s 2 P - i . O M and 3 P -1.0 M-The p o l y s a c c h a r i d e was s o l u b i l i z e d i n 1.OM p o t a s s i u m h y d r o -x i d e and d i a l y s e d u n t i l n e u t r a l - The d i a l y s e d s o l u t i o n was c o n c e n -t r a t e d t c a s m a l l volume and p l a c e d on a DEAE-Sephadex-A-25 column e q u i l i b r a t e d i n p h o s p h a t e b u f f e r (0.01 M, pK 6.5). The m a t e r i a l on the column was e l u t e d w i t h 0.1, 1 and 2 M s o l u t i o n s o f s o d i u m . c h l o -r i d e . The main f r a c t i o n ( a b o u t 70 %) was h y d r o l y s e d and f o u n d t o be v e r y s i m i l a r i n c o m p o s i t i o n t o the s t a r t i n g m a t e r i a l . G e l f i l t r a t i o n and e l e c t r o p h o r e s i s o f dyed p o l y s a c c h a r i d e s . DEAE-Sephadex p u r i f i e d f r a c t i o n s o f the x y l a n s were dyed a c c o r d i n g t o Dudman and B i s h o p (108) w i t h an aqueous s o l u t i o n o f P r o c i o n r e d MX-G . E x c e s s dye was removed on a B i o g e l P 2 column and t h e band c o n t a i n i n g the dyed p o l y s a c c h a r i d e e l u t e d on an A g a r o s e B i o - G e l A-0.5m column w i t h 1.0 M sodium c h l o r i d e s o l u t i o n . A single ban was o b t a i n e d . Sodium c h l o r i d e was d i a l y z e d o u t and t h e dyed x y l a n f r e e z e - d r i e d . H y d r o l y s a t e s o f f r a c t i o n s o b t a i n e d from 2 P s~1.0 M had a c o m p o s i t i o n s i m i l a r t o the s t a r t i n g p o l y s a c c h a r i d e , and showed a s i n g l e band by e l e c t r o p h o r e s i s on c e l l u l o s e a c e t a t e s t r i p s (2.5 X 17 cm) i n 0.1 M sodium t e t r a b o r a t e - s o d i u m c h l o r i d e b u f f e r (25 0-350 V, 7 mA). a E i o - R a d L a b o r a t o r i e s -141-T e s t s o f p u r i t y on f r a c t i o n 2P -1.0 M ; - s = F r a c t i o n , S t a r t i n g x y l a n DEAE-Sephadex A g a r o s e A-0.5 m G a l Moi r a t i o G l c A r a 1 . 1 1 . 3 1 . 3 2.0 3 . 5 3.4 X y l 6.0 90 6.4 89 5.2 90 M e t h y l a t i o n . A l l m e t h y l a f i o n s were c a r r i e d o u t w i t h a s i n g l e t r e a t m e n t a c c o r d i n g t o t h e Hakomori (42) p r o c e d u r e (NaK, DMSO, CH^I f o l l o w e d by 2 o r 3 m e t h y l a t i o n s a c c o r d i n g t o P u r d i e ( 3 8 ) . The p e r m e t h y l a t e d p r o d u c t s were f r a c t i o n a t e d by C H C l ^ = i i g h t p e t r o l e u m m i x t u r e s . A l l o p e r a t i o n s were as d e s c r i b e d p r e v i o u s l y . E x t r a c t i o n o f a c e t y l a t e d p o l y s a c c h a r i d e s . Samples o f t i s s u e s a t t h r e e s t a g e s o f m a t u r i t y were e x t r a c -t e d i n a S o x h l e t a p p a r a t u s w i t h b e n z e n e - e t h a n o l . ( 2 : 1 , v/v) and s u b j e c t e d t o s o d i u m c h l o r i t e d e l i g n i f i c a t i o n . D e l i g n i f i e d m a t e r i a l was e x t r a c t e d w i t h » d i m e t h y l s u l p h o x i d e (2 X 24 h o u r s ) . F i l t r a t e s were p r e c i p i t a t e d w i t h e t h a n o l (3 volumes) a c i d i f i e d w i t h h y d r o c h l o -r i c a c i d (pH 4 ) . The p r e c i p i t a t e s were c o l l e c t e d by c e n t r i f u g a t i o n and d r i e d by s o l v e n t exchange s u c c e s s i v e l y w i t h e t h a n o l , a c e t o n e and e t h y l e t h e r . Y i e l d s f o r s e c t i o n s 1 t o 3 were 1.7 %, 5.2 % and -142-1.9 % o f the d r y m a t e r i a l r e s p e c t i v e l y . • A c e t y l a n a l y s i s The method d e s c r i b e d by M e i e r (109) was employed w i t h a s l i g h t m o d i f i c a t i o n . Samples (1O-10O mg) o f p o l y s a c c h a r i d e s were m e t h a n o l y z e d i n a s e a l e d tube w i t h a n h y d r o u s m e t h a n o l c o n t a i n i n g 2 % h y d r o g e n c h l o r i d e (2 ml) and m e t h a n o l (0.1 - 0.5 ml) c o n t a i n i n g a known amount o f 1 , 4 - d i o x a n as i n t e r n a l s t a n d a r d . Th.e t u b e s were h e a t e d i n a b o i l i n g w a t e r b a t h f o r 30 m i n u t e s . A f t e r c o o l i n g , t h e s amples were a n a l y s e d by g a s - l i q u i d - c h r o m a t o g r a p h y on a 2 m e t r e column (10 % Carbowax 20 M on Chromosorb W - DMCS) h e l d a t 4 0 ° . G l u c u r o n i c a c i d and 4 - 0 - m e t h y l g l u c u r o n i c a c i d d e t e r m i n a t i o n . The p o l y s a c c h a r i d e s were h y d r o l y s e d w i t h t r i f l u o r o a c e t i c a c i d (2 M) f o r 2 h o u r s i n s e a l e d t u b e a t 100° and n e u t r a l and a c i d i c s u g a r s s e p a r a t e d on A m b e r l i t e IR-45 r e s i n . The a c i d i c mate-' r i a l was e l u t e d from the r e s i n w i t h a 10 % s o l u t i o n o f f o r m i c a c i d and t a k e n t o d r y n e s s . The a l d o b i o u r o n i c a c i d s were d i s s o l v e d i n an-h y d r o u s m e t h a n o l c o n t a i n i n g 2 % h y d r o g e n c h l o r i d e and the s o l u t i o n h e a t e d a t 70° f o r 4 h o u r s i n a s e a l e d t u b e . The s o l u t i o n was neu-t r a l i z e d (Ag2 CO^) , ..-..centrif uged and e v a p o r a t e d . The r e s u l t i n g s y -r u p was r e d u c e d o v e r n i g h t w i t h sodium b o r o h y d r i d e and t h e n n e u t r a -l i z e d w i t h 50 % a c e t i c a c i d and s t i r r e d w i t h A m b e r l i t e IR-120 (H +) b e f o r e r e m o v a l o f b o r a t e by e v a p o r a t i o n w i t h m e t h a n o l c o n t a i n i n g 1 % h y d r o c h l o r i c a c i d . The m i x t u r e v/as t h e n h y d r o l y s e d w i t h t r i f l u o -r o a c e t i c a c i d (2 M) -at. 100° f o r 1 h o u r and the r e s u l t i n g n e u t r a l 143-s u g a r s were a n a l y z e d by gas l i q u i d c h r o m a t o g r a p h y as t h e i r a l d i t o l a c e t a t e d e r i v a t i v e s . -1 44-BIB LIOG RAP H Y 1. S t r a s b u r g e r ' s T e x t b o o k o f B o t a n y , Longmans ed , London (1965) 2. J.P. J o s e l e a u , These 3eme c y c l e (1965) U n i v e r s i t e de G r e n o b l e ( F r a n c e ) . 3. M. A r n o u x , Ann . ' Ame l i o r . P l a n t e s , 24_ ( 1974) 349. 4. C R . M e t c a l f e , "Anatomy o f the M o n o c o t y l e d o n s " I 48, C l a r e n d o n P r e s s , O x f o r d (1960) 5. V . I . C h e a d l e , J . A r n o l d A r b o r . , 3_6 (1955) 141. 6 . R.L. W h i s t l e r and C.L. Smart, " P o l y s a c c h a r i d e C h e m i s t r y " , A c a d e m i c P r e s s , New-York ( 1 9 5 3 ) 7. H.O. Bouveng and B. L i n d b e r g , A d v a n ces i n C a r b o h y d r a t e C h e m i s t r y 1 5 : ( 1 9 6 0 ) 5 3 . " ' 8 . T.E. T i m e l l , A d v a n c es i n C a r b o h y d r a t e C h e m i s t r y , 19 (1964) 247. 9. T.E. T i m e l l , A d v a n c e s i n C a r b o h y d r a t e C h e m i s t r y , 2 0 (1965) 409. ' ~ 1 0 . G.0. A s p i n a l l , " P o l y s a c c h a r i d e s " , Pergamon P r e s s , O x f o r d ( 1970) 1 1 . G. Jayme and G. Hanke, C e l l u l o s e Chemie , 2_2 ( 1 9 4 3 ) 102. 12. L . E . Wise, M. Murphy and A.A. D ' A d d i e c o , P a p e r T r a d e J . , 122 (1946) 35. ~ 1 3 . E. H a g g l u n d , B. L i n d b e r g and J . Mc P h e r s o n , A c t a Chem. Sc a n d . , IP (1956) 1160. • 1 4 . T.E. T i m e l l . , T a p p i , 4 4 (19 61) 8 8 . 1 5 . M.H. 0'Dwyer, Biochem. J . , 2p_ (1926) 656. 1 6 . R.L. W h i s t l e r and G.E. L a u t e r b a c h , A r c h . Biochem. B i o p h y s . , 7_7_ ( 1 9 5 8 ) 22 . 17. R.L. W h i s t l e r and J . L . S a n n e l l a , Methods i n C a r b o h y d . Chem . , 5_ ( 1965 ). 3 4 . 1 8 . G.G.S. D u t t o n , B . I . J o s e l e a u and P.E. R e i d , T a p p i 5j5 (1973) 168 . " 19. E. S a l k o w s k y , B e r . , 27 (1894) 497. 145-C . P . J . Glaudemans and T.E. T i m e l l , J . Am... Chem. Soc. , 8 0 ( 1 9 5 8 ) 9 4 1 . II. M e i e r , A c t a Chem. S c a n d . , 12 ( 1958) 144. J.K. H a m i l t o n and H.W. K i r c h e r , J . Am. Chem. Soc. , 8_0 (1 95 8) 4 7 03 . J . E . S c o t t , B iochem. J , , 8J_ (1961) 418. B.N. Stepanenko. and L.B. U z d e n i k o v a , C a r b o h y d . Res., 25 (1972) 526. " H.O. Bouveng and' B. L i n d b e r g , A c t a Chem. S c a n d . , 11 (19 5 8 ) 1 9 7 7 . ' G.G.S. D u t t o n , Symposium I n t . G r e n o b l e ( 1 9 6 4 ) , Imprimeri.es de Chambery ed. G.O. A s p i n a l l a n d R.S. Fanshawe, J'. Chem. S o c . , 42 15 (1961) . J . S . Saeman, W.E. Moore, R.L. M i t c h e l l and M.A. M i l l e r , T a p p i , 37 (1954) 336. J.D. B l a k e and G.N. R i c h a r d s , C a r b o h y d . Res., 14 (1970) P. A l b e r s h e i m , D.J. N e v i n s , P.D. E n g l i s h and A. K a r r , C a r b o h y d . Res., 5 (1967) 340. D.J. N e v i n s , P.D. E n g l i s h and P. A l b e r s h e i m , P l a n t P h y s i o l  42 (1967) 900. ~ J.P . J o s e l e a u and J . Comtat, U n p u b l i s h e d r e s u l t s . C.T. B i s h o p and D.R. W h i t a k e r , Chem. I n d . (London) (1955) 119. ~ " • G.O. A s p i n a l l , I.M. C a i r n c r o s s , R . J . S t u r g e o n and K.C.B. W i l k i e , J'. Chem. Soc. (1960) 3881. A.G. Mc I n n e s , D.H. B a l l , F.P. Coo p e r and C.T. B i s h o p , J . Chromatog., 1 (1958) 556. G.G.S. D u t t o n , A d v a n c e s i n C a r b o h y d r a t e Chem. and P.iochem 28 (1973) 11. W.N. Haworth, J . Chem. Soc. , 107 (191-5) 8 T. P u r d i e and J . C . I r v i n e , J . Chem. Soc. , 8_3_ (1903) 1021. R. Kuhn, H. T r i s c h m a n n and I . Lov?, Ang<-w. Chem. , 6 7 (19 5 5 ) 3 2 . -146-H.C. S r i v a s t ' a v a , P . P . s ingh,. S .w. Ha r s h e and K. V i r k , " T e t r a h e d r o n L e t t . (1964) 493. E . J . C o r e y and M. C h a y k o v s k y , J . Am. Chem. S o c , 04 ( 1962) . ~ S. H a k o m o r i , J . Biochem. (Tokyo) 5_5 ( 1 9 6 4 ) 2 0 5 . N. Handa and R. Montgomery, C a r b o h y d . Res. , 1 1 ( 1 9 6 9 ) 4 6 7 . L. Malaprade-, Comptes-Rendus , 1 8 6 ( 1 9 2 8 ) 3 8 2 . M. B o b b i t t , A d v a n c e s i n C a r b o h y d . Chem., 1 1 ( 1 9 5 6 ) 1 I . J . G o l d s t e i n , G.W. Hay; B.A. Le i v i s and F. S m i t h , A b s t r a c t s P a p e r s Am. Chem. Soc. ( 1 9 5 9 ) 1 3 5 , 3 D . D . P . Sweet, P". A l b e r s h e i m and R.H. S h a p i r o , C a r b o h y d . Res. 4 0 ( 1 9 7 5 ) 1 9 9 . D.P. Sweet, P. A l b e r s h e i m and R.H. S h a p i r o , C a r b o h y d . Res. 40 ( 1 9 7 5 ) 2 1 7 • :  G.A. Adams, Methods i n C a r b o h y d . Chem., _5 ( 1 9 6 5 ) 1 8 5 . 0. S m i d s r o d and A. Haug, J . PjOlymer S c i . P a r t C, 1 6 ( 1 9 6 7 ) 1 5 8 7 . ' J.S.G. R e i d and K.C.B. W i l k i e , P h y t o c h e m i s t r y , 8 (1969) 2045 ~ W.Z. H a s s i d , S c i e n c e , 1 6 5 ( 1 9 . 6 9 ) 1 3 7 . G.G.S. D u t t o n , K.B. G i b n e y , G.D. J e n s e n and P.E. R e i d , J . Chromatog.,, 36 (1968) 152 . M. B y l u n d and A. Donetzhuber,: Svensk P apper s t i d n . , 15 (1968) 505. — — ~ ' P.A. S a n d f o r d and H.E. C o n r a d , B i o c h e m i s t r y , 5_ ( 1 9 6 6 ) 1 5 0 8 H. B j o r n d a l , C.G. H e l l e r q v i s t , B. L i h d b e r g and S. S v e n s s o n Angew. Chem.. I n t e r n a t . E d . , 9_ ( 1 9 7 0 ) 6 1 0 . G.G.S. D u t t o n and S.A. Mc K e l v e y , Can. J . Chem. , 3 9 ( 1 9 6 1 ) 2 5 8 2 . H. B j o r n d a l , B. L i n d b e r g and S. S v e n s s o n , A c t a Chem. Scand 21 (1967) 1801. ~ H. B j o r n d a l , B. L i n d b e r g and S. S v e n s s o n , C a r b o h y d . Res., 5 ( 1967) 433. " ! -147-G. P e t e r s s o n and 0. S a m u e l s o n , Svensk P a p p e r s t i d n . , 71 (1968) 77. O.S. C h i z h o v , N.V. M o l o d t s o v and N.K. K o c h e t k o v , C a r b o h y d . Res. , 4_(1967) 2 7 3. H. G.J. De W i l t and T. T s u c h i y a , Mass S p e c t r o s c o p y , 18, (1970) 1294. G.W. Hay, B.A. Lev/is and F. S m i t h , Methods i n C a r b o h y d . Chem. , 5 ( 1965) 357. "• ~ 1  C O . A s p i n a l l and R.J. F e r r i e r , Chem. I n d . (London) (1957) 1216. M. Abdel-Akhe'r / J.K. H a m i l t o n , R. Montgomery and F. S m i t h , J . Am. Chem. S o c . , 7 4 (1952) 4970. J.N.C. Whyte and J.R. E n g l a r , Can. J . Chem., 49 (1971) 1300. J.N.C. Whyte, C a r b o h y d . Res. , 16 (1 971) 2 20. J . Comtat and F. Barn'oud, C R . A c a d . S c i . P a r i s , 277 (19 7 3 ) 6 1 . ~ A . J . R o u d i e r and L E b e r h a r d , B u l l . Soc. Chim. F r . (1960) 2074. A . J . R o u d i e r , C R . Acad.. S c i . ( P a r i s ) , 237 (1953) 840. C O . A s p i n a l l and K.C.B. W i l k i e , J . Chem. Soc. (1956)1072. G.G.S. D u t t o n and-'.-M.S. K a b i r , Phy to c h e m i s t r y , 11 ( 1972) 779. J.K. H a m i l t o n , E.V. P a r t l o w and N.S. Thompson, T a p p i , 41 (1958) 803 . ^ ~ as 14 T.E. T i m e l l , Methods i n C a r b o h y d . Chem., 5_ ( 1965) 134 . D.W. C l a y t o n , Svensk P a p p e r s t i d n . , 66 (1963) 1 15. G.G.S. D u t t o n , B . I . J o s e l e a u and P.E. R e i d , T a p p i , 56 (1973) 168. ~ A. B e e l i k , R.J,. Con c a , J.K. H a m i l t o n and E.V. P a r t l o w , T a p p i , 50 (1967) 78. J . E . S c o t t , Methods Biochem. A n a l . , 8 (1960) 145. - 1 4 8 -H.O. Bouveng and B. L i n d b e r g , A c t a Chem. S c a n d . , 1_2_ ( 1 9 5 8 ) 1 9 7 7 . D.M.W. A n d e r s o n , I.M. Dea, P.A, Maggs and A.C. Munro, C a r b o h y d . R e s . , , 5 ( 1 9 6 7 ) 4 8 9 . C.W. Mc C l e a r y , D.A. Rees, J.W.B. Samuel and I.W. S t e e l e , C a r b o h y d . Res . , 5_ ( 1 9 6 7 ) 4 9 2 . B. L i n d b e r g , J . L o n n e g r e n and J . L . Thompson, C a r b o h y d . Res 2 8 ( 1 9 7 3 ) 3 5 1 . J . K i s s , A d v a n ces i n C a r b o h y d . Chem. and Biochem., 29_, ( 1 9 7 4 ) 2 2 9 . V.D. Harwood, Svensk P a p p e r s t i d n , 7_6 ( 1 9 7 3 ) 3 7 7 . G.G.S. D u t t o n and N.A. F u n n e l l , Can. J . Chem., 5 1 ( 1 9 7 3 ) 3 1 9 0 . R.H. W a l k e r , M a s t e r Sc. T h e s i s ( 1 9 7 1 ) , U n i v e r s i t y o f B r i t i s h C o l u m b i a - V a n c o u v e r - C a n a d a . G.A. Adams, Can. J . Chem., 39, ( 1 9 6 1 ) 2 4 2 3 . G. A. H e n d e r s o n and G.W. Hay, C a r b o h y d . Res., 2 3 ( 1 9 7 2 ) 3 7 9 N. Abdurahman, G.G.S. D u t t o n , D.M. Mc L a r d y , K.E. P i e r r e and B . I . S t e p h e n s o n , T a p p i , 47_ ( 1 9 6 4 ) 8 1 2 . T.E. T i m e l l , Svensk P a p p e r s t i d n . 1 5 ( 1 9 6 0 ) 4 7 2 . J.K. R o g e r s and N.S. Thompson, Sv e n s k P a p p e r s t i d n , 3_, ( 1 9 6 3 ) 6 1 . H. M e i e r , A c t a Chem. S c a n d . , 1 4 ( 1 9 6 0 ) 7 4 9 . G.G.S. D u t t o n and R.H. W a l k e r , C e l l u l o s e Chem. T e c h n o l . , £ ( 1 9 7 2 ) 2 9 5 . J.K. H a m i l t o n , E.V. P a r t l o w and N.S. Thompson, J . Am. Chem. Soc . , 8 2 ( I 9 6 0 ) 4 5 1 . J . Corn-tat, K. R u e l , J .P. J o s e l e a u and F. B a r n o u d , "Symposium on E n z y m a t i c H y d r o l y s i s o f C e l l u l o s e " , H e l s i n k i March 1 9 7 5 . J . P . T h o r n d e r and D.H. N o r t h c o t e , B i o c h e m . J . , 8 1 ( 1 9 6 1 ) . 4 4 9 . M. M a r x - F i g i n i , N a t u r e , 2 J . 0 ( 1 9 6 6 ) 7 5 4 . J.P. Thornder. and D.H. Nor t h c o t e , Biochem. J . , 8 2 (1962) 3 4 0 '. r ~ ' -149-J.A. Monro, R.W. B a i l e y and P. Penny, Pay tochemi :-. r.rv, 1 1 ( 1 9 7 2 ) 1 5 9 7 . ' G. F r a n z . P1 an ta__( Ber l i n ) , 102 ( 1972) 334 . J . S . G . R e i d and K.C.B. W i l k i e , P h y t o c h c m i s L r y , 8_ (1969) 205 9. A . J . B u c h a l a and K.C.B. W i l k i e , Phy to ch e mir. t r y , 1 2 (1972) 499. F. B a r n o u d and J . P . J o s e l e a u , P l a n t Self-nee L e t t e r s , 4 (1975) 169. J.P. J o s e l e a u and F. B a r n o u d , P h y t o c h e r a i s t r y , 14 ( 1975) 71 . ' M.A. C h r y s l e r , B o t . Gaz., 41 (1906) 16. D.M.W. A n d e r s o n , P.C. B e l l and H.A. K i n g , C a r b o h y d . Res., 22 (1972) 453. K.W. Talmadge, K. K e e g s t r a , W.D. B a u e r and P. A l b c r s h e i m , P l a n t P h y s i o l . , 51 (1973) 166. H. M e i e r , A c t a Chem. S c a n d . , 15 (1961) 1381. M. Z i n b o and T.E. T i m e l l , Svensk P a p p e r s t i d n , 68 (1965) 647 . G. O. A s p i n a l l , i n " B i o g e n e s i s o f P l a n t C e l l W a l l P o l y s a -c c h a r y d e s " , F» Loewus e d . , (1973) 95. H. M e i e r , J . Polym. S c i . , 51 (1961) 11. D.J. N e v i n s , P.D. E n g l i s h and P. A l b e r s h e i m , P l a n t  P h y s i o l . , 43 (1968) 914. J . Comtat and J . P . J o s e l e a u , C e l l . Chem. T a c h n o l . , 7 (1973) 653. C M . S t e w a r t , C e l l . Chem. T e c h n o l . , 1_ ( 1973) 639 . J . Comtat, J.P. J o s e l e a u , C. B o s s o and F. B a r n o u d , C a r b o h y d . Res. 38 ( 1974) 2 17.-R. W a i t e and A.R.N. G o r r o d , J . S c i . Food A g r i c , 10 (1959) 308. A . J . B u c h a l a and K.C.B. W i l k i e , P h y t o c h e m i s t r y , 12 ( 1973) 65 5 . H. Kauss and W.E. Hess i d , J_.__ J3jLol_._j:hei,>. , 242 ( 1967) 1680 K.G. R o s e l l and S. S v e n s s o n , Carbohyd._Res.,42_ (1975) 297 / / PUBLICATIONS -F. Barnoud, T. Higuchi, J.P. Joseleau and A. Mollard. CR. Acad. Sci., T. 259, 3589 (1964) Nature des lignines synthetisees dans les vegetaux cultives in vitro - Etudes des produits de degradation. F. Barnoud, T. Higuchi and J.P. Joseleau. CR. Acad. Sci., T. 259, 4110 (1964) Nature des lignines dans les vegetaux cultives i n vitro - Etude par spectro-scopic dans 1'infra-rouge. F. Barnoud, T. Higuchi, J.P. Joseleau and A. Mollard. CR. Acad. Sci., 259, 4339 (1964) La bio synthese des lignines dans les tissus vegetaux cultives i n vitro - Role de precurseur de l a L-phenylalanine. F. Barnoud, G.G.S. Dutton and J.P. Joseleau. Carbohyd. Res., 27, 215 (1973) La xylanne du roseau Arundo  donax. J. Comtat and J.P. Joseleau. C e l l . Chem. Technol. 7_> 653 (1973) Mise en evidence de 4-0^-methyl-D-glucose forme dans l a 4-0-methyl-glucuronoxylanne extraite de Populus apres reduction in situ. J.P. Joseleau and F. Barnoud. Phytochemistry, 13, 1155 (1974) Hemicelluloses of young internodes of Arundo  donax. J. Comtat, J.P. Joseleau, C Bosso and F. Barnoud. Car-bohyd. Res., 38, 217 (1974) Characterization of structurally similar neutral and acidic tetra-saccharides i n the hydrolysate of a glucuronoxylan. J.P. Joseleau and F. Barnoud. Phytochemistry, 14, 71 (1975) Hemicelluloses of Arundo donax at different stages of maturity. F. Barnoud and J.P. Joseleau. Plant Science Letters, 4., 169 (1975) Changes of the c e l l wall carbohydrates i n the internodes of Arundo donax at different stages of growth. J. Comtat, K. Ruel, J.P. Joseleau and F. Barnoud. Sym-posium on Enzymatic Hydrolysis of Cellulose, M. Bailey, T.M. Enari, M. Linko, Editors (1975) 351 Purification of a xylanase - Its mode of action on xylans and on fibrous tissues. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0061081/manifest

Comment

Related Items