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Some structural features of sapota achras gum Kabir, Mohammad Shahjahan 1971

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OME STRUCTURAL FEATURES OF SAP'OTA' ACHRAS GUM By SHAHJAHAE KABIR .Sc.?The U n i v e r s i t y of B r i t i s h Columbia,1970 THESIS SUBMITTED IH PARTIAL FULFILMENT OP THE REQUIREMENTS POR THE DEGREE OP DOCTOR OP PHILOSOPHY i n the Department of CHEMISTRY We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OP BRITISH COLUMBIA August, 1971 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 The University of Bri t ish Columbia Vancouver 8, Canada Date - i i -TABLE OF CONTENTS Page I n t r o d u c t i o n 1 Object of the pr-esent i n v e s t i g a t i o n 8 D i s c u s s i o n of the methods A. F r a c t i o n a t i o n on d i e t h y l a r a i n o e t h y l ( D S A S ) - c e l l u l o s e 12 B. F r a c t i o n a l p r e c i p i t a t i o n of propionate 13 C. M e t h y l a t i o n s t u d i e s 14-D. G a s - l i q u i d chromatography 18 E. A u t o h y d r o l y s i s 22 F. P e r i o d a t e o x i d a t i o n 24-G. Reduction of the u r o n i c a c i d s 28 K. A c e t o l y s i s 31 I . Hofmann degr a d a t i o n of p o l y s a c c h a r i d e s 32 J . Mass spectrometry 34-R e s u l t s and d i s c u s s i o n F r a c t i o n a t i o n on DEAE—cellulose 4-6 F r a c t i o n a l p r e c i p i t a t i o n of propionate 45 'Total a c i d h y d r o l y s i s 4-6 M e t h y l a t i o n s t u d i e s 4-8 A u t o h y d r o l y s i s 55 P e r i o d a t e o x i d a t i o n 59 Simultaneous e s t i m a t i o n of u r o n i c a c i d s by g a s - l i q u i d chromatography 69 - i i i -T t •rage P e r i o d a t e o x i d a t i o n of carboxyl-reduced p o l y s a c c h a r i d e 73> A c e t o l y s i s 77 Hofmann deg r a d a t i o n 87 Enzymatic degradation 91 Mass spectrometry 95 A p a r t i a l s t r u c t u r e of sapote gum 112 Experimental P u r i f i c a t i o n of the gum 116 Determination of e q u i v a l e n t weight 115 P r o p i o n a t i o n of sapote gum 117 F r a c t i o n a t i o n of the p o l y s a c c h a r i d e propionate 117 P r e p a r a t i o n of DEAE-cellulose column 118 M o l e c u l a r weight d e t e r m i n a t i o n of sapote gum 120 E s t i m a t i o n of sugar by p h e n o l - s u l p h u r i c a c i d method 121 Determination of formaldehyde 125 T o t a l a c i d h y d r o l y s i s of sapote gum 125 'Thin layex* chromatography of a l d o b i o u x o n i c a c i d s 127 M e t h y l a t i o n of sapote gum 126 P r e p a r a t i o n of m e t h y l s u l p h i n y l anion 128 - i v -Generation of the p o l y s a c c h a r i d e a l k o x i d e M e t h y l a t i o n r e a c t i o n H y d r o l y s i s of the methylated sapote gum S e p a r a t i o n of a c i d i c and n e u t r a l components S e p a r a t i o n of n e u t r a l components of methylated n e u t r a l sugars by paper chromatography C h a r a c t e r i s a t i o n of components S e p a r a t i o n of the n e u t r a l components of methylated n e u t r a l sugars by c e l l u l o s e - c o l u m n chromatography C h a r a c t e r i s a t i o n of components A n a l y s i s of the a c i d i c f r a c t i o n s G a s - l i q u i d chromatography of the methylated n e u t r a l sugars A u t o h y d r o l y s i s of sapote gum M e t h y l a t i o n of the degraded gum H y d r o l y s i s of the methylated degraded gum _ v -• Page R e d u c t i on of "the methylated degraded gum 15^ *" P e r i o d a t e uptake of sapote gum 158 Smith degradation 159 I d e n t i f i c a t i o n of the separated components 160 Carboxyl r e d u c t i o n of sapote gum 172 P e r i o d a t e uptake of the carboxyl-reduced p o l y s a c c h a r i d e 177 A c e t o l y s i s of the carboxyl-reduced sapote gum 181 Charcoal column chromatography of the d e a c e t y l a t e d m a t e r i a l 182 P r e p a r a t i o n of the p o l y s a c c h a r i d e amide 191 Cleavage of the p o l y s a c c h a r i d e amide 191 M e t h y l a t i o n of the Hofmann degraded p o l y s a c c h a r i d e 192 Gel f i l t r a t i o n of Hofmann d i f i u s a t e s 195 Enzymatic d e g r a d a t i o n of sapote gum 197 References 199 - v i -LIST OF FIGURES Fi g u r e Page 1 A h y p o t h e t i c a l r e p e a t i n g u n i t of sapote gum 4-2 A l d o t r i o u r o n i c a c i d s i s o l a t e d from sapote gum 5 5 S t e r i c hindrance by s u b s t i t u e n t at C-3 i n borohydride r e d u c t i o n 20 4- Smith de g r a d a t i o n of the x y l a n framework of a p o l y s a c c h a r i d e 26 5 Hof mann degradation of a p o l y s a c c h a r i d e amide 32 6 Flow diagram f o r the a u t o h y d r o l y s i s of sapote gum 37 7 P e r i o d a t e o x i d a t i o n of x y l o s y l - g l y c e r o l 62 8 P e r i o d a t e o x i d a t i o n of x y l o b i o s y l - g l y c e r o l 64 9 Flow diagram f o r the r e d u c t i o n of sapote gum 70 10 Plow diagram f o r the a c e t o l y s i s of the carboxyl-reduced p o l y s a c c h a r i d e 78 11 C h a r a c t e r i s a t i o n of D-Glu 1—^ -2 D-xylose 80 12 C h a r a c t e r i s a t i o n of D-Glu 1---2 D-xylose-1---4 D-xylose 83 P 15 Enzymatic degradation of wheat f l o u r a r a b i n o x y l a n 94-- v i i -F i g u r e Page 14- Mass s p e c t r u m o f 1 , 4 — d i - 0 - a c e t y l - 2 , 3 , 5 -t r i - O - m e t h y l - L - a r a b i n i t o l 96 15 S o u r c e o f t h e most i m p o r t a n t i o n s o f 1,4— d i - 0 - a c e t y l - 2 , 3 , 5 - t r i - O - m e t h y l - L -a r a b i n i t o l 97 16 Mass s p e c t r u m o f 1 , 5 - d i - 0 - a c e t y l - 2 , 3 , 4 — t r i - O - m e t h y l - D - x y l i t o l 98 17 S o u r c e o f t h e most i m p o r t a n t i o n s o f 1 , 5 - d i - 0 - a c e t y l - 2 , 3 , 4 - t r i - 0 - m e t h y l - D -x y l i t o l 99 18 Mass s p e c t r u m o f 1 , 4 - , 5 - t r i - 0 - a c e t y l - 2 , 3 -d i - O - m e t h y l - D - x y l i t o l 100 19 S o u r c e o f t h e most i m p o r t a n t i o n s o f 1 , 4 - , 5 - t r i - 0 - a c e t y l - 2 , 3 - d i - 0 - m e t h y l - D -x y l i t o l 101 20 Mass s p e c t r u m o f m e t h y l 3 , 4 — d i - O - m e t h y l -D - g l u c o s i d e 103 21 S o u r c e o f t h e most i m p o r t a n t i o n o f m e t h y l 3,4— d i - O - m e t h y l - D - g l u c o s i d e 104-22 Mass s p e c t r u m o f m e t h y l 2 ,3 ,4— t r i - 0 -r n e t h y l - p - D - g l u c o s i d e 105 23 S o u r c e o f t h e most i m p o r t a n t i o n s o f m e t h y l 2 , 3 , 4 - t r i - O - m e t h y l - f - B -g l u c o s i d e 106 - v i i i -F i g u r e Page 24- Pass spectrum of x y l o s y l - g l y c e r o l 108 2 5 Mass spectrum of t r i m e t h y l s i l y l d e r i v -a t i v e of 3-0-rnethyl-D-xylose 1 1 0 26 O r i g i n of the most important i o n s of t r i m e t h y l s i l y l d e r i v a t i v e of 5-0-methyl-D-xylose 1 1 1 2 7 A p o s s i b l e p a r t i a l s t r u c t u r e of sapote gum 113 28 Standard curve f o r p h e n o l - s u l p h u r i c a c i d r e a c t i o n of x y l o s e 1 2 2 2 9 Standard curve f o r formaldehyde by the chromotropic a c i d method 124-3 0 S e p a r a t i o n of methyl g l y c o s i d e s of p a r t i a l l y methylated sugars by g a s - l i q u i d chromatography ( g . l . c . ) * 14-6 31 S e p a r a t i o n of methylated a l d i t o l a c e t a t e s by g . l . c . 148 3 2 I n f r a r e d a b s o r p t i o n spectrum of methylated degraded gum 1 5 2 3 3 I n f r a r e d a b s o r p t i o n spectrum of c a r b o x y l -reduced methylated degraded gum 1 5 5 3ZI- S e p a r a t i o n of methylated a l d i t o l a c e t a t e s from methylated c a r b o x y l reduced degraded gum 1 5 7 - i x -F i g u r e Page 55 S e p a r a t i o n of t r i m e t h y l s i l y l d e r i v a t i v e of x y l o s y l - g l y c e r o l by g . l . c . 16-'; 56 S e p a r a t i o n of a l d i t o l a c e t a t e s of L-arab-i n o s e , D-xylose, D-glucose and 4 - 0-methyl-D-glucose by g . l . c . 174-57 S e p a r a t i o n of a l d i t o l - a c e t a t e s of 4-0-methyl-D-glucose and g a l a c t o s e by g . l . c . 175 38 S e p a r a t i o n of products of p e r i o d a t e o x i d i z e d c a r b o x y l - r e d u c e d sapote gum 179 39 S e p a r a t i o n of methanolyzed products of a methylated t r i s a c c h a r i d e by g . l . c . 189 40 S e p a r a t i o n of methanolysed products of a methylated t e t r a s a c c h a r i d e by g . l . c . 190 41 S e p a r a t i o n of a l d i t o l a c e t a t e s of p a r t i a l l y methylated monosaccharides from methylated Hofmann degraded p o l y s a c c h a r i d e 194 42 E l u t i o n p a t t e r n f o r the i s o l a t i o n of an o l i g o s a c c h a r i d e on Sephadex G-10 196 - X -LI80?. OF TABLES Table Page 1 Percent r e d u c t i o n of the f u n c t i o n a l groups on a l g i n i c a c i d by d i f f e r e n t r e d u c t i o n procedures 29 2 A n a l y t i c a l data f o r p u r i f i e d sapote gum 45 3 Approximate molar r a t i o s from p a r t i a l l y methylated monosaccharides from methylated sapote gum 54-4 P r o p e r t i e s of the degraded sapote gum 55 5 Examination of g l y c e r o l - g l y c o s i d e s 68 6 Simultaneous e s t i m a t i o n of p o l y o l s and sugars 73 7 C h a r a c t e r i s a t i o n of o l i g o s a c c h a r i d e s ( i - i v ) 86 8 Gas chromatography of methylated sugar d e r i v a t i v e s 86 9 F r a c t i o n a l p r e c i p i t a t i o n of sapote gum p r o p i o n a t e 118 10 S e p a r a t i o n of n e u t r a l components of methylated sapote gum by paper chromatography 131 11 S e p a r a t i o n of n e u t r a l sugars of methylated sapote gum by c e l l u l o s e - c o l u m n chroma-tography 137 - x i -Table Page 12 P e r i o d a t e uptake of sapote gum p o l y s a c c h a r i d e 158 13 Column chromatographic s e p a r a t i o n of Smith-degraded p o l y o l 162 14 P e r i o d a t e uptake of the c a r b o x y l -reduced p o l y s a c c h a r i d e 177 - X l l -ABSTRACT Sapote gum p o l y s a c c h a r i d e was examined f o r homogeneity by ion-exchange chromatography on d i e t h y l a m i n o e t h y l ( D E A E ) - c e l l u l o s e and by f r a c t i o n a l p r e c i p i t a t i o n of i t s p r o p i o n a t e d e r i v a t i v e . The p o l y s a c c h a r i d e showed e s s e n t i a l homogeneity and was t h e r e f o r e s u i t a b l e f o r s t r u c t u r a l a n a l y s i s . The p o l y s a c c h a r i d e showed a low n e g a t i v e s p e c i f i c r o t a t i o n and c o n t a i n e d r e s i d u e s of L-arabinose, D-xylose, D-glucuronic and 4-O-methyl-D-glucuronic a c i d s . A new method was developed f o r the simultaneous e s t i m a t i o n of 4-0-methyl-D-glucuronic a c i d and other u r o n i c a c i d s by g a s - l i q u i d chromatography. Uronic a c i d s i n p o l y s a c c h a r i d e s may be reduced by r e a c t i o n w i t h l i t h i u m borohydride i n t e t r a h y d r o f u r a n . A f t e r h y d r o l y s i s and r e d u c t i o n of the monosaccharides the a c e t y l a t e d a l d i t o l s may be separated by g a s - l i q u i d chromatography on a column of b u t a n e d i o l s u c c i n a t e . The method permits the simultaneous e s t i m a t i o n of 4-0-methyl-D-glucuronic a c i d , D-glucuronic a c i d and D - g a l a c t u r o n i c a c i d s . I t was found by t h i s method t h a t L - a r a b i n o s e , D-xylose, D-glucuronic and 4-0-methyl-D-g l u c u r o n i c a c i d s were present i n the r a t i o of 1:2.8:0.46:0. An examination of the O - m e t h y l - d e r i v a t i v e of the gum y i e l d e d 2,5,5- (+++) and 2,3,4- (+)- 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 - 0 - , 2,3- (+)- d i - 0 - , 3-0- (+++)-- x i i i -methyl-D-xylose, 3 , 4 - ( - 1 - + ) - di-O-methyl-D-glucuronic and 2 ,3 ,4— ( - ) - - : - ) - t r i - O - m e t h y l - D - g l u c u r o n i c a c i d . The m e t h y l a t i o n data i n d i c a t e s the h i g h degree of branching i n the p o l y s a c c h a r i d e . The presence of 3 , 4 - d i - O - m e t h y l -D-glucuronic a c i d a l s o i n d i c a t e s t h a t a p a r t of the t o t a l u r o n i c a c i d s i s branched at C - 2 . A u t o h y d r o l y s i s of the gum y i e l d e d a s e r i e s of o l i g o u r o n i c a c i d s and a l e s s e r branched p o l y s a c c h a r i d e . The p o l y -s a c c h a r i d e a f t e r m e t h y l a t i o n , r e d u c t i o n and h y d r o l y s i s a f f o r d e d 2 , 3 , 4 - t r i - O - m e t h y l , 2 , 3 - d i - 0 - m e t h y l , 3 -0-methyl-D-x y l o s e and 2 , 3 , 4 ~ t r i - 0 - m e t h y l - D - g l u c o s e i n a molar r a t i o of 2 : 1 . 3 : 1: 2 i n d i c a t i n g t h a t the degraded p o l y -s a c c h a r i d e has a x y l o s e backbone. Sapote gum p o l y s a c c h a r i d e a f t e r s u c c e s s i v e p e r i o d a t e o x i d a t i o n , borohydride r e d u c t i o n and m i l d a c i d h y d r o l y s i s y i e l d e d a s e r i e s of homologous g l y c o s i d e s , ( x y l o s y l ) 0 -g l y c e r o l , which were f u l l y i d e n t i f i e d by m e t h y l a t i o n and p e r i o d a t e o x i d a t i o n . The occurrence of these g l y c o s i d e s s i g n i f i e s a random branching i n the p o l y s a c c h a r i d e . P e r i o d a t e o x i d a t i o n and subsequent complete a c i d h y d r o l y s i s of the c a r b o x y l reduced p o l y s a c c h a r i d e a f f o r d e d ethylene g l y c o l , g l y c e r o l , 2 - 0 - m e t h y l - D - e r y t h r i t o l , D-xylose and 4 - 0-methyl-D-glucose whose molar r a t i o s were determined by g a s - l i q u i d chromatography ( g . l . c ) . The presence of 4 - 0-methyl_D-glucose i n d i c a t e s t h a t a major p a r t . o f 4 - 0 -- x i v -methyl-D-glucuronic a c i d i s branched at C-2 (a c o n c l u -s i o n supported by the m e t h y l a t i o n data) and 2-0-methyl-D - e r y t h r i t o l a r i s e s from a minor p a r t of 4-0-methyl-D-g l u c u r o n i c a c i d w i t h no s u b s t i t u e n t at C-2. P a r t i a l a c e t o l y s i s of the carboxyl-reduced p o l y s a c c h a r i d a f f o r d e d a new s e r i e s of o l i g o s a c c h a r i d e s 2-0-<<-D-gluco-p y r a n o s y l - ( 4 - 0 - f J - D - x y l o s y l ) n Q ^ 2 ~ xJ^-ose a n c i 2-0-4-0-methyl-D-glucopyranosyl -D-xylose. The r e s u l t s c o n f i r m the presence of the o l i g o u r o n i c a c i d s i n the p o l y s a c c h a r i d e . A new method f o r the s e l e c t i v e cleavage of g l y c o s i d u -r o n i c a c i d s , Ilofmann. d e g r a d a t i o n , was a p p l i e d to sapote gum i n order to study the r e s u l t i n g degraded p o l y s a c c h a r i d e and to f i n d out the nature of the s u b s t i t u e n t on 4-0-methyl-D-glucuronic a c i d . The degraded p o l y s a c c h a r i d e a f t e r m e t h y l a t i o n and h y d r o l y s i s a f f o r d e d 2,3>4-tri-0-methyl-, 2,3-di-0-methyl-, and mono-O-methyl-D-xylose i n a molar r a t i o of 3: 1 1 : 2. An o l i g o s a c c h a r i d e o r i g i n a l l y a t t a c h e d at C-2 of 4-0-methyl-D-p;lucuronic a c i d was i s o l a t e d . D i g e s t i o n of the p o l y s a c c h a r i d e w i t h a r a b i n o f u r a n o s i d a s e y i e l d e d f r e e arabinose i n d i c a t i n g t h a t a major p a r t of the t e r m i n a l non-reducing arabinose u n i t s are i n the furanose form and l i n k e d w i t h x y l o s e i n 1 c < 1 c o n f i g u r a t i o n Some enzymes w i t h known 'xylanase' a c t i v i t y d i d not degrade - X V -the gum because of the h i g h degree of branching of the p o l y s a c c h a r i d e . The. s t r u c t u r a l evidence suggests t h a t sapote gum p o l y s a c c h a r i d e possesses a h i g h l y branched x y l a n framework to which are a t t a c h e d L-arabinose u n i t s , D-glucuronic a c i d and 4-0-methyl-D-glucuronic a c i d c o n t a i n i n g s i d e c h a i n s . I n t r o d u c t i o n . a ) H i s t o r i c a l background. Sapote gum i s a v a i l a b l e i n Peru where i t i s c o l l e c t e d from Sapote t r e e s which grow i n a semi-t r o p i c a l climate- near the equator'at an e l e v a t i o n of 3 , 0 0 0 f e e t . The t r e e s are tapped f o r l a t e x from which c h i c l e i s prepared. The gum s l o w l y forms i n the wounds a f t e r the f l o w of l a t e x ceases.- The gum i s used as a s i z i n g agent f o r c l o t h and f e l t , as a glue and has been t e s t e d as a beat e r a d d i t i v e i n the manufacture of paper. The e a r l i e s t study on the composition of sapote gum was c a r r i e d out by Anderson and L e d b e t t e r ( 1 ) . These, authors found t h a t sapote gum was a mixture of 9 0 - 9 4 $ water s o l u b l e a c i d i c p o l y s a c c h a r i d e , and 6—10$ water s o l u b l e r e s i n * The two substances were separated by d i s s o l v i n g the p o l y u r o n i d e i n hot water and f i l t e r i n g o f f the r e s i n . . The gum c o n s i s t e d of D-xylose, L - a r a b i n o s e , D-glucuronic a c i d and one methoxyl group was present f o r every 2 u r o n i c a c i d u n i t s and 7 pentose u n i t s . The two pentoses were present i n a r a t i o of 1:8.-5 (L-arabinose and D-xylose r e s p e c t i v e l y ) . The gum was p u r i f i e d by p r e c i p i t a t i n g i t t w i c e from an a c i d i c aqueous s o l u t i o n w i t h a l c o h o l . The composition of the p u r i f i e d gum as o b t a i n e d by Anderson and L e d b e t t e r (1) i s g i v e n i n Table 2 . Although these authors c o u l d not i d e n t i f y the u r o n i c a c i d , they e s t a b l i s h e d the presence of a methoxyl group e t h e r l i n k e d to some of the u r o n i c a c i d r e s i d u e s by the methods of Deniges ( 2 ) and von F e l l e n b e r g ( 3 ) . D-Xylose and L-arabinose were i s o l a t e d from the a c i d h y d r o l y s a t e of the gum and i d e n t i f i e d . No hexose sugar was i d e n t i f i e d i n the a c i d h y d r o l y s a t e of the gum. Anderson and L e d b e t t e r (1) a l s o found t h a t p a r t i a l a c i d h y d r o l y s i s y i e l d e d a mixture of a l d o b i o u r o n i c and a l d o t r i o u r o n i c a c i d s which c o n t a i n r e s i d u e s of D - g l u c o p y r a n o s y l u r o n i c acid.and a monomethyl ether t h e r e o f , l i n k e d to xylopyranose units.-The s t r u c t u r e of sapote gum has been s t u d i e d by White ( 4 , 5 , 6 ) u s i n g m e t h y l a t i o n techniques., M e t h y l a t i o n was c a r r i e d out by u s i n g d i m e t h y l sulphate and sodium hydroxide.. Upon meth a n o l y s i s , methylated sapote gum y i e l d e d as the g l y c o s i d e s , 2 , 3 , 4 — t r i - O -methyl-D-xylose, 2 , 3 , 4 - t r i - O - m e t h y l - L - a r a b i n o s e , 3 - 0-methyl-D-xylose and an a c i d f r a c t i o n . The l a t t e r c o n t a i n e d , 5,4-di-O-methyl-D-glucuronic a c i d i d e n t i f i e d by the r e d u c t i o n of i t s methyl e s t e r methyl g l y c o s i d e w i t h l i t h i u m aluminium hydride to the methyl g l y c o s i d e of 5,4-di-O-methyl-jJ-glucose and a l s o 2 , 3 , 4 -t r i - O - r a e t h y l - D — g l u c u r o n i c a c i d i d e n t i f i e d as 2,3,4— t r i - O — m e t h y l D-glucose. The two methylated d e r i v a t i v e s of u r o n i c a c i d s are each g l y c o s i d i c a l l y l i n k e d to 3-0-methyl-D-xylose.. A q u a n t i t a t i v e a n a l y s i s of the compo s i t i o n of methylated sapote gum was c a r r i e d out by s u b j e c t i n g the gum to p a r t i a l methanolysis and r e d u c i n g the r e s u l t i n g mixture w i t h l i t h i u m aluminium h y d r i d e . The reduced m a t e r i a l was h y d r o l y s e d to the corresponding r e d u c i n g sugars. The mixture of methylated sugars, was found (6) t o c o n s i s t of 3-0-methyI-D-xylose (3 moles) 3,4~di-0-raethyl-D-glucuronic a c i d , determined as 3,4— di-O-methyl—D-glucose (1 mole), 2,3, 4 r - t r i - 0 - m e t h y l -D-glucuronic a c i d , determined as 2 ,3,4—tri-O-methy1-D-glucose (1 mole), 2,3,4— tri - O - m e t h y l - D - x y l o s e (1 mole), and 2 , 3,4~tri-O-methyl-L-arabinose (1 mole). From the above r e s u l t s , White (6) a p p l i e d the concept of the r e p e a t i n g u n i t r e g a r d i n g the s t r u c t u r e of sapote gum and drew the f o l l o w i n g c o n c l u s i o n s z, i j The main c h a i n probably c o n s i s t s of (1—4-) l i n k e d xylopyranose u n i t s . i i ) I n an average r e p e a t i n g u n i t of seven sugar r e s i d u e s , 2 , 3,4—tri-O-methyl-L-arabinose,2 , 3,4— t r i - O - m e t h y l - D - x y l o s e and 2 , 3,4—tri-O-methyl-D-g l u c u r o n i c a c i d r e s i d u e s are terminal.- i i i ) Between the two u r o n i c a c i d s one i s nonterminal and i s l i n k e d - 4- -F i g u r e 1 . A h y p o t h e t i c a l r e p e a t i n g u n i t of sapote gum - 5 -O H ACID (A) : 4 - 0 - M E T l l Y L - D - G L U C O P V R A N O S Y L U R O N I C ACID ( l - £ ^ - 2 ) D - X Y L O P Y R A N O S E ( l - ^ - 4 ) D - X Y L O P Y R A N O S E O H A C I D ( B ) : D-GLUCOPYRANOSYLURONI IC ACID ( 1 - ^ * 2 ) D - X Y L O P Y R A N O S E (l--—<» 4) D - X Y L O P Y R A N O S E ' Figure 2 . A l d o t r i o u r o n i c acids i s o l a t e d from sapote gum through £-1 and C-2 i n a s i d e c h a i n of • r e p e a t i n g u n i t , i v ) C o n s i d e r i n g the methoxyl v a l u e of the o r i g i n a l p o l y s a c c h a r i d e , approximately h a l f of u r o n i c a c i d s bear a methoxyl group.. Cn the b a s i s of the above i n f o r m a t i o n , Smith and Montgomery (7) have assigned a s t r u c t u r a l formula f o r a r e p e a t i n g u n i t of gum as shown i n F i g . 1. Although White (6) found t h a t the u r o n i c a c i d u n i t s i n sapote gum are u n i t s of D-glucuronic a c i d and t h a t they are j o i n e d bya g l y c o s y l bond to C-2 of D-xylose u n i t s , the c o n f i g u r a t i o n of t h i s bond v/as not determined and the exact l o c a t i o n of the methoxyl group had not been assigned.. A l s o , i t was not u n e q u i v o c a l l y proved t h a t D-xylose u n i t s were j o i n e d by a g l y c o s y l (1—^-4) bond. In 1c}68, Lambert, Dickey and Thompson (8) r e p o r t e d the i s o l a t i o n and i d e n t i f i c a t i o n of some of the a c i d i c o l i g o s a c c h a r i d e s i n a h y d r o l y s a t e of sapote gum... The gum gave two s e r i e s of a c i d i c fragments c o n t a i n i n g 4-0-methyl-D-glucuronic a c i d and" D-glucuronic a c i d r e s p e c t i v e l y ( F i g - 2 ) . Two a l d o t r i o u r o n i c a c i d s , 4-O-methyl-D-glucopyranosyluronic a c i d (1-^—2) D-xylopyranose (1-|~4) D-xylopyranose (A) and D-gluco-p y r a n o s y l u r o n i c a c i d (1--—2) D-xylopyranose (1-^-4) D-xylopyranose (B) were i s o l a t e d by paper chromato-grphy. ' The i d e n t i f i c a t i o n of A was c a r r i e d out by - 7 -comparison w i t h an a u t h e n t i c sample. The a c i d (3) on p a r t i a l a c i d h y d r o l y s i s .yielded D-glucopyranosyl-u r o n i c a c i d (1---2)-J-xylopyranose which was i d e n t i f i e d as the c r y s t a l l i n e a c e t y l a t e d methyl e s t e r methyl g l y c o -s i d e . The two a c i d s (A) and (B) had s i m i l a r s p e c i f i c r o t a t i o n s i n d i c a t i n g t h a t the x y l o s e t o x y l o s e bond of the a c i d was i n the {J - L V c o n f i g u r a t i o n . From the above r e s u l t s Lambert et a l (8) made the f o l l o w i n g d e d uctions: i ) The methoxyl group i s at C-4 of the u r o n i c a c i d r e s i d u e s , i i ) A l l of the u r o n i c a c i d r e s i d u e s p r e s e n t i n the gum are p r o b a b l y e i t h e r D-glucuronic or 4~0-methyl-D-glucuronic a c i d and the bond between u r o n i c a c i d and the x y l o s e backbone i s i n ^ n e c < - D - c o n f i g u r a t i o n . - 8 -b) Object of the present i n v e s t i g a t i o n : X y l o s e c o n t a i n i n g p l a n t gums are not at a l l common i n n a t u r e . Only tv/o gums are known t o have x y l o s e as the p r i n c i p a l sugar component. They are known as sapote gum and brea gum ( 9 ) . The s t r u c t u r e of sapote gum has on l y been s t u d i e d by m e t h y l a t i o n and many f i n e p o i n t s i n the s t r u c t u r e have not been e l u c i d a t e d . From the m e t h y l a t i o n d a t a of V&ite (4,5,6), Smith and Montgomery ( 7 ) have proposed a r e p e a t i n g u n i t s t r u c t u r e , of sapote gum as shown i n F i g . 1 . L a t e r when Lambert e t a l (8) s t u d i e d the u r o n i c a c i d s of sapote gum, a doubt was. c a s t on the r e p e a t i n g u n i t concept of sapote gum. These authors, however, d i d not pursue "the matter f u r t h e r and l i m i t e d t h e i r i n -v e s t i g a t i o n to the nature of the tv/o a l d o t r i o u r o n i c a c i d s obtained from sapote gum. I t appears t o the present i n v e s t i g a t o r t h a t a few que s t i o n s ought to be answered r e g a r d i n g the s t r u c t u r e of sapote gum p o l y s a c c h a r i d e . These are as f o l l o w s : i ) Whether the gum has the same r e p e a t i n g u n i t s t r u c t u r e as proposed by Smith (7) on the b a s i s of Wlii t e 1 s r e s u l t s ? . i i ) I t appears t h a t u r o n i c a c i d s are branched... This type of branching i s not very common. Which of - 9 -the u r o n i c a c i d s or whether both of them are branched? I f so, what i s the nature of the s u b s t i t u e n t ? i i i ) The r a t i o s between d i f f e r e n t monosaccharide u n i t s i n sapote gum as found by the pr e v i o u s workers do not agree w i t h one another. What i s then the molar r a t i o between the d i f f e r e n t sugar u n i t s ? i v ) . The gum has a h i g h degree of branch i n g . What i s the nature of t h i s branching? In recent y e a r s , there has been a very c o n s i d e r a b l e development i n methods of s t r u c t u r a l , p o l y s a c c h a r i d e chemistry.. M e t h y l a t i o n , a conventional, method f o r s t r u c t u r a l a n a l y s i s of p o l y s a c c h a r i d e s , has been made more convenient due to improvements i n procedure (10) and g a s - l i q u i d chromatography has become a r o u t i n e o p e r a t i o n f o r s e p a r a t i n g p a r t i a l l y methylated mono-sacc h a r i d e d e r i v a t i v e s , and other sugar d e r i v a t i v e s . Mass spectrometry 01,12,13,14,15,16,17) has been s u c c e s s f u l l y a p p l i e d f o r the i d e n t i f i c a t i o n of the v a r i o u s sugar d e r i v a t i v e s . . A p p l i c a t i o n of the Smith degradation (a sequence of r e a c t i o n s i n v o l v i n g p e r i o d a t e o x i d a t i o n , borohydride r e d u c t i o n and m i l d a c i d h y d r o l y s i s ) to p o l y s a c c h a r i d e s has pr o v i d e d v a l u a b l e i n f o r m a t i o n on the f i n e s t r u c t u r e of complex p o l y s a c c h a r i d e s ( 1 5 ) , S t r u c t u r a l m o d i f i c a t i o n i . e . , r e d u c t i o n of u r o n i c a c i d s to hydroxymethyl groups b r i n g s a marked change i n - 1 0 -the p r o p e r t y of the polymer ( , ' 1 9 , 2 0 , 2 1 , 2 2 , 2 $ ) . S e l e c t i v e cleavage., of u r o n i c acid's, by Hoffman degradation o f f e r s a very good o p p o r t u n i t y to analyse a c i d i c p o l y s a c c h a r i d e s (24). Except m e t h y l a t i o n and p a r t i a l , .acid h y d r o l y s i s none of the methods has.been a p p l i e d on sapote gum. Moreover p a r t i a l l y methylated monosaccharides, had to be i s o l a t e d by White (.4) by f r a c t i o n a l d i s t i l l a t i o n , because d u r i n g the p e r i o d of i n v e s t i g a t i o n a n a l y t i c a l techniques were not so w e l l developed, as they are now. This i s probably the main reason t h a t White c o u l d not o b t a i n some of the b a s i c i n f o r m a t i o n i n h i s i n v e s t i g a t i o n . . Hence, one of the; main o b j e c t s of the present i n v e s t i g a t i o n i s to analyse the s t r u c t u r e of sapote gum a p p l y i n g these r e c e n t chemical methods and experimental t e c h n i q u e s . DISCUSSION OF THE METHODS - 12 -A. F r a c t i o n a t i o n on d i e t h y l a m i n o e t h y l (DEAE)-celluIose  columns: The p u r i f i c a t i o n and f r a c t i o n a t i o n of p o l y s a c c h a r i d e s i n t o more or l e s s homogeneous i n d i v i d u a l components have been one of the most important and d i f f i c u l t steps i n p o l y s a c c h a r i d e chemistry. F r a c t i o n a t i o n has been accomplished by f r a c t i o n a l p r e c i p i t a t i o n i n organic s o l v e n t s w i t h ' o r without the a d d i t i o n of i n o r g a n i c s a l t . The ion-exchanger, D E A E - c e l l u l o s e , has been used i n s e p a r a t i n g mixtures of p r o t e i n s , n u c l e i c a c i d s and p o l y s a c c h a r i d e s . A c i d i c p o l y s a c c h a r i d e s are r e a d i l y adsorbed on the DEA E - c e l l u l o s e columns at pH near 6 and are e l u t e d , depending on t h e i r content of a c i d i c groups, (a) by i n c r e a s i n g the b u f f e r c o n c e n t r a t i o n at the same pH ( o n l y f o r weakly a c i d i c p o l y s a c c h a r i d e s ) , (b) by a l k a l i n e s o l u t i o n s of i n c r e a s i n g s t r e n g t h , ( c ) by a c i d i c s o l u t i o n s of i n c r e a s i n g s t r e n g t h . The a d s o r p t i o n of p o l y s a c c h a r i d e s on DEAE - c e l l u l o s e i s s t r o n g l y i n f l u e n c e d by 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 s t o be f r a c t i o n a t e d . I n g e n e r a l , the a d s o r p t i o n on DEAE-cellulose i s enhanced w i t h increasing-amounts of a c i d i c groups. - 13 -3. F r a c t i o n a l p r e c i p i t a t i o n of sapote gum p r o p i o n a t e : P o l y s a c c h a r i d e s t h a t are d i f f i c u l t to separate by complex f o r m a t i o n or by f r a c t i o n a l p r e c i p i t a t i o n from aqueous s o l u t i o n s w i t h a l c o h o l , because of very c l o s e s i m i l i a r i t y of s t r u c t u r e or a s s o c i a t i o n by hydrogen bonding can sometimes be f r a c t i o n a t e d a f t e r a c e t y l a t i o n or p r o p i o n a t i o n , f o r by t h i s means the h y d r o x y l c h a r a c t e r i s o b l i t e r a t e d and there i s much l e s s tendency for. m o l e c u l a r a s s o c i a t i o n . This method has been a p p l i e d to p u r i f y many p o l y s a c c h a r i d e s . Thus, f o r example, c e r e a l pentosan has been separated from contaminating glucans such as s t a r c h , s i n c e the pentosan a c e t a t e s are much l e s s s o l u b l e than the hexosan a c e t a t e s (25). A f t e r r e s o l u t i o n of the p o l y s a c c h a r i d e i n t h i s way, the a c e t y l or the p r o p i o n y l group can be r e a d i l y s a p o n i f i e d " and the p o l y s a c c h a r i d e recovered unchanged. This method can a l s o serve as a t e s t f o r the homogeneity of the p o l y s a c c h a r i d e . - 14- -G . M e t h y l a t i o n s t u d i e s : M e t h y l a t i o n a n a l y s i s i s an important method i n the s t r u c t u r e d e t e r m i n a t i o n of p o l y s a c c h a r i d e s . The purpose of the m e t h y l a t i o n i s to achieve an e t h e r -i f i c a t i o n of a l l of the f r e e h y d r o x y l groups i n the p o l y s a c c h a r i d e . A. f u l l y methylated p o l y s a c c h a r i d e on t o t a l a c i d h y d r o l y s i s y i e l d s a mixture of monomeric methylated sugars which are then separated, i d e n t i f i e d and q u a n t i t a t i v e l y e s t i m a t e d . The p o s i t i o n s of the g l y c o s i d i c l i n k a g e s i n the p o l y s a c c h a r i d e molecule correspond to the p o s i t i o n s of u n s u b s t i t u t e d h y d r o x y l groups i n these methylated sugars. However, a methyl-a t i o n study does not p r o v i d e i n f o r m a t i o n on the r e l a t i v e order of the sugar r e s i d u e s or on t h e i r anomeric n a t u r e . The procedures f o r methyl e t h e r f o r m a t i o n are l i m i t e d . i n scope. The m e t h y l a t i n g agents used are e i t h e r d i m e t h y l sulphate or methyl i o d i d e ; the former u s u a l l y i n c o n j u n c t i o n w i t h a s t r o n g a l k a l i , which serves to promote i o n i z a t i o n of the p e r t i n e n t h y d r o x y l f u n c t i o n , and the l a t t e r w i t h an agent such as s i l v e r o x i d e . However, these m e t h y l a t i n g agents are not always very e f f e c t i v e , because the o p e r a t i o n has to be repeated s e v e r a l times before a complete m e t h y l a t i o n i s achieved. An e f f e c t i v e and r a p i d m e t h y l a t i o n technique has - 15 -been developed by Hakomori ( 1 0 ) , wherein the methyl s u l p h i n y l anion (26, 27) i s used to generate the p o l y s a c c h a r i d e a l k o x i d e p r i o r to the a d d i t i o n of methyl i o d i d e . Dimethyl sulphoxide i s used as the s o l v e n t wherein m e t h y l a t i o n r e a c t i o n s proceed at f a v o u r a b l e r a t e s under r e l a t i v e l y m i l d c o n d i t i o n s . A h i g h degree of m e t h y l a t i o n i s achieved i n one treatment. R e a c t i o n sequence can be shown as f o l l o w s : 0 I 1. CH 7 S CH 3 + NaH * 0 _> GIL, S£ Na h + H, '2 r 2. ROH + CE 3 0 b s>- i'i a JJL RO' ia + L5 ¥ cw 3 . KG Na + + CK 7I 5 ROCK + K a l Sodium m e t h y l s u l p h i n y l c a r b a n i o n i s u s u a l l y ana most c o n v e n i e n t l y prepared by h e a t i n g f i n e l y powdered sodium hydride i n dime t h y l sulphoxide under n i t r o g e n at 7 0 ° - 80° f o r one hour, or u n t i l hydrogen e v o l u t i o n ceases. Longer p e r i o d s of h e a t i n g , or h i g h e r temperatures - 16 -cause s i g n i f i c a n t (28), at times t o t a l decomposition of the carban i o n (29). S o l u t i o n s of m e t h y l s u l p h i n y l c a r b a n i o n are r e l a t i v e l y s t a b l e at room temperature and l o s e o n l y approximately By> of t h e i r a c t i v i t y per vie ok ( 3 0 ) . Anion c o n c e n t r a t i o n s may be determined by t i t r a t i o n s w i t h f o r m a n i l i d e u s i n g t r i p h e n y l methane as i n d i c a t o r ( 28). The i o n i s a t i o n of the h y d r o x y l groups i n the p o l y -s a c c h a r i d e i s a co m p a r a t i v e l y slow r e a c t i o n ( 1 0 ) . The p o l y s a c c h a r i d e i s t h e r e f o r e l e f t i n c o n t a c t w i t h d i m e t h y l s u l p h i n y l anion f o r a p e r i o d of at l e a s t f o u r hours before the methyl i o d i d e i s added. R e a c t i o n of a l k o x i d e i o n s w i t h methyl i o d i d e i s presumably much f a s t e r . The base a l s o consumes methyl i o d i d e . I f the m e t h y l a t i o n I s not complete i n one s t e p , i t i s a d v i s a b l e not t o remethylate by the Hakomori method ( 1 0 ) . A .few s i d e r e a c t i o n s might cause c o m p l i c a t i o n s d u r i n g r e m e t h y l a t i o n . The r e a c t i o n of m e t h y l s u l p h i n y l c a r b a n i o n w i t h e s t e r s a f f o r d s ^ - k e t o Q s u l p h o x i d e s (28). i R-C-OR + CH-.-S-GH" v R-G-CH0-S-CH., + R 0 II P || 2 > n 2 5 0 o 0 CH-.-3-CH0 'J II 2 0 R-C-CII0-S-CH.. H + R-C-Cfl-S-Cl „ 2 „ p < „ - | 0 0 0 0 - 17 -An e s t e r i f i e d u r o n i c a c i d r e s i d u e might a l s o undergo ^ - e l i m i n a t i o n i n the s t r o n g l y b a s i c medium y i e l d i n g an u n s a t u r a t e d r e s i d u e (31) As f u l l y methylated p o l y s a c c h a r i d e s are g e n e r a l l y i n s o l u b l e i n hot water, methanolysis ( b o i l i n g w i t h methanolic hydrogen c h l o r i d e ) , f o r m o l y s i s ( b o i l i n g w i t h 9G> f o r m i c a c i d ) or treatment w i t h 72# s u l p h u r i c a c i d at room temperature i s u s u a l l y performed before hydro-l y s i n g w i t h hot d i l u t e a c i d . A comparison has been made by L i n d b e r g (32) c o n s i d e r i n g the extent of d e m e t h y l a t i o n and d e g r a d a t i o n o c c u r r i n g i n s e v e r a l types of h y d r o l y t i c procedures. H y d r o c h l o r i c a c i d , both aqueous and methanolic, caused c o n s i d e r a b l y more deme t h y l a t i o n than e i t h e r f o r m o l y s i s or treatment w i t h 72/5 s u l p h u r i c a c i d f o l l o w e d by h y d r o l y s i s i n d i l u t e s u l p h u r i c a c i d . - 18 -D. G a s - l i q u i d chromatography: G a s - l i q u i d chromatography i s an i n d i s p e n s a b l e t o o l i n the a n a l y s i s of carbohydrate d e r i v a t i v e s i n g e n e r a l and p a r t i a l l y methylated d e r i v a t i v e s i n p a r t i -c u l a r . I n the present i n v e s t i g a t i o n of p a r t i a l l y methylated monosaccharide u n i t s o b t a i n e d from the methylated p o l y s a c c h a r i d e s three types of d e r i v a t i v e s ( a l d i t o l a c e t a t e s , methyl g l y c o s i d e s and 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 ) have been used. The most w i d e l y used d e r i v a t i v e s were a l d i t o l a c e t a t e s of p a r t i a l l y methylated monosaccharides. A l d i t o l a c e t a t e s of p a r t i a l l y methylated mono-sa c c h a r i d e s on g a s - l i q u i d chromatography g i v e s i n g l e peaks f o r each component. This i s because of the fact-t h a t r e d u c t i o n of sugars to a l d i t o l s w i t h borohydride removes the p o s s i b i l i t y of a n o m e r i z a t i o n and a c e t y l a t i o n of f r e e h y d r o x y l groups g i v e s a v o l a t i l e d e r i v a t i v e capable of being r e s o l v e d . The s e p a r a t i o n was c a r r i e d out on a column c o n s i s t i n g of a y - i l i q u i d phase of an o r g a n o s i l i c o n e p o l y e s t e r of ethylene g l y c o l s u c c i n a t e chercics.lly combined w i t h a s i l i c o n e of the cyanoethyl type. I t was found t h a t s e p a r a t i o n s of d i f f e r e n t components were b e t t e r achieved and s o l v e n t t a i l i n g e f f e c t was g r e a t l y reduced i f the columns were h e l d i s o t h e r m a l l y at 160 f o r 3 minutes and then programmed at 2° per minute to h o l d at 180°, r a t h e r than i s o t h e r -m a l l y at 180°. The s e p a r a t i o n curves were found to be q u i t e r e p r o d u c i b l e . The time r e q u i r e d f o r r e d u c t i o n of p a r t i a l l y methylat sugars w i t h sodium borohydride r e q u i r e s b r i e f comment. Although L i n d b e r g (33) has r e p o r t e d a r e d u c t i o n p e r i o d of 2 hours f o r borohydride r e d u c t i o n of p a r t i a l l y methylated sugars, i t was found i n an e a r l y i n v e s t i g a t i o n by the author (34) t h a t a s h o r t e r r e d u c t i o n time gave r i s e t o many peaks on the chromatogram which was probably due t o incomplete r e d u c t i o n . Hence a l o n g e r r e d u c t i o n time ( c a . 12 hours) was-employed f o r the p r e p a r a t i o n of p a r t i a l l y methylated a l d i t o l a c e t a t e s . This obser-v a t i o n i s i n agreement w i t h a r e p o r t of Bragg and Hough (35) on q u a n t i t a t i v e aspects of borohydride r e d u c t i o n of carbohydrates. The r e d u c t i o n of an aldose to a g l y c i t o l would be preceded by r i n g opening of the c y c l i c m o d i f i c a t i o n t o the aldose of the aldehydo-form i n the a c y c l i c staggered z i z - z a g conformation. A bulky s u b s t i t u e n t at C-3 of t h i s conformation causes the approach of borohydride i o n to the aldehyde group to be s t e r i c a l l y h i n d e r e d . This i s i l l u s t r a t e d i n P i g . 3« Although t r i m e t h y l s i l y l d e r i v a t i z a t i o n i s simple - 2 0 -F i g u r e 3 S t e r i c hindrance by s u b s t i t u e n t at C-5 i n borohydride r e d u c t i o n . - 21 -and r a p i d , i t s u f f e r s from c e r t a i n shortcomings. In s o l u t i o n a sugar may e x i s t as an e q u i l i b r i u m mixture of the anomers of the furanose and pyranose forms. Hence each monosaccharide can giv e r i s e to as many as f o u r d e r i v a t i v e s d u r i n g i t s c o n v e r s i o n t o 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 . These d e r i v a t i v e s , which are formed through anomeric and r i n g i s o m e r i z a t i o n , . each produce a peak on the chromatogram. Thus a complex mixture of carbohydrates c o u l d g i v e a m u l t i p l i c i t y of bands which would make t o t a l r e s o l u t i o n q u i t e c o m p l i c a t e d . However, i n c e r t a i n cases, 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 p a r t i a l l y methylated sugars have one advantage over c o r r e s p o n d i n g p a r t i a l l y methylated a l d i t o l a c e t a t e s . A l d i t o l a c e t a t e s of 2 - 0-methyl, and J-O-methyl-D-xyloses c o u l d not be separated s i n c e they have i d e n t i c a l r e t e n t i o n tirae on a g a s - l i q u i d chromatogram ( 3 6 ). The anomeric p a i r s of 2 - 0-methyl and 3 - 0 - m e t b y l - 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 x y l o s e s are w e l l separable from each other when examined by g a s - l i q u i d chromatography ( 3 7 ) . Hence i d e n t i f i c a t i o n s and q u a n t i t a t i v e e s t i m a t i o n s of mono-O-methyl x.yloses i n a mixture can be accomplished by t h i s procedure. - 2 2 -E. A u t o b y d r o l y s i s Many p o l y s a c c h a r i d e s c o n t a i n d i f f e r e n t g l y c o s i d i c l i n k a g e s which are h y d r o l y s e d at d i f f e r e n t r a t e s (38). For example, f u r a n o s i d e s are h y d r o l y s e d much f a s t e r than the p y r a n o s i d e s . Again, g l y c o s i d u r o n i c a c i d l i n k a g e s ( i . e . , l i n k a g e s i n which u r o n i c a c i d s are g l y c o s i d i c a l l y l i n k e d to n e u t r a l sugar u n i t s ) s u r v i v e c o n d i t i o n s of h y d r o l y s i s which cause o t h e r g l y c o s i d i c l i n k a g e s to break down. I f a p o l y s a c c h a r i d e c o n t a i n i n g the v a r i o u s g l y c o s i d i c l i n k a g e s i s s u b j e c t e d to the a c t i o n of d i l u t e (0.01-0.1N) m i n e r a l a c i d , the a c i d - l a b i l e sugar r e s i d u e s L-arabofuranose, L-rhamnopyranose and 3,6-anhydro-D- and L- g a l a c t o s e undergo p r e f e r e n t i a l cleavage. I f the p o l y s a c c h a r i d e gum c o n t a i n s u r o n i c a c i d u n i t s , the f r e e gum a c i d may undergo a u t o h y d r o l y s i s when i t s aqueous s o l u t i o n s are heated, there being a s u f f i c i e n t c o n c e n t r a t i o n of hydrogen i o n s from the c a r b o x y l groups (39, 40). The advantage of t h i s method i s t h a t the degraded molecules are l e s s c o m p l i c a t e d and more amenable to c o n s t i t u t i o n a l s t u d i e s . A f t e r removal of the l a b i l e r e s i d u e s , i t i s g e n e r a l l y found t h a t the r e s i d u a l p o r t i o n or p o r t i o n s of the molecule are r e l a t i v e l y s t a b l e to a c i d i c reagents and much more v i g o r o u s - 23 -treatment i s necessary to e f f e c t complete h y d r o l y s i s , This s t a b i l i t y may a r i s e from the f a c t t h a t the r e s i d u e s , which compose the s t a b l e s e c t i o n s of the p o l y s a c c h a r i d e , are of pyranose or s i x membered r i n g type or from the presence of u r o n i c a c i d r e s i d u e s which c o n f e r s t a b i l i t y e s p e c i a l l y on those l i n k a g e s which j o i n C-1 of the u r o n i c a c i d s t o other sugar r e s i d u e s . I f there are no l a b i l e r e s i d u e s i n the p o l y s a c c h a r i d e , m i l d a c i d treatment i s without e f f e c t on a gum molecule. - 24 -I'1. P e r i o d a t e o x i d a t i o n : In 1928, Halaprade found t h a t p e r i o d i c a c i d and i t s s a l t s q u a n t i t a t i v e l y c l e a v e d the carbon-carbon bond of 1,2 d i o l s (41): R 2C(0H)CR 20H + H^IO^ > R2C=0 +EIO^+ 2H 20 L a t e r P l e u r y (42) showed t h a t the h y d r o x y l groups had to be on adjacent carbon atoms, and e s t a b l i s h e d p e r i o d i c a c i d and i t s s a l t s as u s e f u l a n a l y t i c a l . reagents.. G - OH - CHO | + I 0z). > + + I 0 3 + G - OH CHO Compounds c o n t a i n i n g three or more h y d r o x y l group£ on adjacent carbon atoms are o x i d i z e d by n-1 moles of p e r i o d a t e , where n i s the number of adjacent h y d r o x y l groups, and are c l e a v e d to y i e l d two aldehyde groups and n-2 moles of f o r m i c a c i d . The formic a c i d may be es t i m a t e d by t i t r a t i o n and can provide i n f o r m a t i o n about the arrangements of h y d r o x y l groups. - 25 -P e r i o d a t e o x i d a t i o n s are q u a n t i t a t i v e and reasonably f a s t at room temperature. They c o u l d be c a r r i e d out over a wide range of pH-. P e r i o d a t e i o n c o u l d be e s t i m a t e d i n the presence of i o d a t e i o n s and o ther r e a c t i o n p r o d u c t s , because i t o x i d i s e s i o d i d e i o n to i o d i n e i n n e u t r a l and m i l d l y a l k a l i n e s o l u t i o n . . P e r i o d a t e o x i d a t i o n i s a 2 e l e c t r o n o x i d a t i o n r e a c t i o n , r e q u i r i n g one molecule of p e r i o d a t e i n which the i o d i n e atom i s reduced from the +7 to the +5 v a l e n c y s t a t e w i t h the f o r m a t i o n of i o d a t e i o n s . As e a r l y as 1933, a mechanism f o r p e r i o d a t e o x i d a t i o n of g l y c o l s suggested by Criegee (43) i n v o l v e d the f o r m a t i o n of a c y c l i c p e r i o d a t e complex. The evidence c o n s i s t s of d i r e c t s p e c t r o p h o t o m e t r y o b s e r v a t i o n s on the complexes (44,45) and more e x t e n s i v e l y , of k i n e t i c evidence (46,47,48). A l s o a study of the s t e r e o c h e m i c a l requirements f o r d i o l . cleavage by p e r i o d a t e has g i v e n p o w e r f u l support to t h i s h y p o t h e s i s (49). The r a t e of p e r i o d a t e o x i d a t i o n i s s t r o n g l y dependent on the d i h e d r a l angle between the two h y d r o x y l groups. A c c o r d i n l y , compounds w i t h v i c i n a l c i s - h y d r o x y l . groups are o x i d i s e d more r a p i d l y than those w i t h t r a n s - h y d r o x y l groups (49). F i g u r e 4 . Smith de g r a d a t i o n of the x y l a n framework of a p o l y s a c c h a r i d e - 27 -P e r i o d a t e o x i d a t i o n has been employed to a great extent i n the s t r u c t u r a l s t u d i e s of p o l y s a c c h a r i d e s . The procedure i n v o l v e s p e r i o d a t e o x i d a t i o n of the polymer, r e d u c t i o n of the newly formed aldehyde groups to a l c o h o l s w i t h sodium bor o h y d r i d e , a c i d h y d r o l y s i s of the r e s u l t i n g p o l y o l , and i d e n t i f i c a t i o n of the p r o d u c t s . Because of the marked d i f f e r e n c e i n s t a b i l i t y between t r u e a c e t a l s and g l y c o s i d e s , i t i s p o s s i b l e by m i l d a c i d h y d r o l y s i s to .cleave the a c e t a l l i n k a g e s i n p o l y a l c o h o l s and to leave any g l y c o s i d i c l i n k a g e s i n t a c t . Those u n i t s which have been c l e a v e d by p e r i o d a t e are t r u e a c e t a l s and ve r y s e n s i t i v e to a c i d , whereas any u n i t which has not been o x i d i z e d by p e r i o d a t e and i s j o i n e d t o a c l e a v e d u n i t appears as a g l y c o s i d e which i s r e l a t i v e l y s t a b l e to a c i d . This p r i n c i p l e i s i l l u s t r a t e d i n P i g . 4. A p p l i c a t i o n of t h i s technique to the complex p o l y s a c c h a -r i d e s has p r o v i d e d v a l u a b l e i n f o r m a t i o n on the r e l a t i v e p o s i t i o n of d i f f e r e n t u n i t s and d i f f e r e n t types of l i n k a g e s i n the macromolecule (18). - 28 -G. Reduction of the u r o n i c a c i d s : S t r u c t u r a l m o d i f i c a t i o n s can be a p p l i e d to o b t a i n new i n f o r m a t i o n about the complex p o l y s a c c h a r i d e s such as gums. One of these methods i s the r e d u c t i o n of the c a r b o x y l groups (-COOH) to the hydroxymethyl (-CHo0R) groups. There are d i f f e r e n t methods f o r the r e d u c t i o n of u r o n i c a c i d s . Manning and Green ( 5 0 ) have compared the e f f i c i e n c y of the d i f f e r e n t r e d u c i n g methods on a l g i n i c o.cid polymers. The f l o w diagram f o r the r e d u c t i o n of a l g i n polymers i s i l l u s t r a t e d below. Di-O-propionyl alginic acid I I I dibc I diazomethane diborane I gent w w win si Methyl di-O-propionyl alginate D l D2 I lithium I lithium I borohydride I borohydride Bl B2 Flow diagram for the reduction of algin polymers orane eratcd Thus a p o l y s a c c h a r i d e c o n t a i n i n g u r o n i c a c i d s can i ) be a c y l a t e d or p r o p i o n y l a t e d and then e s t e r i f i e d , and the l a t t e r may be reduced w i t h l i t h i u m borohydride ( 5 0 ) ; i i ) be a c y l a t e d and reduced w i t h diborane generated i n s i t u ( 5 1 ) ; i i i ) be e s t e r i f i e d w i t h propylene oxide and then reduced w i t h l i t h i u m borohydride ( 5 2 ) ; i v ) be - 29 -s i l y l a t e d and then reduced w i t h l i t h i u m borohydride ( 5 3 ) . Percent r e d u c t i o n of the o r i g i n a l f u n c t i o n a l groups as expressed by Manning and Green (50) are quoted i n Table 1 . Table 1 Percent r e d u c t i o n of the f u n c t i o n a l groups on a l g i n i c a c i d by d i f f e r e n t r e d u c t i o n procedures Reduced p o l y s a c c a r i d e s „ , , . „ . . , B1 B2 P/1 D2 Reauction of u r o n i c a c i d / • / \ ^3 5 -5 3 9 . 0 8 5 . 7 carooxy-groups (>o) Reduction of e s t e r i f i e d u r o n i c a c i d carboxy-groups (#) 100 Reductive cleavage of 1 Q 0 ? 4 ^ n - p r o p i o n y l e s t e r ($) Reduction of n - p r o p i o n y l e s t e r to n-propyl ether CO 0 . 0 0 .0 52 18 Prom Table 1 i t i s q u i t e evident t h a t there i s a marked d i f f e r e n c e between r e d u c t i o n w i t h a Lev/is base l i t h i u m borohydride, and w i t h a Lewis a c i d , diborane The r e l a t i v e r e a c t i v i t y of d i - O - p r o p i o n y l a l g i n i c a c i d towards diborane, a Lewis a c i d , i s : u r o n i c a c i d carboxy-g r o u p > n - p r o p i o n y l e s t e r group. In c o n t r a s t , towards l i t h i u m borohydride, a Lev/is base, the order i s r e v e r s e d n-propicmyl e s t e r group > u r o n i c a c i d carboxy group. The f a c t t h a t the u r o n i c a c i d carboxy group i s the most r e a c t i v e f u n c t i o n a l group toward diborane and the l e a s t r e a c t i v e f u n c t i o n a l group toward l i t h i u m borohydride i s e x p l a i n e d i n terms of e l e c t r o p h i l i c i t y . A u r o n i c a c i d carboxy group, w i t h a centre of h i g h e l e c t r o n d e n s i t y w i l l a t t r a c t a s t r o n g e l e c t r o p h i l e such as diborane and r e p e l a n u c l e o p h i l e such as boro h y d r i d e . On the c o n t r a r y , the r e d u c t i o n w i t h borohydride i n v o l v e s t r a n s f e r of the hy d r i d e i o n to the e l e c t r o n d e f i c i e n t c e n t r e of the f u n c t i o n a l group. Diborane r e d u c t i o n has disadvantages of e t h e r f o r m a t i o n and incomplete r e d u c t i o n of the u r o n i c a c i d carboxy group. In order to achieve the complete r e d u c t i o n of an a c i d i c p o l y s a c c h a r i d e , the l i t h i u m borohydride procedure has been recommended (50) and t h method does not i n v o l v e i n the f o r m a t i o n of any ether l i n k a g e s . - 31 -H. A c e t o l y s i s The d e p o l y m e r i z a t i o n of p o l y s a c c h a r i d e s w i t h reagents such as a c e t i c a n h y d r i d e - a c e t i c a c i d - s u l p h u r i c a c i d has been known f o r almost a century (54-). A c e t o l y s i s of p o l y s a c c h a r i d e s at lev/ temperature y i e l d s a c e t y l a t e d o l i g o s a c c h a r i d e s ; these may be d e a c e t y l a t e d w i t h methanolic sodium methoxide, before or a f t e r f r a c t i o n a t i o n , to y i e l d f r e e sugars. The u s e f u l n e s s of a c e t o l y s i s i s t h a t , as a method, i t i s complementary to a c i d h y d r o l y s i s . The major i n t e r e s t i n a c e t o l y s i s , however, l i e s i n i t s d i f f e r e n t s p e c i f i c i t y , as compared w i t h m i n e r a l a c i d , towards the g l y c o s i d i c l i n k a g e s of the u n s u b s t i t u t e d p o l y s a c c h a r i d e s . Thus, whereas (1 6 ) - l i n k a g e s between hexose r e s i d u e s are the most r e s i s t a n t to a c i d h y d r o l y s i s , t h i s type of l i n k a g e .is r a p i d l y c l e a v e d by a c e t o l y s i s . B a l l o u (55) has s t u d i e d the p a r t i a l a c e t o l y s i s of yeast mannans and o b t a i n e d h i g h e r o l i g o s a c c h a r i d e s c o n t a i n i n g mannose. This i s a very good method f o r the i s o l a t i o n of h i g h e r o l i g o s a c c h a r i d e ' s . - 32 -I . Hoffman de g r a d a t i o n of p o l y s a c c h a r i d e s : There are many processes a v a i l a b l e f o r the s e l e c t -i v e f r a g m e n t a t i o n of biopolymers such as p o l y p e p t i d e s , p r o t e i n s and s o l u b l e r i b o n u c l e i c a c i d s (24). Unfortun-a t e l y , t h e r e are not too many methods a v a i l a b l e f o r the s e l e c t i v e d e g r a d a t i o n of p o l y s a c c h a r i d e chains probably because of the complex s t r u c t u r e of these polymers. R e c e n t l y , t h e r e have been r e p o r t s of methods f o r the s e l e c t i v e f r a g m e n t a t i o n of p o l y s a c c h a r i d e s ( 5 6 , 5 7 } 5 8 , 5 9 ) • Such methods are of i n t e r e s t and importance, s i n c e they c o n s t i t u t e a new stage i n s t u d i e s of the s t r u c t u r e of p o l y s a c c h a r i d e s and conjugated biopolymers. One of these methods i s the a p p l i c a t i o n of Hoffman d e g r a d a t i o n t o p o l y s a c c h a r i d e a c i d amides. This method i n v o l v e s the co n v e r s i o n of u r o n i c a c i d r e s i d u e s t o amides f o l l o w e d by t h e i r t r a n s f o r m a t i o n i n t o 5-amiriopentopyranoses by Hoffman r e a c t i o n . The l a t t e r compounds which are s t r u c t u r a l l y analogous to g l y c o s y l amines, are h y d r o l y s e d by a c i d under very m i l d c o n d i t i o n s . F i g u r e 3» The Hoffman degradation of a p o l y s a c c h a r i d e a c i d amide + R O H + N H , + - 33 -Kochetkov e t . a l . , (57) f i r s t s t u d i e d t h i s r e a c t i o n on model compounds such as methyl and (-)-menthyl-D-glucopyranosiduronamide. The p e n t o d i a l d o s e s formed by t h i s r e a c t i o n ( P i g . 5) have been c h a r a c t e r i z e d as p e n t i t o l a c e t a t e s by mass spectrometry f o l l o w i n g t h e i r r e d u c t i o n w i t h borohydride and a c e t y l a t i o n . To prove the v a l i d i t y of the Hofmann rearrangement f o r s t u d y i n g the s t r u c t u r e of p o l y s a c c h a r i d e s c o n t a i n i n g u r o n i c a c i d s i t s a p p l i c a t i o n to b i r c h x y l a n , one of the s i m p l e s t p o l y s a c c h a r i d e s of t h i s type, was s t u d i e d (57). B i r c h x y l a n c o n s i s t s of a l i n e a r c h a i n of ( 1 - ^ - 4)-linked xylopyranose u n i t s . I t c o n t a i n s 11.7$ of u r o n i c a c i d s which are attach e d to the main x y l o s e backbone. A p p l i c a t i o n of Hofmann degradation r e s u l t e d i n the removal of about 94$ of the t e r m i n a l u r o n i c a c i d r e s i d u e s , without e f f e c t i v e h y d r o l y s i s of other g l y c o s i d i c l i n k a g e s . _ 34 -J . Mass s p e c t r o m e t r y : Mass s p e c t r o m e t r y h a s now become an i m p o r t a n t t e c h n i q u e f o r t h e s t r u c t u r a l a n a l y s i s o f c a r b o h y d r a t e d e r i v a t i v e . I m p r o v e d m e t h y l a t i o n p r o c e d u r e s and s u b s e q u e n t s e p a r a t i o n o f p a r t i a l l y m e t h y l a t e d m o n o s a c c -h a r i d e d e r i v a t i v e s by g a s - l i q u i d c h r o m a t o g r a p h y have made t h e m a s s - s p e c t r a l t e c h n i q u e a u s e f u l s u p p l e m e n t t o t h e c h e m i c a l methods f o r t h e a n a l y s i s o f p o l y s a c c h a r i d e s . C a r b o h y d r a t e s b e i n g p r a c t i c a l l y n o n - v o l a t i l e , mass s p e c t r a l s t u d i e s on them have been c a r r i e d o u t on t h e i r more v o l a t i l e d e r i v a t i v e s , s u c h as m e t h y l e t h e r s (11), a c e t a t e s ( 1 2 ) , a l d i t o l a c e t a t e s ( 1 3 ) , t r i m e t h y l s i l y l e t h e r s ( 1 4 ) , t r i f l u o r o a c e t a t e s ( 1 5 ) . A l t h o u g h mass s p e c t r o m e t r y h a s been u s e d i n o r g a n i c c h e m i s t r y f o r m o l e c u l a r w e i g h t d e t e r m i n a t i o n , u n f o r t u n a t e l y o w i n g t o t h e e x t r e m e i n s t a b i l i t y o f t h e c a r b o h y d r a t e m o l e c u l e , t h e m o l e c u l a r i o n ( M + ) c a n h a r d l y be t r a c e d i n t h e mass s p e c t r a o f m o n o s a c c h a r i d e d e r i v a t i v e s . 1 . P a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s : Prom mass s p e c t r a one c a n d i s t i n g u i s h b e t w e e n p r i m a r y and s e c o n d a r y f r a g m e n t s o f a l d i t o l a c e t a t e s . P r i m a r y f r a g m e n t s a r i s e by f i s s i o n be tween two c a r b o n atoms i n t h e a l d i t o l c h a i n ; e i t h e r o f t h e two f r a g m e n t s c o u l d c a r r y t h e p o s i t i v e c h a r g e . The s e c o n d a r y f r a g m e n t s - 35 -are observed from the primary fragments by s i n g l e or c o n s e c u t i v e e l i m i n a t i o n of a c e t i c a c i d ( 6 0), ketene ( 4 2 ) , methanol (32) or formaldehyde (50). The most i n t e n s e s i g n a l i n the mass spectrum of p a r t i a l l y methylated a l d i t o l a c e t a t e s i s g e n e r a l l y m/e 4-3. The a c e t y l i u m i o n i s g e n e r a l l y d e r i v e d from the acetate groups by c<-cleavage. I t i s reasonable to assume t h a t the charge of the molecul a r i o n o b t a i n e d from p a r t i a l l y methylated a l d i t o l a c e t a t e i s e s s e n t i a l l y l o c a t e d on an ether oxygen atom (as i n s t r u c t u r e s 1 and 2) and not on an e s t e r oxygen atom (as i n s t r u c t u r e s 3 and 4-). HC-'0*Me HC-OMe HC-OMe HC-OMe K —> . K —* . HC-OMe HC-OMe HC-OAc H^-OAc 1 2 HC-OMe HC-OMe HC-0 +Ac HC*OAc k t —> K —> . HC-OAc HC-OAc HC-OAc HC-OAc ' 1 I I In agreement w i t h t h i s assumption, the f i s s i o n s i n d i c a t e d f o r 1 and 2 are s i g n i f i c a n t but not those i n d i c a t e d f o r 3 and 4-. F u r t h e r , the f i s s i o n of 1 i s p r e f e r r e d over f i s s i o n of 2 because the methoxyl r a d i c a l - 56 -seems t o be b e t t e r s t a b i l i z e d than the a c e t o x y l r a d i c a l . T h e r e f o r e , i n s t r u c t u r e 5> the cleavage between the methoxylated carbon atoms predominates. HC-OAc H C - O A c KC-OMe . = = > HC-OMe HC-OMe HC-OMe I 1 5 H C - O A c HC*OMe <-I HC-OMe The primary fragment A, m/e 4-5, i s gi v e n by substances having a primary methoxyl group, e.g., a h e x i t o l methy-l a t e d at the C-6 p o s i t i o n and a p e n t i t o l methylated at the C - 3 p o s i t i o n . The primary fragment 3, m/e 59? i s obtained from a 6-deoxyhexitol methylated at the C-5 p o s i t i o n . H 2 C * O M e H p O M c C H 3 A B -m/e AO m/f 59 The primary fragment C, m/e 89» i s obt a i n e d when a primary and a secondary methoxyl group are adjacent ( h e x i t o l s methylated i n the 5- and 6 - p o s i t i o n ; e.g, 2,3,5,6-tetra-O-methyl-D-glucitol), by f i s s i o n between the methoxylated and the a c e t o x y l a t e d carbon atoms (C-5 and C-4). This f i s s i o n - i s favoured over t h a t between the methoxylated carbon atoms (C-5 and C-6) and only has been r e p o r t e d to occur i n the present s i t u a t i o n (16). H 2 C-OMe * " HCftoM. C m/e 89 A secondary fragment c^ , m/e 5 9 , i s formed from 0 by l o s s of formaldehyde. The formaldehyde i s probably favoured by the f o r m a t i o n of an oxironium i o n as i n d i c a t e d below. c ,/e 89 The primary fragment D, m/e 117, i s o b t a i n e d from a l d i t o l d e r i v a t i v e s having an a c e t o x y l group at 0-1 and a methoxyl group at C-2. + HG^OMe D H 2C-OAc „ 7 The primary fragments and F^, m/e 161, would be expected from 1 , 5 - d i - 0 - a c e t y l - 2 , 5 > 4,6-tetra - O-methyl-D - g l u c i t o l by f i s s i o n between C-3 and C-4. - 38 -i 2 3 A 5 6 H,C-OAc HC-OMe I MeO-<jH HC-OMc I H^-OAc H2C-OMe F,, m/e 1G1 F „ m/e 161 H2C-OMe Two secondary fragments, m/e 129 ( f ? ) and m/e 101 ( f ^ ) } are obtained from the primary fragments and r e s p e c t i v e l y . This i s i l l u s t r a t e d below. H^C-O-J-C HC-OMe H-rC-OAc H, m/e 161 H(>0Me MeO-C) 70 H»VSH, HC-OMe C H 2 ^ , C H 2 H <2 m/e 129 HOOMe I C-OMe 1 CH, HC-OMe 0=0 C H 3 «2 m/e 87 r1 m/e 161 'l m/e 101 The secondary fragment, m/e 129 ( f 0 X undergoes an e l i m i n a t i o n of ketene (42) to y i e l d f ' 0 , m/e 87. The primary fragment H, m/e 189, i s d e r i v e d from a l d i t o l s a c e t y l a t e d i n the 1- and 2 - p o s i t i o n s and methylated i n the 3 - p o s i t i o n , e.g., from 1,2,4,5,6-penta O - a c e t y l - 3 - C - m e t h y l - D - g l u c i t o l . The secondary fragment h^, m/e 129, i s formed from H by e l i m i n a t i o n of a c e t i c a c i d . HC-OMe HC-OAc H 2C-OAc ^ " 2 H h 1 m/e 189 ™ ' e 1 2 The primary fragments and K p, m/e 205, are formed f o r example, from 1 , 3-di - 0-acetyl - 2 , 3,4-,6-tetra O- m e t h y l h e x i t o l and 1,4-di-0-acetyl-2,3,5,&-'fce'tra-0-m e t h y l h e x i t o l r e s p e c t i v e l y . HC-OMe H,C-0Me I u HC-OMe =. <>OMe | I HC-OAc HC-OAc H 2 C-OMe H 2 C-OMe K 1 HC"OMe I HC-OAc -OMe HC-C H 2 C-OMe K 2 m/e 205 - 4-0 -probably e x i s t s i n two tautomeric forms and produces a secondary peak, k,j, m/e 14-5, by e l i m i n a t i o n of a c e t i c a c i d . Two secondary fragments, k ^ , m/e 173? and k^, m/e 14-5, are formed from ]L-> by e l i m i n a t i o n of methanol and a c e t i c a c i d , r e s p e c t i v e l y . ii A secondary fragment k 0 , m/e 101, i s observed i n the mass spectrum of 1 , 2 , 4 - - t r i - 0 - a c e t y l - 5 , 5 , 6 - t r i - 0 -m e t h y l - D - g l u c i t o l which a r i s e s due t o the cleavage between C-5 and G-6. HC'^ OMe J> CH + CH-.COOCH-OCH, il KC-OMe m/e 101 HC-OMe H < j ^ x c / C H 3 MeO-CH) MeO-CH 2 m/e 205 i - 4-1 -2. Methyl g l y c o s i d e s of p a r t i a l l y methylated  sugars: Fragmentation p a t t e r n s of methyl g l y c o s i d e s of p a r t i a l l y methylated sugars have been put forward by Kochetkov and Chizhov (11). These authors designate a s e r i e s of i o n s as A, B, C, D, F, J , H, K e t c . A c c o r d i n g to t h a t scheme, 'A' s e r i e s of i o n s are produced by the l o s s of s u b s t i t u e n t s from C-1, w i t h subsequent e l i m i n a t i o n of other s u b s t i t u e n t s . 'B' s e r i e s of i o n s are produced w i t h the e l i m i n a t i o n of C-5 and oxygen as formaldehyde. Ions of the C, D, F, and J s e r i e s are i n i t i a t e d by cleavage of the C-1 to C-2 bond and subse-quent f r a g m e n t a t i o n and d i s t r i b u t i o n of charges on the fragments. A conjugated e l e c t r o n i c s h i f t g i v e s r i s e to i o n s of the K and K s e r i e s , depending on the charge l o c a l i s a t i o n . The o r i g i n of most of these i o n s i s i l l u s t r a t e d i n F i g . 23. 3. 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 sugars: Because of t h e i r ease of p r e p a r a t i o n and v o l a t i l i t y , 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 mono- and d i s a c c h a r i d e s appear to be one of the most s u i t a b l e d e r i v a t i v e s f o r mass s p e c t r o m e t r i c a n a l y s i s . The t x ' i m e t h y l s i l y l group i s not a c o n s i s t e n t d i r e c t o r of c h a r a c t e r i s t i c f r a g m e n t a t i o n and cleavage i s much the same f o r s i l y l as f o r the methyl ethers of monosacc-h a r i d e s (81). - 42 -The molec u l a r i o n s of most s i l y l a t e d compounds are weak or n o n e x i s t e n t . The i o n K-15, r e s u l t i n g from the l o s s of a methyl group from a t r i m e t h y l s i l y l r e s i d u e has the h i g h e s t m/e v a l u e . The peak has a r a t h e r low i n t e n s i t y . The peaks of K-(15+90) and K-(15+90+90) a r i s i n g from the K-15 i o n by s p l i t t i n g of one ( 9 0 m.u.) or two (180 m.u.) t r i m e t h y l s i l a n o l molecules, are more i n t e n s e . The most i n t e n s e peaks i n a l l of the s p e c t r a are those at m/e 204 corresponding t o the i o n (Me^,Si 0-CK=CH-0 3 i l v i e ^ ) + . This i o n o r i g i n a t e s from C-2—C-5 or C - 5 — C-4 of the monosaccharide u n i t . The peak at m/e 1 9 1 present i n the mass s p e c t r a of t r i m e t h y l s i l y l e t h e r s of monosaccharides a l s o has a h i g h + i n t e n s i t y . The corresponding i o n (Me^SiO-CH-OSiKe^) pr o b a b l y a r i s e s due t o rearrangement. Fragments at m/e $05 and 217 are common from 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 carbohydrates. These peaks r e t a i n G-2, C-3 and C-4, mainly of the monosacc-h a r i d e u n i t . OTMoi I • CH=CK CH=CCH"r Cih-CIICir I I I I / I ThSiO 0TM8i TMoiO CTHSi TMoiG 0TM8i m/e 204 m/e 3 0 5 m/e 2 1 7 where TMSi - ( C H 7 ) 7 S i - 4-3 -In the mass s p e c t r a of t r i m e t h y l s i l y l e t h e r s of monosaccharides the f o l l o w i n g i n t e n s e peaks are observed which are a l s o c h a r a c t e r i s t i c of s i l y l a t e d a l c o h o l s ( 8 2 ) . - r + ( C H ^ S i * HO=Si(CH 5) 2 CH^OSiHtCR. ) 2 m/e 73 m/e 73 m/e 8 9 + + CH 2=OSi(CH 5) 5 ( C H 5 ) 2 S i = C 3 i ( C H 5 ) 5 m/e 103 m/e 14-7 RESULTS AND DISCUSSION _ 4-5 -The n a t u r a l gum was obtained as brown nodules. I t was i s o l a t e d by a d d i t i o n of an a c i d i f i e d (pH ca 2.5) aqueous s o l u t i o n of the gum to e t h a i r o l . A p u r i f i e d product was obtained by the repeated a p p l i c a t i o n of the procedure. A n a l y t i c a l data of the p u r i f i e d gum i s r e p o r t e d i n Table 2. Table 2 A n a l y t i c a l data f o r p u r i f i e d sapote gum According Anderson to (1) According to present study S p e c i f i c r o t a t i o n -6° -8.2° E q u i v a l e n t weight &79 67& N i t r o g e n , yi — 0.5 Methoxyl, $ 2.80 2.40 Uronic anhydride, p 27.5 27. 5 B 27-4C a A l l weights are based on ash f r e e s o l i d s k By gas l i q u i d chromatography c By average value determined by d e c a r b o x y l a t i o n of ten nodules chosen at random by Lambert et a l (8) - 46 -F r a c t i o n a t i o n on d i e t h y l a m i n o e t h y l ( D E A E ) - c e l l u l o s e  columns: Samples of sapote gum were examined by ion-exchange chromatography on D E A E - c e l l u l o s e . Most of the m a t e r i a l which was e l u t e d i n one peak had the same p r o p e r t i e s as those of the o r i g i n a l p o l y s a c c h a r i d e . This i n d i c a t e s t h a t the p o l y s a c c h a r i d e under i n v e s t i g a t i o n was homoge-neous. F r a c t i o n a l p r e c i p i t a t i o n of sapote gum p r o p i o n a t e : P r o p i o n a t e d sapote gum, which was e x t e n s i v e l y used f o r the p r e p a r a t i o n of carboxyl-reduced p o l y s a c c h a r i d e and p o l y s a c c h a r i d e amide, was f r a c t i o n a t e d from c h l o r o f o r m s o l u t i o n w i t h petroleum e t h e r . About 90/J of the p o l y -s a c c h a r i d e propionate was p r e c i p i t a t e d at a petroleum ether c o n c e n t r a t i o n of 37-41/J, and t h i s behaviour i s i n d i c a t i v e of e s s e n t i a l homogeneity i n a p o l y s a c c h a r i d e . T o t a l a c i d h y d r o l y s i s : The presence of u r o n i c a c i d r e s i d u e s i n the gum c o n f e r s r e s i s t a n c e t o a c i d h y d r o l y s i s and t h i s method was not s u i t a b l e f o r use i n the q u a n t i t a t i v e a n a l y s i s of the c o n s t i t u e n t sugars; f o r example h y d r o l y s i s w i t h 1 H s u l p h u r i c a c i d ( 8 hours at 100°) gave x y l o s e , arabinose ana a mixture of a c i d i c o l i g o s a c c h a r i d e s . However, the q u a n t i -t a t i v e a n a l y s i s of the c o n s t i t u t e n t sugar was c a r r i e d out - 4 7 -by a c i d h y d r o l y s i s of the carboxyl-reduced p o l y s a c c h a r i d e as d e s c r i b e d l a t e r (page 7 1 ) • The mixture of a c i d i c sugars c o n s i s t e d of D-glucuronic, 4-0-methyl-D-glucuronic, 4-0-methyl-D-glucopyi >anosyluronic a c i d (1 « 2)D-xylopyranose and D-glucopyranosyluronic a c i d ( 1 -- 2 ) D - x y l o p y r a n o s e . The compounds were i d e n t i f i e d by t h e i r o p t i c a l r o t a t i o n s and chromatographic m o b i l i t i e s on paper. Thin l a y e r chromatography of a l d o b i o u r o n i c a c i d s have not been r e p o r t e d i n the l i t e r a t u r e . The two a l d o b i o u r o n i c a c i d s i s o l a t e d from sapote gum were s p o t t e d on t h i n l a y e r s of s i l i c a g e l i n the presence of sodium mono- and dihydrogen phosphate and e x c e l l e n t s e p a r a t i o n was o b t a i n e d . The v a l u e s of the two a l d o b i o u r o n i c a c i d s , D-glucopyranosyl-u r o n i c a c i d ( 1 -- 2 ) D - x y l o p y r a n o s e and 4-0-methyl-D-gluco~ p y r a n o s y l u r o n i c a c i d ( 1 -- 2 ) D - x y l o p y r a n o s e are 0 . 1 5 and 0 . 5 B r e s p e c t i v e l y . - 48 -M e t h y l a t i o n s t u d i e s : M e t h y l a t i o n ox sapote gum p o l y s a c c h a r i d e was c a r r i e d out a c c o r d i n g to the method d e s c r i b e d by Halcomori ( 1 0 ) . R e a c t i o n was found to be complete i n one treatment. Methylated p o l y s a c c h a r i d e was h y d r o l y s e d a c c o r d i n g to the procedure of Li n d b e r g ( 3 2 ) . M i x t u r e s of methylated sugars were separated i n t o the a c i d i c and n e u t r a l components by passage through ion-exchange r e s i n s . The components were analysed by the f o l l o w i n g methods: i ) paper chromatography, i i ) c e l l u l o s e -column chromatography, i i i ) g a s - l i q u i d chromatography and i v ) mass spectrometry. Paper chromatographic examination of the n e u t r a l p o r t i o n s showed the presence of 7 components (Table 1 0 ) . A p a r t of the methylated n e u t r a l sugars was separated on a c e l l u l o s e : h y d r o c e l l u l o s e column u s i n g butanone-water azeotrope as e l u a n t . Component 1 : 2 , 3 , 5 - T r i - O - m e t h y l - L - a r a b i n o s e . — I t was i d e n t i f i e d i ) by i t s chromatographic m o b i l i t y on paper, i i ) by comparing the r e t e n t i o n time of i t s a c e t y l a t e d a l d i t o l d e r i v a t i v e on g a s - l i q u i d chromato-graphy w i t h t h a t of an a u t h e n t i c sample, and i i i ) by the c h a r a c t e r i s t i c mass spectrum of i t s a c e t y l a t e d a l d i t o l d e r i v a t i v e . - 49 -Component 2 : 2 , 3 , 4 — T r i - O - m e t h y l - D - x y l o s e . — I t was i d e n t i f i e d i ) as i t s c r y s t a l l i n e methyl - f-D-g l y c o s i d e , i i ) by comparing the r e t e n t i o n time of i t s a c e t y l a t e d a l d i t o l d e r i v a t i v e on g a s - l i q u i d chromato-graphy, and i i i ) by the c h a r a c t e r i s t i c mass spectrum of the a c e t y l a t e d d e r i v a t i v e . Component :• 2 , 3 , 4 - T r i - O - m e t h y l - L - a r a b i n o s e . — I t was i d e n t i f i e d i ) by q u a l i t a t i v e paper chromatography, i i ) by d e m e t h y l a t i c n r e s u l t i n g i n the f o r m a t i o n of a r a b i n o s e , i i i ) by comparing the r e t e n t i o n time of i t s a c e t y l a t e d a l d i t o l d e r i v a t i v e on g a s - l i q u i d chromatography w i t h t h a t of an a u t h e n t i c sample^and i v ) by the charac-t e r i s t i c mass spectrum of i t s a c e t y l a t e d a l d i t o l d e r i v a t i v e . Component 4 : 2,3-Di-O-methyl-D-xylose.— I t was i d e n t i f i e d i ) by q u a l i t a t i v e paper chromatography > i i ) by g a s - l i q u i d chromatography of i t s a c e t y l a t e d a l d i t o l d e r i v a t i v e , a n d i i i ) by the c h a r a c t e r i s t i c mass spectrum of i t s a l d i t o l a c e t a t e d e r i v a t i v e . Component 5: 4 - 0 - ( 2 , 3 - M - 0 - m e t h y l - p - D - x y l o s y l ) - D - x y l o s e . The s t r u c t u r e of t h i s compound which i s a d i s a c c h a r i d e was proved as f o l l o w s : i ) on a c i d h y d r o l y s i s i t gave D-xylose and 2,3-di-O-methyl-D-xylose i n an equimolar r a t i o , i i ) the s p e c i f i c r o t a t i o n of the compound [©<3^  - 2 5 . 3 ° suggested t h a t the l i n k a g e between the tv/o sugar u n i t s i s of t h e ^ - D - t y p e , i i i ) h y d r o l y s i s of the - 5 0 -borohydride reduced m a t e r i a l gave x y l i t o l and 2 , 3 - d i -0- methyl-D-xylose i n d i c a t i n g t h a t x y l o s e was the r e d u c i n g end of the d i s a c c h a r i d e , and i v ) m e t h y l a t i o n of the d i s e x c h a r i d e f o l l o w e d by methanolysis gave only 2,3-cLi-O-methyl-D-xylosides i n d i c a t i n g t h a t i t was a 1 — 4- l i n k e d d i s a c c h a r i d e . Component 6: 3 - 0 - X e t h y l - D - x y l o s e . — I t was i d e n t i f i e d i ) by q u a l i t a t i v e paper chromatography, i i ) by comparing the r e t e n t i o n time of i t s t r i m e t h y l s i l y l d e r i v a t i v e w i t h t h a t of a known a u t h e n t i c sample, and i i i ) as the c r y s t a l l i n e sugar. Component 7- D - X y l o s e . — I t was i d e n t i f i e d i ) by paper chromatography,and i i ) as i t s t r i m e t h y l s i l y l d e r i v a t i v e on g a s - l i q u i d chromatography. The mixture of a c i d i c sugars was converted by r e f l u x i n g w i t h methanolic hydrogen c h l o r i d e ( 3 w ) i n t o methyl e s t e r methyl g l y c o s i d e s which on r e d u c t i o n w i t h l i t h i u m aluminum hydride gave r i s e to n e u t r a l sugars. A f t e r h y d r o l y s i s , t h r e e components were observed, these being 2 , 5 , 4 — t r i - O - m e t h y l - D - g l u c o s e , 3,4--di-0-methyl-L>-glucose and 3-0-methy1-D-xylose. 2,3,4— Tri-O-methyl-D-glucose was i d e n t i f i e d i ) by paper chromatography, i i ) by comparing the r e t e n t i o n times of the methyl g l y c o s i d e s w i t h those of a u t h e n t i c - 51 -samples on g a s - l i q u i d chromatography, i i i ) as the c r y s t a l l i n e ji-methyl g l y c o s i d e , i v ) by r e d u c t i o n to 2 , 3 , 4 — t r i - O - m e t h y l - D - g l u c i t o l which on o x i d a t i o n w i t h p e r i o d a t e gave 2 , 5 , 4 — t r i - O - m e t h y l - L - x y l o s e , and v) by the c h a r a c t e r i s t i c mass spectrum of I t s methyl g l y c o s i d e s . The mixture of 5-0-methyl-D-xylose and 5 , 4 — di-O-methyl-D-glucose p a r t l y overlapped on a paper chroma.to-gram and the mixture of a l d i t o l a c e t a t e s of these sugars gave r i s e t o a s i n g l e peak on the g a s - l i q u i d chromato-gram. But the mixture of methyl g l y c o s i d e s of 3-0-methyl-D-xylose and 3 , 4 —di-O-methyl-D-glucose was w e l l r e s o l v e d by g a s - l i q u i d chromatography. 3-O-Kethyl-D-xylose was i d e n t i f i e d by comparing the r e t e n t i o n time of i t s g l y c o s i d e s w i t h an a u t h e n t i c sample. A p a r t of the c o l l e c t e d methyl-3-0-methyl-D-x y l o s i d e was h y d r o l y s e d i n t o f r e e sugar which was then converted i n t o 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 which gave a c h a r a c t e r i s t i c mass spectrum. The i d e n t i f i c a t i o n of 5 , 4 — di-O-methyl-D-glucose was c a r r i e d out by i s o l a t i n g m e t h y l - 5 , 4 — d i - O - m e t h y l - g l u c o s i d e s by g a s - l i q u i d chromatography and s u b j e c t i n g a p a r t of the c o l l e c t e d m a t e r i a l to mass-spectrometric i n v e s t i g a t i o n . A p a r t of the sample was h y d r o l y s e d to generate f r e e - 5 2 -r e d u c i n g sugar which was dernethylated to g i v e g l u c o s e . From the m e t h y l a t i o n data c e r t a i n s t r u c t u r a l f e a t u r e s can be i n f e r r e d . I t i s ev i d e n t t h a t the 2 , 3 , 4 — t r i - O -methyl-D-xylose, 2,5,4— t r i - O - m e t h y l - L - a r a b i n o s e and 2,3,5-"fcri-0-methyl-L—arabinose occur as t e r m i n a l non-r e d u c i n g ends of the p o l y s a c c h a r i d e . The 3-0-methyl-D-x y l o s e i s d e r i v e d from u n i t s of x y l o s e which form branch p o i n t s i n the molecule. S i m i l a r l y , i s o l a t i o n of D-xylose i n d i c a t e s t h a t these u n i t s are f u l l y s u b s t i t u t e d . The i s o l a t i o n of 2,5-di-O-methyl-D-xylose i n s m a l l amounts i n d i c a t e s t h a t the p o l y s a c c h a r i d e has r e l a t i v e l y few x y l o s e u n i t s which do not c a r r y any s u b s t i t u e n t at C-2 and C-5. The i d e n t i f i c a t i o n of 2,5,4— tri-O-methyl-D-glucose and 5,4—di-O-methyl-D-glucose from the a c i d i c fragments a f t e r r e d u c t i o n i n d i c a t e s t h a t these m o i e t i e s occur' 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 as u r o n i c a c i d s . The i s o l a t i o n of 5,4— di-O-methyl-D-glucose i s i n agreement w i t h White's (6) c o n c l u s i o n t h a t one of the u r o n i c a c i d s i s branched. But u n l i k e 'White's q u a n t i t a t i v e e s t i m a t i o n of u r o n i c a c i d s the present i n v e s t i g a t i o n r e c o r d s t h a t the r a t i o between t e r m i n a l u r o n i c a c i d and branched u r o n i c a c i d i s 5 : 2 . According to White (6) i t was 1 : 1 . The p o s i t i o n of branching i n the u r o n i c a c i d was ob t a i n e d through the p e r i o d a t e o x i d a t i o n of the - 53 -carboxyl-reduced p o l y s a c c h a r i d e as d i s c u s s e d on page 7 1 . The i s o l a t i o n of 3-0-methyl-D-xylose from the a c i d i c fragments a l s o i n d i c a t e s t h a t the u r o n i c a c i d s are l i n k e d t o the main c h a i n at p o s i t i o n 2. , p h i s has been confirmed by the i s o l a t i o n of o l i g o u r o n i c a c i d s by p a r t i a l a c i d h y d r o l y s i s and a l s o by the i s o l a t i o n of o l i g o s a c c h a r i d e s by p a r t i a l a c e t o l y s i s as d i s c u s s e d l a t e r (page 77)» From m e t h y l a t i o n data i t i s q u i t e e v i d e n t t h a t sapote gum p o l y s a c c h a r i d e has a very h i g h l y branched s t r u c t u r e . Mole r a t i o s were c a l c u l a t e d by a combination of g a s - l i q u i d , paper and c e l l u l o s e column chromatographic methods. - 5 4 -Table Approximate rnolar r a t i o s of p a r t i a l l y methylated  monosaccharides from methylated sapote gum Sugar d e r i v a t i v e Hole r a t i o 2 , 3 , 5 - T r i - O - m e t h y l - L - a r a b i n o s e 4 2 , 3 , 4 - T r i - O - m e t h y l - D - x y l o s e 1 2 , 3 , 4 - T r i - O - m e t h y l - L - a r a b i n o s e 1 2 , 5-Di-O-methyl-D-xylose 1. 5 3 - 0 - i ' i e t h y l - j J - x y l o s e 4 D-Xylose 3 . 5 2 , 3,4-Tri-O-methyl-D-glucose 3 3,4-I)i - 0-methyl-I)-glucose 2 - 55 -A u t o h y d r o l y s i s The p o l y s a c c h a r i d e was autohydrolysed f o r a p e r i o d of 60 hours over a steam bath. The s o l u t i o n was d i a l y s e d a g a i n s t d i s t i l l e d water and the content i n s i d e the d i a l y s i s bag was f r e e z e - d r i e d . The d i f f u s a t e s were co n c e n t r a t e d to a t h i c k syrup. The f r e e z e - d r i e d m a t e r i a l w i l l be r e f e r r e d t o as the degraded p o l y s a c c h a r i d e . The p r o p e r t i e s of t h i s degraded p o l y s a c c h a r i d e are l i s t e d i n Table 4. Table 4 P r o p e r t i e s of the degraded sapote gum S p e c i f i c r o t a t i o n E q u i v a l e n t weight M o l e c u l a r weight 14 , 3 0 0 (number average) A p a r t of the degraded p o l y s a c c h a r i d e was methylated a c c o r d i n g to the procedure of Hakomori (10). A p o r t i o n of the methylated m a t e r i a l was h y d r o l y s e d a c c o r d i n g to the procedure of L i n d b e r g (32). The n e u t r a l sugars were reduced w i t h sodium borohydride and converted i n t o a l d i t o l a c e t a t e s which were then examined by g a s - l i q u i d chromatography. .The n e u t r a l sugars were found to c o n s i s t of 2,5,4-tri-O-methyl-U-xylose, 2,5-di-O-methyl-iJ-xylose and mono-0-methyl-i)-xylose i n a molar r a t i o of 2:1.3:1• -8 ( c , 1.0 i n water) 850 - 56 -Since 2 - 0 - , and 3 - 0 - m e t h y l - D - x y l i t o l a c e t a t e s co-chromatograph, a s m a l l amount of methylated n e u t r a l sugars was separated from a. p r e p a r a t i v e paper chromato-gram. The p o r t i o n of the n e u t r a l sugars corresponding to the mono-O-methyl r e g i o n was s i l y l a t e d and the r e t e n t i o n time was found to be i d e n t i c a l w i t h t h a t of the s i l y l d e r i v a t i v e of a u t h e n t i c 3-0-methyl-D-xylose. A p o r t i o n of the methylated reduced m a t e r i a l was h y d r o l y s e d and converted i n t o a l d i t o l a c e t a t e s . Pour peaks corresp o n d i n g to the a l d i t o l a c e t a t e s of 2,3,4— t r i - O - m e t h y l - D - x y l o s e , 2,3-di-O-methyl-P-xylose, 2,3,4— tri-O-methyl-D-glucose and a mono-O-methyl-D-xylose i n a molar r a t i o of 2:1.3:1:2 r e s p e c t i v e l y were observed. The h i g h l y branched nature of the i n t e r i o r p o r t i o n of the gum was c l e a r l y shown w i t h r e s i d u e s of u r o n i c a c i d as end groups. The degraded p o l y s a c c h a r i d e was dev o i d of any arabinose r e s i d u e s i n d i c a t i n g t h a t arabinose m o i e t i e s occur on the p e r i p h e r y of the polymer. Prom the m e t h y l a t i o n data i t i s p o s s i b l e to deduce the s t r u c t u r e of the degraded p o l y s a c c h a r i d e as f o l l o w s : 1 UA X y i 1 n where UA D-Glucuronic or 4— O-Hethyl-D-G l u c u r o n i c a c i d - 57 -Sapote gum Autohydrolysis Degraded gum i )DMSo, NaH Methylated degraded gum H + U A I H 4 , THF Reduced methylated degraded gum H + i) Neutral methylated sugars fi) Acidic methylated sugars Neutral methylated sugars F i g u r e 6. Flow diagram f o r the a u t o h y d r o l y s i s of sapote gum - 58 -The d i f f u s a t e from the autohydrolysed mixture was c o n c e n t r a t e d to a syrup which was separated i n t o n e u t r a l and a c i d i c p a r t s by means of ion-exchange r e s i n s . The n e u t r a l p o r t i o n c o n s i s t e d of monosaccharides such as arabinose and x y l o s e . The a c i d i c fragment was found to c o n s i s t of two s e r i e s of o l i g o u r o n i c a c i d s : (A) one s e r i e s c o n s i s t i n g of a c i d s c o n t a i n i n g r e s i d u e s of D-glucuronic a c i d and D-xylose, (B) the other s e r i e s c o n s i s t i n g of a c i d s c o n t a i n i n g r e s i d u e s of 4— 0-methyl-D-glucuronic a c i d and D-xylose. Lambert et a l (8). i s o l a t e d the aforementioned s e r i e s of a c i d s from the p a r t i a l a c i d h y d r o l y s i s of sapote gum. The present i n v e s t i g a t i o n of the d i f f u s a t e s from autohydrolysed sapote gum a l s o i n d i c a t e d compounds w i t h s i m i l a r chromatographic m o b i l i t y on paper as d e s c r i b e d by Lambert et a l ( 8 ) . Thus s e r i e s A c o n s i s t s of the f o l l o w i n g a c i d s : i ) y+-O-methyl-D-glucuronic a c i d , i i ) 4-O-methyl-D-glucuronosyl (1---2)-D-xylose, i i i ) 4— 0-methyl-D-glucuronosyl (1--2)-D-xylopyranosyl . (1 - Q-4)-D-xylose. In the s i m i l a r way s e r i e s B c o n s i s t s of the f o l l o w i n g a c i d s : i ) D-glucuronic a c i d , i i ) g l u c u r o n o s y l (1^-2) D-xylose, i i i ) g l u c u r o n o s y l ( 1 — 2 ) D-xylopyranosyl (1-^-4) D-xylose. As these components have been f u l l y s t u d i e d by Lambert ( 8 ) , they were not f u r t h e r s t u d i e d . - 59 -P e r i o d a t e o x i d a t i o n Sapote gum p o l y s a c c h a r i d e was o x i d i z e d w i t h p e r i o d a t e to g i v e polyaldehyde which was reduced to the c o r r e s p o n d i n g p o l y a l c o h o l w i t h sodium borohydride. Graded h y d r o l y s i s of the p o l y a l c o h o l ( l i . s u l p h u r i c a c i d f o r 3 hours at room temperature) p r o v i d e d a mixture of components which was r e s o l v e d by ion-exchange r e s i n s and by sheet paper chromatography. •The f o l l o w i n g components from sapote gum p o l y a l c o h o l were i d e n t i f i e d : (1) ethylene g l y c o l (2) g l y c e r o l (3) 2 - 0 - f - D - x y l o p y r a n o s y l - g l y c e r o l (40 2-0-($-D-x y l o b i o s y l - g l y c e r o l (5) x y l o b i o s e (6) 2-0-£-D-xylo-t r i o s y l g l y c e r o l . The a p p l i c a t i o n of graded a c i d h y d r o l y s i s to the p o l y a l c o h o l i s a l s o known as Smith d e g r a d a t i o n a f t e r the name of the o r i g i n a l i n v e s t i g a t o r , P . S m i t h (18). The a p p l i c a t i o n of Smith de g r a d a t i o n to the branched x y l a n type p o l y s a c c h a r i d e would be expected t o g i v e r i s e to i n t a c t x y l o s e r e s i d u e s s i n c e a branch at e i t h e r C-2 or C-3 of a x y l o s e u n i t w i t h i n the 1-^ -4- l i n k e d c h a i n would render t h a t u n i t r e s i s t a n t to p e r i o d a t e o x i d a t i o n . On the other hand, the two x y l o s e u n i t s i n the polymer f l a n k i n g the r e s i d u e which had a branch attac h e d t o i t , i n a d d i t i o n to a l l of the other l-jj-4- l i n k e d x y l o s e u n i t s which do not c a r r y a branch, are a t t a c k e d by p e r i o d a t e - 60 -because i n a l l these r e s i d u e s adjacent h y d r o x y l groups e x i s t at C-2 and C-3. A branching of t h i s type i n the polymer c h a i n a f t e r the r e d u c t i o n and the m i l d a c i d h y d r o l y s i s steps g i v e r i s e to the f o r m a t i o n of 2 - 0 - ^ - h - x y l o p y r a n o s y l - g l y c e r o l . A p e r i o d a t e r e s i s t a n t x y l o s e u n i t would be d e r i v e d from a 1 — 3 l i n k e d x y l o s e w i t h i n the x y l a n backbone i n the absence of a branch s i n c e i n t h i s case, as i n the case of a 1—4- l i n k e d x y l o s e r e s i d u e w i t h a branch at C-2 or C-3, there are no adjacent h y d r o x y l groups on the x y l o s e u n i t under c o n s i d e r a t i o n . The s i t u a t i o n i s i l l u s t r a t e d below. OH OH OK A 1—3- l i n k a g e would gi v e r i s e t o 2,4~di-0-methyl-D-xylose i n m e t h y l a t i o n s t u d i e s , a component which was not d e t e c t e d i n the m e t h y l a t i o n s t u d i e s . o f sapote gum p o l y s a c c h a r i d e . I t may be concluded, t h e r e f o r e , t h a t 2 - 0 - j J - D - x y l o p y r a n o s y l - g l y c e r o l was not d e r i v e d from a 1 — 3 l i n k a g e but from a l i n k e d x y l o s e u n i t i n the - 61 -x y l a n c h a i n which c o n t a i n e d a branch at C-2 or C-y. The c o n s t i t u t i o n of 2 - 0 ~ ( J - D - x y l o p y r a n o s y l - g l y c e r o l was proved as f o l l o w s : i ) On a c i d h y d r o l y s i s i t gave x y l o s e and g l y c e r o l almost i n an equimolar r a t i o . The molar r a t i o v/as estimated by g a s - l i q u i d chromatography as t h e i r s i l y l d e r i v a t i v e s , i i ) The s p e c i f i c r o t a t i o n was s i m i l a r t o the v a l u e s quoted i n the l i t e r a t u r e (60). i i i ) 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 of the compound gave a s i n g l e symmetrical peak on a g a s - l i q u i d chromato-gram. S e p a r a t i o n of t h i s compound has not been r e p o r t e d i n the l i t e r a t u r e . Hence t h i s i n v e s t i g a t i o n i s the f i r s t example f o r the s e p a r a t i o n of t h i s compound by g a s - l i q u i d chromatography. ' i v ) The compound gave no formaldehyde upon p e r i o d a t e o x i d a t i o n . This i n d i c a t e d t h a t the g l y c e r o l moiety was 2 - 0 - s u b s t i t u t e d . v) The compound on p e r i o d a t e o x i d a t i o n consumed 2 moles of p e r i o d a t e per mole of the compound i n d i c a t i n g t h a t a nonreducing xylopyranose end group was present i n the pyranose form, v i ) P e r i o d a t e o x i d i z e d and borohydride reduced product of the compound on a c i d h y d r o l y s i s gave ethylene g l y c o l and g l y c e r o l almost i n an equimolar r a t i o . v i i ) F u r t h e r evidence f o r the presence of a xylopyranose end group was obtained by the examination of methanolysed products from the methylated d e r i v a t i v e s by g a s - l i q u i d chromatography. - 62 -O K C H 2 O H 1.IQ 2 . B H , HOH 2C H O H 2 C C H p O H H CF-UOH I C H 2 O H C H O I C R p O H C f - U D H I 2 C H O H I C H g O H F i g u r e 7. P e r i o d a t e o x i d a t i o n of 2-0 - 0-D-xylosyl g l y c e r o l Components having the c h a r a c t e r i s t i c r e t e n t i o n times of 2,3,4— tr i - O - m e t h y l - D - x y l o s e were r e c o g n i s e d , v i i i ) The mass spectrum 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 of the compound i s i n agreement w i t h the assigned s t r u c t u r e (page 107 ). By reason of the f a c t t h a t x y l o s e u n i t s i n a 1—4- l i n k e d x y l a n are r e s i s t a n t to p e r i o d a t e o n l y when branches are att a c h e d at C-2 or C-3, i t f o l l o w s t h a t 2 - 0 - 0-D-xylo-b i o s y l g l y c e r o l must have a r i s e n from two adjacent 1-y4- l i n k e d xylopyranose u n i t s i n the x y l a n c h a i n , each of which had a p e r i o d a t e o x i d i z a b l e sugar r e s i d u e a t t a c h e d to i t by a g l y c o s i d i c bond. The g l y c e r o l moiety of 2 - 0 - P - D - x y l o b i o s y l - g l y c e r o l , s i m i l a r to the g l y c e r o l of 2 - 0 - ^ - D - x y l o p y r a n o s y l - g l y c e r o l , was d e r i v e d from a x y l o s e u n i t . The c o n s t i t u t i o n of 2 - 0 - f - D - x y l o b i o s y l - g l y c e r o l was proved as f o l l o w s : i ) The s p e c i f i c r o t a t i o n -4-6° ( c , 1.0 i n water) of the compound i n d i c a t e d t h a t the g l y c o s i d i c l i n k a g e s are of the P-type. I t s o p t i c a l r o t a t i o n i s s i m i l a r to t h a t r e p o r t e d i n the l i t e r a t u r e (60). i i ) The compound on a c i d h y d r o l y s i s gave x y l o s e and g l y c e r o l . The molar r a t i o of x y l o s e to g l y c e r o l was shown to be 2:1 by the f o l l o w i n g methods: a) Xylose was determined by the p h e n o l - s u l p h u r i c a c i d method and g l y c e r o l by p e r i o d a t e o x i d a t i o n and subsequent e s t i m a t i o n of - 64 -C hLOH C HO CH2OH CH2OH C HpOH CHOH CHOH 2 CHO I CHOH CH2OH CHOH i CH2OH F i g u r e 6. Pe r i o d a t e o x i d a t i o n of 2-0-p - D - x y l o b i o s y l -g l y c e r o l formaldehyde by the chromotropic a c i d reagent, b) The molar r a t i o of x y l o s e to g l y c e r o l was determined 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 by g a s - l i q u i d chromato-graphy, i i i ) M e t h y l a t i o n of the compound f o l l o w e d by a c i d h y d r o l y s i s gave r i s e to 2 spots on a paper chromatogram which had i d e n t i c a l m o b i l i t i e s w i t h 2,3,4— tri - O - m e t h y l - D - x y l o s e and 2,3-di-O-methyl-D-xylose. The m a t e r i a l was converted i n t o a l d i t o l a c e t a t e s and gave r i s e to 2 peaks which had i d e n t i c a l r e t e n t i o n times w i t h those of a u t h e n t i c samples of 1 , 5 - d i - 0 - a c e t y l -2 , 3,4-tri-O-methyl- and 1 , 4 , 5 - t r i - 0 - a c e t y l - 2 , 3 - d i - 0 -methyl x y l i t o l s . The molar r a t i o was found to be 1:1. i v ) The compound on p e r i o d a t e o x i d a t i o n consumed almost 3 moles of p e r i o d a t e per mole of the compound. Subsequent r e d u c t i o n of the dialdehyde and h y d r o l y s i s gave r i s e to ethylene g l y c o l and g l y c e r o l i n a molar r a t i o of 1:2 approximately. The r e a c t i o n . i s i l l u s t r a t e d i n i ' i g . v) Since no formaldehyde was produced on p e r i o d a t e o x i d a t i o n i t i s evi d e n t t h a t g l y c e r o l was 2 - 0 - s u b s t i t u t e d . By the same reason as before i t follows t h a t 2-O-f-D-x y l o t r i o s y l g l y c e r o l has a r i s e n from t h a t p a r t of the polymer c h a i n which had 3 adjacent x y l o s e u n i t s carrying-p e r i o d a t e o x i d i a a b l e side c h a i n s and f l a n k e d by x y l o s e - 66 -u n i t s . The chromatographic m o b i l i t y of t h i s compound i n d i c a t e s t h a t t h i s should be x y l o t r i o s y l g l y c e r o l . I t s c o n s t i t u t i o n was proved as f o l l o w s : i ) On a c i d h y d r o l y s i s i t gave x y l o s e and g l y c e r o l i n a molar r a t i o of 1:3. The molar r a t i o was determined by the f o l l o w i n g methods: a) The molar r a t i o between these two components was determined by g a s - l i q u i d chromatography 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 , b) By a combination of p h e n o l - s u l p h u r i c a c i d and chromotropic a c i d methods as d e s c r i b e d f o r x y l o b i o s y l g l y c e r o l . i i ) Since no f o r m a l -dehyde was produced on p e r i o d a t e o x i d a t i o n g l y c e r o l i s s u b s t i t u t e d at p o s i t i o n C-2. i i i ) Reduction of the p e r i o d a t e o x i d i z e d m a t e r i a l and subsequent a c i d h y d r o l y s i s gave ethylene g l y c o l , and g l y c e r o l i n a molar r a t i o of 1:3. A compound w i t h R , 0.30 was o b t a i n e d i n a very x y l o s e ' J s m a l l amount. On a c i d h y d r o l y s i s i t gave x y l o s e which i n d i c a t e d t h a t i t was o r i g i n a l l y x y l o b i o s e . A s p i n a l l (60) has a l s o i s o l a t e d x y l o b i o s e from the Smith degrada-t i o n of b arley-husk a r a b i n o x y l a n . Ethylene g l y c o l can a r i s e o n l y from C-4- and C-5 of the t e r m i n a l nonreducing xylopyranose and arabopyranose u n i t s a f t e r p e r i o d a t e o x i d a t i o n , r e d u c t i o n and graded a c i d - 67 -h y d r o l y s i s of a p o l y s a c c h a r i d e . I t was i d e n t i f i e d as the c r y s t a l l i n e p - d i n i t r o - b e n z o a t e d e r i v a t i v e . G l y c e r o l may be d e r i v e d from C-3, 0-4- and C-5 of the 1 LY l i n k e d pentose u n i t s , which have adjacent h y d r o x y l groups at C-2 and C-5 and t h e r e f o r e are a t t a c k e d by the p e r i o d a t e reagent. G l y c e r o l may a l s o be o b t a i n e d from t e r m i n a l nonreducing arabinofuranose r e s i d u e s (C-3, C-4- and C - 5 ) . The s e p a r a t i o n of the components ob t a i n e d by the Smith d e g r a d a t i o n was found to be very e f f e c t i v e on b a s i c anion exchange r e s i n s such as Dowex 1 ( O i l - ) f o l l o w i n g the procedure d e s c r i b e d by A u s t i n et a l ( 6 1 ) . The i s o l a t i o n of 2 - 0 - P - D - x y l o p y r a n o s y l - g l y c e r o l , x y l o b i o s y l - g l y c e r o l . and x y l o t r i o s y l - g l y c e r o l from the d e g r a d a t i o n of sapote gurn p o l y s a c c h a r i d e i n d i c a t e s t h a t the p o l y s a c c h a r i d e has i r r e g u l a r branching, r a t h e r than an a l t e r n a t i o n of branched and unbranched x y l o s e r e s i d u or the presence of b l o c k s of r e s i d u e s a l l c a r r y i n g s i d e c h a i n s w i t h corresponding b l o c k s of unbranched u n i t s . Table 5 Examination of g l y c e r o l g l y c o s i d e s G l y c o s i d e H y d r o l y s i s products (mol. prop.) X y l G l y c e r o l Koles of 10 uptake 4 2-G - p-D-Xylosyl g l y c e r o l 2 - 0 - ^ - h - X y l o b i o s y l g l y c e r o l X y l o t r i o s y l g l y c e r o l Products of 10^ oxdn. (mole r a t i o ) G l y c e r o l Ethylene g l y c o l 0.90 1.80 2.80 1.00 1.00 1.00 2 . 1 0 3 . 1 5 4.10 0 . 9 5 1.00 1.00 1.00 2.00 2.80 - 69 -Simultaneous e s t i m a t i o n of 4—O-methyl-D-glucuronic a c i d  and o t h e r u r o n i c a c i d s by g a s - l i q u i d chromatography (52): The e s t i m a t i o n of the u r o n i c a c i d content of a p o l y s a c c h a r i d e may be c a r r i e d out by t i t r a t i o n (6$), d e c a r b o x y l a t i o n ( 6 3 ) , c o l o r i m e t r i c a l l y (64) or by weighing the a c i d i c f r a c t i o n a f t e r h y d r o l y s i s . Each of these methods pres e n t s c e r t a i n problems and none of them i s s u i t a b l e f o r s e p a r a t e l y e s t i m a t i n g mixtures of u r o n i c a c i d s such as occur' , f o r example, i n gums (65). When zr-0-methyl-D-glucuronic a c i d i s present t o g e t h e r w i t h o t h e r a c i d s the amount may be determined by comparing the v a l u e s f o r t o t a l c a r b o x y l and methoxyl but t h i s i s u n r e l i a b l e when r e s i d u a l l i g n i n i s a p o s s i b l e contaminant (66). Any method of e s t i m a t i n g u r o n i c a c i d a f t e r h y d r o l y s i s i s c o m p l i c a t e d by the d i f f i c u l t y of h y d r o l y s i n g the g l y c u r o n o s y l l i n k a g e s as w e l l as the tendency of the a c i d s to lactoni'se (67). A more p r o f i t a b l e route i n v o l v e s r e d u c t i o n of the u r o n i c a c i d f u n c t i o n before h y d r o l y s i s . S e v e r a l methods proposed f o r t h i s t r a n s f o r m a t i o n have been d i s c u s s e d before (page 2 8 ) . A r e c e n t study (68) d i r e c t e d towards the e s t i m a t i o n of •'!— C—mothyl-D-glucuronic a c i d i n x y l a n s employed e ^ t e r i f i c - t i o n w i t h propylene oxide and r e d u c t i o n w i t h sodium borohydride. The r e s u l t s showed an a c i d content - 70 -Sapote gum Propionic anhydride Propionylated gum Diazomethethane Ester i f ied propionylated gum Li th ium borohydride Carboxyl -reduced gum : H + arabinose v x y l o s e , 4-0-methyl-glucose, glucose j) sod ium borohydride y ii)acetic anhydride: pyridine Alditol acetates of arabinose xylose 4-0-methy l -gl ucose glucose F i g u r e 9 . Flow diagram f o r the r e d u c t i o n of sapote gum - 71 -of 5# although the sample was known to c o n t a i n 10. Recoveries of 17-20/5 have a l s o been reported, i n the case of a h y d r o l y z e d x y l a n by r e d u c t i o n of methyl e s t e r s by sodium borohydride (66 ). These low va l u e s are l i k e l y due to competing s a p o n i f i c a t i o n of the e s t e r and d i f f i c u l t y i n o b t a i n i n g complete r e a c t i o n w i t h propylene o x i d e . B e t t e r r e c o v e r i e s , of the order of 70-90/0, have been o b t a i n e d by diborane r e d u c t i o n of an a c e t y l a t e d p a r t i a l l y h y d r o l y z e d x y l a n ( 66 ). The purpose of the present i n v e s t i g a t i o n was t o demonstrate t h a t e x c e l l e n t q u a n t i t a t i o n c o u l d be achieved by r e d u c t i o n of prop i o n a t e d methyl e s t e r by l i t h i u m borohydride i n t e t r a h y d r o f u r a n . Sapote gum c o n t a i n s 27•4$ u r o n i c a c i d w i t h D-gluc-u r o n i c a c i d and 4—O-methyl-D-glucuronic a c i d i n a p p r o x i -mately equal amounts ( 8 ). During the course of the present i n v e s t i g a t i o n a sample of the gum was pr o p i o n a t e d , e s t e r i f i e d w i t h diazomethane and reduced w i t h l i t h i u m borohydride i n t e t r a h y d r o f u r a n . The product recovered by d i a l y s i s and l y o p h i l i s a t i o n gave a negati v e t e s t f o r u r o n i c a c i d w i t h c a r b a z o l e reagent (64- ). H y d r o l y s i s of the n e u t r a l p o l y s a c c h a r i d e and e s t i m a t i o n of the components as t h e i r a l d i t o l a c e t a t e s (69 ) on a column of b u t a n e d i o l s u c c i n a t e gave L-arabinose, D-xylose, D-glucose and 4~0-methyl-D-glucose i n the r a t i o of 1.0:2.8:0.4-8:0.52. - 72 -These f i g u r e s correspond to a t o t a l u r o n i c anhydride content of 27.3$ i n e x c e l l e n t agreement w i t h t h a t (27.4$) o b t a i n e d by Lambert, Dickey and Thompson by d e c a r b o x y l -a t i o n ( 8 ). Furthermore the i d e n t i f i c a t i o n of D - g l u c i t o l hexacetate and 4 - 0 - i i i e t h y l - D - g l u c i t o l (3-0-methyl-L-g u l i t o l ) pentaacetate a l s o served to i d e n t i f y the compon-ent a c i d s (68). The s e p a r a t i o n of g a l a c t i t o l , D - g l u c i t o l and D-mannitol hexaacetates has been demonstrated i n s e v e r a l l a b o r a t o r i e s and thus the corresponding u r o n i c a c i d s may be e s t i m a t e d s i n g l y or i n m i x t u r e s . Although the author has confirmed the o b s e r v a t i o n of Richards ( 7 0 ) t h a t on a column of .SCNSS-M g a l a c t i t o l hexaacetate o v e r l a p s w i t h 4 - O - m e t h y l - D - g l u c i t o l pentaacetate _ a model experiment showed t h a t they were separable on a column of b u t a n e d i o l s u c c i n a t e ( F i g . 3 7 ) . N e i t h e r column, however, w i l l r e s o l v e a mixture of D-mannitol hexaacetate and 4 - 0 - m e t h y l - D - g l u c i t o l p e n t a a c e t a t e . The r e s u l t s on sapote gum demonstrate t h a t r e d u c t i o n of u r o n i c a c i d s coupled w i t h g a s - l i q u i d chromatography i s a convenient method f o r t h e i r e s t i m a t i o n and i d e n t i -f i c a t i o n and t h a t the method i s of p a r t i c u l a r value when more than one u r o n i c a c i d i s p r e s e n t . F u r t h e r , the r e s u l t s suggest t h a t l i t h i u m borohydride i s the r e d u c i n g agent of c h o i c e , a c o n c l u s i o n s u b s t a n t i a l l y i n agreement w i t h Rees ( 5 3 ) and Green ( 5 0 ) . - 75 -P e r i o d a t e o x i d a t i o n of c a r b o x y l reduced p o l y s a c c h a r i d e From m e t h y l a t i o n data i t appears t h a t approximately one t h i r d of the u r o n i c a c i d s are branched at the 0-2 p o s i t i o n of u r o n i c a c i d s . Nov/ the q u e s t i o n a r i s e s as t o what type of u r o n i c a c i d s are branched. The answer was o b t a i n e d by the p e r i o d a t e o x i d a t i o n , borohydride r e d u c t i o n and complete a c i d h y d r o l y s i s of the c a r b o x y l reduced p o l y s a c c h a r i d e . T o t a l a c i d h y d r o l y s i s gave ethylene g l y c o l , g l y c e r o l , 2 - 0 - m e t h y l - D - e r y t h r i t o l , x y l o s e and 4-0-methyl-D-glucose.' A q u a n t i t a t i v e e s t i m a t i o n of the monosaccharides and p o l y o l s was c a r r i e d out s i m u l t a n e o u s l y by g a s - l i q u i d chromatography as t h e i r t r i m e t h y l s i l y l e t h e r s . The molar re,tios of these components are shown i n Table 6. Table 6 Simultaneous e s t i m a t i o n of p o l y o l s and sugars Component R e l a t i v e molar r a t i o Ethylene g l y c o l 1.00 G l y c e r o l 4.00 2 - 0 - i ' i e t h y l - B - e r y t h r i t o l 0.69 D-Xylose 2.15 4- 0- P' e t hy 1 - D- g 1 u c o s e - 74 -I t i s q u i t e evident t h a t ethylene g l y c o l conies from the p e r i o d a t e o x i d a t i o n of the t e r m i n a l xylopyranose and arabinopyranose u n i t s . These components on methy-l a t i o n g i v e 2 , 3 , 4 - t r i - 0 - m e t h y l - D - x y l o , and I — a r a b i n o -pyranose. G l y c e r o l comes from the t e r m i n a l a r a b i n o -furanose u n i t s and a l s o from glucose u n i t s o b t a i n e d a f t e r the r e d u c t i o n of g l u c u r o n i c a c i d . These components a f t e r m e t h y l a t i o n g i v e 2 , 3 , 5 - t r i - O - m e t h y l - L - a r a b i n o s e and 2 , 3 , 4 - t r i - O - m e t h y l - D - g l u c o s e . G l y c e r o l a l s o comes from x y l o s e u n i t s f r e e at C - 2 and C - 3 i n a (1 4) l i n k e d x y l a n c h a i n . Hence any r a t i o between ethylene g l y c o l and g l y c e r o l s h o u l d agree w i t h the r e l a t i v e r a t i o s of the combined 2 , 3 , 4 — t r i - O - m e t h y l - D - x y l o s e and 2 , 3 , 4 - t r i - O - m e t h y l - L -arabinose w i t h the combined 2 , 3 , 5 - t r i - O - m e t h y l - L — a r a b i n o s t h a t p a r t of 2 , 3 , 4 — t r i - O - m e t h y l - D - g l u c o s e coming from g l u c u r o n i c a c i d and 2 , 3-di-O-methyl-D-xylose u n i t s . The l a t t e r r a t i o i s 1:4 which i s i n e x c e l l e n t agreement w i t h the r a t i o betv/een ethylene g l y c o l and g l y c e r o l (1:4). The presence of 4-0-methyl-D-glucose a f t e r p e r i o d a t e o x i d a t i o n i n d i c a t e s t h a t t h i s p a r t o r i g i n a t e s from 4-0-methyl-D-glucuronic a c i d s u b s t i t u t e d at C - 2 or- C - 5 . The p o s s i b i l i t y of s u b s t i t u t i o n at C - 5 i s r u l e d out because of the i s o l a t i o n of 3,4-di-0-methyl-D-glucose - 75 -from the a c i d i c fragments of the methylated sapote gum a f t e r r e d u c t i o n and h y d r o l y s i s . Hence, i t was immune to p e r i o d a t e a t t a c k . 2 - 0 - i ' i e t h y l - D - e r y t h r i t o l o r i g i n a t e d from t h a t 4—O-methyl-D-glucose which d i d not c a r r y any s u b s t i t u e n t at C-2. This i s i l l u s t r a t e d below C H 2 O H > n o r e a c t i o n C H O H 2 H - C - O H I H - C - O C H C H 2 O H A r a t i o between 2 - 0 - m e t h y l - D - e r y t h r i t o l and 4~0-methyl-D-glucose o b t a i n e d a f t e r p e r i o d a t e o x i d a t i o n would g i v e the r a t i o between 4-0-rnethyl-I)-g].ucuronic a c i d s which c a r r y a s u b s t i t u e n t at C-2 and which do not. In t h i s present i n v e s t i g a t i o n the molar r a t i o between 2 - 0 - r n e t h y l - D - e r y t h r i t o l and 4-G-methyl-D-glucose i s 1:2.7-- 7 6 -In the o r i g i n a l p o l y s a c c h a r i d e 5 2 / of the u r o n i c a c i d s have a -OCH~ group at C-4-. From m e t h y l a t i o n data about 3 7 / of the u r o n i c a c i d s have a s u b s t i t u e n t at C-2. Hence the r a t i o between 4~O-methyl-D-glucuronic a c i d c a r r y i n g a s u b s t i t u e n t at C-2 and t h a t without a. s u b s t i t u e n t at C-2 i s 1:2.5. This i s i n e x c e l l e n t agreement w i t h the r e s u l t o b tained from p e r i o d a t e o x i d a t i o n d a t a . - 77 -A c e t o l y s i s By the a p p l i c a t i o n of p a r t i a l a c e t o l y s i s to the carboxyl-reduced sapote gum p o l y s a c c h a r i d e i t has been p o s s i b l e to i s o l a t e o l i g o s a c c h a r i d e s which c o n f i r m the experimental r e s u l t s obtained from the p a r t i a l a c i d 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 and i t has been p o s s i b l e to i s o l a t e a s e r i e s of o l i g o s a c c h a r i d e s which have not been r e p o r t e d p r e v i o u s l y . P a r t i a l a c e t o l y s i s of the carboxyl-reduced p o l y -s a c c h a r i d e was c a r r i e d out ac c o r d i n g to the procedure d e s c r i b e d by B a l l o u ( 55 ) • rfhe i s o l a t i o n of the o l i g o -s a c c h a r i d e s was c a r r i e d out by c h a r c o a l column chromatography on a buchner f u n n e l . This method has been found to be very e f f e c t i v e s i n c e i t i s very time s a v i n g . Monosaccharides and o l i g o s a c c h a r i d e s were i s o l a t e d by stepwise e l u t i o n w i t h i n c r e a s i n g amounts of e t h a n o l . Apart from monosaccharides such as arabinose and x y l o s e , the f o l l o w i n g o l i g o s a c c h a r i d e s were i s o l a t e d ; i ) D-Glu 1-7-2 1)-xylose i i ) 4--0-Methyl-D-glu 1~-2 .D-xylose i i i ) D-Glu 1-^-2 D-xyl 1-y4- D-xylose i v ) D-Glu 1-^2 D-xyl 1 - y4 D-xy 1-1—^-4- D-xylose The 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 o l i g o s a c c h a r i d e s - 78 -.Carboxyl reduced sapote gum i) formamide ii) pyr idine, acetic anhydride Carboxy l reduced sapote gum acetates i) acetic anhydride, acetic acid ii) sulphuric acid Part ia l ly acetolysed material charcoal column chromatography Oligosaccharides F i g u r e 10. Plow diagram f o r the a c e t o l y s i s of the carboxyl - r e d u c e d sapote gum - 79 -was c a r r i e d out as f o l l o w s : a) The nature of the c o n s t i t u e n t sugars was found by paper chromatography of the products of the a c i d h y d r o l y s i s of the o l i g o -s a c c h a r i d e , b) The r a t i o of the c o n s t i t u e n t sugars was determined by g a s - l i q u i d chromatography of the cleavage products of the o l i g o s a c c h a r i d e , c) The nature of the re d u c i n g end group W a s " determined by paper chromato-graphy of the products of a c i d h y d r o l y s i s of the d e r i v e d g l y c i t o l . d) The l i n k a g e among the d i f f e r e n t sugar u n i t s i n the o l i g o s a c c h a r i d e was determined by the a n a l y s i s of methanolyzed products of the methylated o l i g o s a c c h a r i d e . O l i g o s a c c h a r i d e ( i ) : D-Glu 1---2 D-xylose N e i t h e r the i s o l a t i o n nor the s y n t h e s i s of t h i s d i s a c c h a r i d e has been r e p o r t e d i n the l i t e r a t u r e . I t s o p t i c a l r o t a t i o n 0 0 D +102.5° i s very much d i f f e r e n t from t h a t of D-glu 1---2 D-xylose,. i . e . , 0° (71). I'he P i d e n t i f i c a t i o n of t h i s compound was c a r r i e d out as f o l l o w s : a) From the s p e c i f i c r o t a t i o n i t appears t h a t the l i n k a g e between the two sugar m o i e t i e s would be <=< . b) On a c i d h y d r o l y s i s i t gave glucose and x y l o s e i n an equimolar r a t i o . c) The d i s a c c h a r i d e a l d i t o l obtained by r e d u c i n g with sodium borohydride produced on a c i d h y d r o l y s i s glucose and x y l i t o l i n d i c a t i n g t h a t x y l o s e was the r e d u c i n g group, d) A p a r t of the methylated - 81 -p r o d u c t was meth'anolysed. and examined by g a s - l i q u i d chromatography whereby peaks corresponding to the methyl g l y c o s i d e s of standard 2,3,4,6-tetra-O-methyl-D-glucose and 3,4-di-O-methyl-L-xylose were •observed. This i n d i c a t e d t h a t the glucose moiety was l i n k e d through the 0-1 p o s i t i o n to the C-2 p o s i t i o n of x y l o s e . From arguments as produced before i t i s q u i t e evident t h a t t h i s d i s a c c h a r i d e i s present as the a l d o b i o u r o n i c a c i d D-gluA ( 1 _ — 2 ) D-xylose 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 . O l i g o s a c c h a r i d e ( i i ) : 4—O-Hethyl-D-glu (1-^-2) D-xylose The p r e p a r a t i o n of 4-O-methyl-D-glucosyl (1 2 ) -D-xylose by the r e d u c t i o n of the a l d o b i o u r o n i c a c i d by diborane has been reported, by T i m e l l and Roy ( 7 2 ) . The i d e n t i f i c a t i o n of t h i s compound was c a r r i e d out as f o l l o w s : a) The o p t i c a l r o t a t i o n W ^ + 1 0 9 ° and the chromatographic m o b i l i t y on paper are i d e n t i c a l w i t h those r e p o r t e d by Roy and T i m e l l ( 7 2 ) . b) On a c i d h y d r o l y s i s i t gave 4-0-methyl-D-glucose and D-xylose i n an equimolar r a t i o . c) The d i s a c c h a r i d e g l y c i t o l on a c i d h y d r o l y s i s gave 4-0-methyl-D-glucose and x y l i t o l i n d i c a t i n g x y l o s e was the r e d u c i n g end. d) The r e s u l t s of the methanolyzates of the methylated o l i g o s a c c h a r i d e are the same as d e s c r i b e d i n the s e c t i o n s (c) and (d) on page 7 9 . - 82 -As b e f o r e , i t I s a l s o concluded t h a t t h i s d i s a c c h a r i d e e x i s t s 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 as the a l d o b i o u r o n i c a c i d 4-0-methyl-D-glucA (1-^-2) D-x y l o s e . O l i g o s a c c h a r i d e ( i i i ) : D - G l u ( 1 — 2 ) - D - x y l (1~£-4)-D-xylose: n e i t h e r the i s o l a t i o n nor the s y n t h e s i s of t h i s o l i g o s a c c h a r i d e has been r e p o r t e d i n the l i t e r a t u r e . I t s i d e n t i f i c a t i o n was c a r r i e d out as f o l l o w s : a) On a c i d h y d r o l y s i s i t gave glucose and x y l o s e i n a molar r a t i o of 1:2. b) The methylated o l i g o s a c c h a r i d e on methanolysis and subsequent examination by g a s - l i q u i d chromatography showed products which had the r e t e n t i o n times of g l y c o s i d e s of 2,3,4,6-tetra-O-methyl-D-glucose, 3,4—di-O-methyl-L-xylose and 2,3-di-O-methyl-D-xylose. c) The o l i g o s a c c h a r i d e a l d i t o l o b t a i n e d by r e d u c i n g w i t h sodium borohydride gave on h y d r o l y s i s g l u c o s e , x y l o s e and x y l i t o l i n d i c a t i n g t h a t x y l o s e was the r e d u c i n g end group. This o l i g o s a c c h a r i d e i s present i n the p o l y s a c c h a r i d e as the a l d o t r i o u r o n i c a c i d GlueA (1-^-2) D-xyl ( 1 - y 4 ) -D-xylose and the next h i g h e r homolog of the d i s a c c h a r i d e ( i ) . The assignment of the s t r u c t u r e i s i n agreement w i t h the m e t h y l a t i o n data and a l s o on the s p e c i f i c - 84 -r o t a t i o n which i n d i c a t e s t h a t the two x y l o s e u n i t s must be l i n k e d by a l i n k a g e of the (3 - t y p e . The s p e c i f i c r o t a t i o n of oligosaccharide ( i i i ) i s +50° which i s lower than t h a t of the o l i g o s a c c h a r i d e ( i ) . I t would have been more p o s i t i v e i f the l i n k a g e would had been i n the o C - c o n f i g u r a t i o n . O l i g o s a c c h a r i d e ( i v ) : D-Glu (1-^-2)-D-xyl ( 1 - ^ - 4 ) -D-xyl (1---4)-D-xylose L i k e the o l i g o s a c c h a r i d e ( i i i ) i t s s y n t h e s i s or i s o l a t i o n has not been r e p o r t e d i n the l i t e r a t u r e . I t s s t r u c t u r e was e s t a b l i s h e d as f o l l o w s : a) Cn a c i d h y d r o l y s i s i t gave x y l o s e and glucose i n a molar r a t i o of 3:1 i n d i c a t i n g t h a t i t was a t e t r a s a c c h a r i d e . b) The gas l i q u i d chromatographic examination of the methanoly-sed products of the methylated d e r i v a t i v e showed peaks correspo n d i n g to the methyl g l y c o s i d e s of 2,3,4 ,6 -tetra-O-methyl-D-glucose, 3,4-di-O-methyl-L-xylose and 2,3-di-O-methyl-D-xylose. The approximate molar r a t i o between combined tetra-O-methyl-D-glucose and 3,4-di-0-methyl-L-xylose and 2,3-di-O-methyl-D-xylose i s 1:1. c) The o l i g o s a c c h a r i d e a l d i t o l on a c i d h y d r o l y s i s gave x y l i t o l , x y l o s e and glucose i n d i c a t i n g x y l o s e was the r e d u c i n g end group. This o l i g o s a c c h a r i d e ( i v ) i s probably present i n the p o l y s a c c h a r i d e as the a l d o t e t r a o u r o n i c a c i d . I t i s a l s o - 85 -the h i g h e r homolog of the o l i g o s a c c h a r i d e ( i i i ) . The s p e c i f i c r o t a t i o n i s lower than t h a t of o l i g o -s a c c h a r i d e ( i i i ) , i n d i c a t i n g the l i n k a g e between the x y l o s e u n i t s are of the (3 - t y p e . A p o s s i b l e o b j e c t i o n to the acceptance of the s t r u c t u r e ( F i g . 1) of sapote gum as proposed by .Smith and Montgomery (7) i s t h a t the s t r u c t u r e can not account f o r the p r o d u c t i o n of o l i g o s a c c h a r i d e ( i v ) by p a r t i a l a c e t o l y s i s of the carboxyl-reduced p o l y s a c c h a r i d e . A p o s s i b l e arrangement i n the gum molecule which would produce o l i g o s a c c h a r i d e ( i v ) i s shov/n below. X x- - x X -X •X A A where X r e p r e s e n t s a x y l o s e u n i t and /\ r e p r e s e n t s a u r o n i c a c i d u n i t . - 86 -Table 7 C h a r a c t e r i s a t i o n of o l i g o s a c c h a r i d e s ( i — i v ) O l i g o H y d r o l y s i s products Reducing Sugars formed on (molar r a t i o ) group cleavage of X y l Glu 4-0-Methyl- methylated derv. _Glu i 1 1 x y l o s e 2,3,4, 6-MeZ)_-glu 3,4-Me ?-xyl i i 1 1 x y l o s e 2,3,4,6-, o,.-.:lu 5, zl--He 2-xyl i i i 2 1 x y l o s e 2,3,4,6-Me^-glu 3,4-He 2-xyl 2 .3- P e 2 - x y l i v 3 1 x y l o s e 2,3,4,6-Me^-glu 3.4- M e ? - x y l 2,3---o 0-::yl Table 8 Gas chromatograplry of methylated sugar d e r i v a t i v e s * Methyl g l y c o s i d e s of I t e l a t i v e r e t e n t i o n times  2 , 3/l - , 6-'cetra-0-methyl-glucose 1.00, 1.33 3,4-di-0-methyl-xylose 1.14 1.33 2,3-di-0-methyl-xylose 1.22 1.41 * on a column (4'x/'.") of 5>? b u t a n e d i o l s u c c i n a t e on 80-100 mesh D i a t o p o r t 3. - 87 -Eofmann d e g r a d a t i o n : Erom the m e t h y l a t i o n of sapote gum and subsequent r e d u c t i o n of the a c i d i c methylated product, White (4,5,6) was able to i s o l a t e 5,4-di-O-methyl-D-glucose and 2,3,4-•tri-O-methyl-D-glucose which i n d i c a t e d the presence of 2 - 0 - s u b s t i t u t e d u r o n i c a c i d r e s i d u e s i n the p o l y s a c c h a r i d e c h a i n . The occurrence of t h i s type of branching i n the p o l y s a c c h a r i d e i s not ve r y common. Two ques t i o n s which appear are as f o l l o w s : i ) What type of u r o n i c a c i d ( g l u c u r o n i c or 4-0-methyl-D-glucuronic) i s i n v o l v e d i n t h i s branching? i i ) What i s the nature of the s u b s t i t u e n t ? The f i r s t problem was s o l v e d by s t u d y i n g the p e r i o d a t e o x i d a t i o n on the carboxyl-reduced p o l y s a c c h a r i d e and a n a l y s i n g the h y d r o l y s a t e of the borohydride reduced m a t e r i a l . The i s o l a t i o n of 4-0-methyl-D-glucose (as d e s c r i b e d on page 7 3 ) i n d i c a t e d the presence of 4-0-methyl-D-glucuronic a c i d being i n v o l v e d i n branching. The second problem was r a t h e r d i f f i c u l t to s o l v e . Erom m e t h y l a t i o n data (page 54) i t was found t h a t arabinose was present as a t e r m i n a l non-reducing end because of the presence of 2,5,5-tri-O-methyl-arabincse. This o b s e r v a t i o n has been confirmed by the enzymatic d e g r a d a t i o n of the p o l y s a c c h a r i d e w i t h c<-1-arabofurano-p o — O u — siclase r e s u l t i n g i n the r e l e a s e of arabinose (page 9 4 - ) . As d e s c r i b e d b e f o r e , a u t o h y d r o l y s i s , p a r t i a l a c i d h y d r o l y s i s and p a r t i a l a c e t o l y s i s gave r i s e to o l i g o -s a c c h a r i d e s w h e r e u r o n i c a c i d r e s i d u e s are the t e r m i n a l non-reducing ends. Hence i t appeared t h a t the a p p l i c a t i o n of Hofmann degradation would be a r a t h e r good approach to s o l v e t h i s problem. According to the F i g u r e • 5 i f there i s any s u b s t i t u e n t at C-2, C-3 or C-4- the r e s u l t i n g penta-d i a l d o s e s would a l s o be s u b s t i t u t e d at those p a r t i c u l a r p o s i t i o n s . Kochetkov et a l ( 5 7 ) s t u d i e d the degraded p o l y s a c c h a r i d e but not the low molecular weight fragments ob t a i n e d by the Hofmann de g r a d a t i o n . In the present i n v e s t i g a t i o n a t t e n t i o n was d i r e c t e d to both of these fragments i . e . , degraded p o l y s a c c h a r i d e and the low molecular weight f r a c t i o n s a f t e r the Hofmann r e a c t i o n , s i n c e the l a t t e r fragment -would p r o v i d e i n f o r m a t i o n as to the nature of the s u b s t i t u t e n t at the C-2 p o s i t i o n of a •'•'r-O-methyl-D-glucuronic a c i d r e s i d u e . The present i n v e s t i g a t i o n r e p o r t s the f i r s t d e t a i l e d study of the a p p l i c a t i o n of Hofmann degradation to p o l y s a c c h a r i d e s i n g e n e r a l and to a complex p o l y s a c c h a r i d e such as sapote gum i n p a r t i c u l a r . - 89 -In order to o b t a i n sapote gum amide, the p o l y s a c c -h a r i d e was p r o p i o n a t e d , e s t e r i f i e d w i t h diazomethane i n t e t r a h y d r o f u r a n and f i n a l l y t r e a t e d w i t h ammonia. I n f r a r e d spectroscopy of the r e s u l t i n g p o l y s a c c h a r i d e d e r i v a t i v e d i d not -show any f r e e c a r b o x y l i c a c i d or e s t e r groups. The amide was t r e a t e d w i t h 4$ sodium h y p o c h l o r i t e f o r 1 hour at pH 12 and at 0°. The mixture was a c i d i -f i e d w i t h a c e t i c a c i d t o pH 5 and l e f t f o r 16 hours. The m a t e r i a l was d i a l y s e d a g a i n s t water. The contents i n the d i a l y s i s bag were recovered by e v a p o r a t i o n . The p o l y s a c c h a r i d e thus recovered w i l l be c a l l e d as the 1 I-i of mann degraded p o l y s a c c h a r i d e 1 . The Hofmann degraded p o l y s a c c h a r i d e had an e q u i v a l e n t weight of 44-82 i n d i c a t i n g t h a t about 85$ of the o r i g i n a l u r o n i c a c i d s had been s e l e c t i v e l y c l e a v e d . The Hofmann degraded m a t e r i a l was methylated a c c o r d i n g to the method developed by Hakomori (10). The f u l l y methylated m a t e r i a l was h y d r o l y s e d i n the u s u a l way and the n e u t r a l sugars were examined both by g a s - l i q u i d chromatography and paper chromatography. The p a r t i a l l y methylated sugars obtained are as f o l l o w s : 2 , 3 , 4 — t r i - 0 -methyl-D-xylose (3 moles), 2 ,3-di-0-methy1-D-xylose (11 moles), mono-O-metbyl-h-xylose (2 moles). The i d e n t i t y - 90 -of these components was r e v e a l e d i n the u s u a l way by comparing the r e t e n t i o n times w i t h those of the standards under i d e n t i c a l c o n d i t i o n s . The s t r u c t u r e of the Hofmann degraded p o l y s a c c h a r i d e •can be w r i t t e n as f o l l o w s : X —x—x—x—x—x—x—x—x—x—x—x—x—x X X where X = x y l o s e n The h i g h degree of branching of sapote gum i s a l s o r e v e a l e d by the presence of mono-O-methyl-xyloses i n the methylated Hofmann degraded.polysaccharide. Hofmann d i f f u s a t e m a t e r i a l was f r e e d from i n o r g a n i c s a l t s by g e l - f i l t r a t i o n w i t h sephadex G-10. A symmetrical peak was obtained as shown i n the e l u t i o n programme ( F i g . 4 2 ) . The o l i g o s a c c h a r i d e was h y d r o l y s e d and the r e s u l t i n g components were analysed by g a s - l i q u i d chromatography as t h e i r a l d i t o l a c e t a t e s . As 2-0-methyl-L-x y l i t o l has been obtained from the 4-0-methyl-D-glucuronic a c i d p a r t which c a r r i e d a branch at C-2, a r a t i o between 2 - 0 - m e t h y l - L - x y l i t o l and other a l d i t o l s would i n d i c a t e the degree of p o l y m e r i s a t i o n . The average r a t i o among 2 - 0 - m e t h y l - L - x y l i t o l , a r a b i n i t o l and x y l i t o l was found to be 1:2:4. - 91 -Enzymatic Degradation of Sapote Gum P o l y s a c c h a r i d e : Although much i n f o r m a t i o n concerning the s t r u c t u r e of s t a r c h and glycogen has r e s u l t e d from s t u d i e s of the s y n t h e t i c and degradative a c t i o n of enzymes (73), very l i t t l e by comparison has been accomplished i n the study of gums by the use of enzymes. Very fev; enzymes have been found to degrade p l a n t gums ( 74 ) • In the present i n v e s t i g a t i o n on the s t r u c t u r e of sapote gum attempts were made to i s o l a t e o l i g o s a c c h a r i d e s c o n t a i n i n g a r a b i n o s e . Since arabinose r e s i d u e s i n sapote gum occur as the t e r m i n a l nonreducing end as evidenced by the m e t h y l a t i o n data and are h i g h l y a c i d l a b i l e , an a l t e r n a t e method would be the enzymatic d e g r a d a t i o n of the p o l y s a c c h a r i d e . Bishop ( 75 ) has r e p o r t e d the p r e f e r e n t i a l h y d r o l y s i s of l i n k a g e s i n the main c h a i n of wheat straw x y l a n by a xylanase from'Kyrothecium v e r r u c a r i a ' l e a d i n g t o the i s o l a t i o n of the t r i s a c c h a r i d e O-L-arabinof uranosy 1- (1 —5 )-0-f* - D - x y l o p y r a n o s y l - (1 — '4-) -D-xylopyranose. . - 92 -Attempts were made to degrade sapote gum p o l y s a c c a r i d e w i t h enzymes such as ' p e c t i n a s e 1 , ' h e m i c e l l u l a s e 1 and ' c e l l u l a s e ' a l l of which have been repox^ted to have xylanase a c t i v i t y ( 7 3 , 7 6 , 7 7 ) . These enzymes were not s u c c e s s f u l i n degrading the p o l y s a c c h a r i d e . Thus when the p o l y s a -c c h a r i d e was d i g e s t e d w i t h p e c t i n a s e o n l y t r a c e s of x y l o s e and arabinose were l i b e r a t e d . In a s i m i l i a r way, i n c u b a t i o n w i t h ' h e m i c e l l u l a s e ' and ' c e l l u l a s e ' gave o n l y s m a l l amounts of x y l o s e . I n a c t i v i t y of the enzymes on gums such as sapote gum needs some e x p l a n a t i o n . Gums are g e n e r a l l y h i g h l y a c i d i c complex p o l y s a c c h a r i d e s . In i t s b i o s y n t h e t i c p r o c e s s , a h i g h l y o r g a n i z e d , complex system of enzymes must have been i n v o l v e d which c o n t a i n a d i v e r s i t y of b u i l d i n g u n i t s and l i n k a g e s t h a t i s r a r e l y encountered i n p o l y s a c c h a r i d e s . This c o m p l e x i t y i s perhaps necessary i n order t h a t the p l a n t may s e a l o f f i t s wounds w i t h m a t e r i a l immune to the d e g r a d a t i v e enzymes present i n b a c t e r i a or f u n g i ( 7 8 ) . A gain, the dimensions of the s u b s t r a t e s i t e i n v o l v e d i n the enzymolysis of a p o l y s a c c h a r i d e might have something to do i n the o v e r a l l p r o c e s s . This phenomenon has been s t u d i e d by P e r l i n and Reese ( 7 9 ) . The s p e c i f i c i t y of an enzyme t h a t degrades a'poly s a c c h a r i d e i s c o r r e l a t e d g e n e r a l l y w i t h a p a r i c u l a r k i n d of g l y c o s y l u n i t or l i n k a g e i n the s u b s t r a t e . However, very l i t t l e i s known as to what p o r t i o n of the p o l y s a c c h a r i d e molecule i s i n v o l v e d i n the r e a c t i o n . P e r l i n and Reese ( l o c c i t ) o b t a i n e d i n f o r m a t i o n about the s p a t i a l requirements i n one s p e c i f i c i n s t a n c e , i . e . , f o r the a c t i o n of a 'xylanase' on an a r a b i n o x y l a n from wheat f l o u r . Prom the products which were i s o l a t e d ( P i g . 1 $ ) i t appears probable t h a t the enzyme c l e a v e s o n l y g l y c o s i d i c l i n k a g e s between unbranched 1 4 l i n k e d (* -D-xylopyranose r e s i d u e s . The r e l a t i v e l y low y i e l d of o l i g o s a c c h a r i d e s p o i n t s to the presence of a ' l i m i t e d number of "open areas i n the p o l y s a c c h a r i d e w i t h contiguous unbranched x y l o s e r e s i d u e s . Since sapote gum i s a h i g h l y branched p o l y s a c c h a r i d e , i t has very few "open ar e a s " f o r the a t t a c k by enzyme molecule. This i s supported by the m e t h y l a t i o n data as very l i t t l e 2,3-di-0-methyl-D-x y l o s e i s o b t a i n e d (page 54). The h i g h l y branched nature of sapote gum p o l y s a c c -h a r i d e i s a l s o demonstrated by the i n t e r a c t i o n of i o d i n e w i t h the p o l y s a c c h a r i d e . Thompson et a l ( 80 ) have s t u d i e d the g e n e r a l nature of the i n t e r a c t i o n of p o l y -s a c c h a r i d e s w i t h i o d i n e . L i k e the h i g h l y branched p o l y s a c c h a r i d e s such as c h e r r y gum, sapote gum d i d not r e a c t w i t h i o d i n e at a l l whereas other l e s s branched p o l y s a c c h a r i d e s d i d i n t e r a c t w i t h i o d i n e . Hence, the _ 94 -h i g h l y branched nature of sapote gum p o l y s a c c h a r i d e i s p r o b a b l y r e s p o n s i b l e f o r the i n a c t i v i t y of d i f f e r e n t enzymes. The a c t i o n of oC-L-arabinofuranocidase on sapote gum polysaccharide, has y i e l d e d some v a l u a b l e i n f o r m a t i o n on the nature of l i n k a g e s ( » C or ? ) between arabinose and the o t h e r sugar u n i t s , and a l s o on the nature of arabinose i t s e l f i . e . , D or L type. A f t e r prolonged i n c u b a t i o n of the p o l y s a c c h a r i d e w i t h <<-L-arabinofuranosidase arabinose was r e l e a s e d . The i s o l a t i o n of 2 , 3 , S - t r i - 0 -methyl-L-arabinose as the major p a r t i a l l y methylated arabinose c o n t a i n i n g sugar suggests t h a t most of the arabinose i s present as the furanose form. This c o n c l u s i o n i s f u r t h e r supported by the a c t i o n of t h i s enzyme. Hence the a c t i o n of oC-L-arabinofuranosidase s t r o n g l y suggests t h a t most of the arabinose r e s i d u e s are terminated by cC-L—linkages. A A I -x-x X—\— chain cleavage A , I - X - X T X X - T X - X - — X - X t : A - X - X - X - X - X x-x- - x-x-x vX A A I x-x-x- X - x-x-x A A A A A A - X - X - X - X - X - X - X - X - X - X - no action F i g . 1 3 . Enzymatic degradation of wheat f l o u r ar a b i n oxylan - 9 5 -Mass spectrometry: a) P a r t i a l l y methylated, a l d i t o l a c e t a t e s : The present i n v e s t i g a t i o n i n c l u d e s analyses of mass s p e c t r a of the a l d i t o l a c e t a t e s of 2,3-di-O-methyl-D-x y l o s e , 2 , 3 , 4 - t r i - 0 - m e t h y l - h - x y l o s e and 2 , 5 , 5 - t r i - 0 -m e thyl-L-arabinose. M o l e c u l a r i o n (M +) c o u l d not be t r a c e d i n any of these cases. The base peak i n a l l cases was at m/e 4-5 which i s due to a c e t y l i u m i o n . Peaks of low i n t e n s i t i e s ( < 5 $ of the base peak) were not co n s i d e r e d i n i n t e r p r e t i n g mass s p e c t r a . Some r e s u l t s are evi d e n t on i n s p e c t i o n of the fra g m e n t a t i o n p a t t e r n s shown i n P i g s . 1 5 , 1? and 1 9 . Primary fragments m/e 1 1 7 and m/e 161 are common to a l l compounds. However, the peak at m/e 161 r e p r e s e n t s a d i f f e r e n t i o n i c s p e c i e s having acetoxy and methoxy groups at d i f f e r e n t carbon atoms as d i s c u s s e d b e f o r e . The promi-nence of the peak at m/e 4 5 i n Pig.14 i n comparison to oth e r compounds can be accounted f o r by the presence of a methoxyl group at the t e r m i n a l carbon atom as i n the case of 2 , 3 , 5 - t r i - O - m e t b y l - L - a r a b i n i t o l d i a c e t a t e . A peak of ve r y h i g h i n t e n s i t y i s observed at m/e 145 i n P i g . 16 . This i s a secondary fragment a r i s i n g from the primary fragment at m/e 2 0 5 . The primary fragment by the l o s s of a c e t i c s c i d ( 6 0 ) , g i v e s r i s e to a sharp i i 9 6 ;100 •"SR. -p • H CQ •FJ 0 -P Pi H 0) t> • H • P Cj r H 43 45 -i r 71 .-117 101 100 129 161 150 m/e F i g u r e 14.' Mass spectrum of 1 , 4 - d i - 0 - a c e t y l - 2 , 3 , 5 - t r i - O -methyl-L - a r a b i n i t o l - 97 -C H O C O C H -2 3 H C O C H -C H O C H C H O C O C H 3 C H O C H 3 » H C - O C H . H 2 C O C O C H m/e 117 C H 0=C H 3 C H O C O C H 3 C H 2 O C H 3 m/e 161 H C - O C H . C H H C O C H 3 m/e 101 H C = O C H . C 0 C H p ^ > C H 2 H m/e 129 H C ^ O C H 3 c = o C H . m /e Q7 F i g u r e 15* Source of the most important i o n s of 1,4--&i-0-a c e t y l - 2 , ; ) , 5 - t r i - O - m e t h y l - L - a r a b i n i t o l - 98 -I Figure 18. Mass spectrum of 1 , 5 - d i - 0 - a c e t v l - 2 , $ , 4 - t r i - 0 -methyl-D-xylitol - 99 -CHOCOCH ' 2 3 H COCH CH O CH 3 I HCOCH. H C=£=OCH 3 CH 2OCOCH m/e 117 CH 2OCOCH 3 H C^-OCH. H C=t^OCH. H^J" O COCH^ H | / C H 2 C H 3 ° m/e 161 HCiOCH. C OCH. CHO C H 3 | H COCH. CH OCOCH., 2 3 m/e 205 H C ^ O C K . HCOCH 3 C O C H . C H 2 m/e 101 F i g u r e 17. 3 CH 2 m/e 145 S o u r c e o f the most i m p o r t a n t i o n s o f 1 , 5 - d i - 0 -a c e t y l - 2 , 3 , 4 - t r i - 0 - m e t h y l - D - x y l i t o l - 100 -|43 117 87 101 129 189 5 0 100 150 2 0 0 m/e F i g u r e 18. Mass spectrum of 1 , 4 , 5 - t r i - 0 - a c e t y l - 2 , 3 - a i - 0 -m e t h y l - D - x y l i t o l - 101 -C H 2 O C O C H 3 HCOCH. C H 2 0 C O C H 3 ^ H C ^ O C H . m/e 117 C H O C H HCOCOCH. CH OCOCH H C ™ O C H . H HC-OCH. c = o CH. O A t c O C O C H ^ II | 3 m/e 189 H C i O C H o C H ^ O C 9 o C H C H 2 H C ^ O C H . HCOCH. H 2 C 0 C Q C H 3 m/e 161 H C ^ O C H , C O C H . C H 2 m/e 101 '3 m/e 87 m/e 129 F i g u r e 19. Source of the most important i o n s of 1 , 4 , 5 - t r i - 0 -ace tyl-2,3-d.i-O-me t h y l - D - x y l i t o l - 102 -peak at m/e I V . Hence prominence of the secondary fragment at m/e 145 i s c h a r a c t e r i s t i c of 2 , 3 , 4 - t r i - 0 - m e t h y l pentose d e r i v a t i v e s . 'The primary fragment at m/e 1 8 9 ( F i g . 1 9 ) ^ s c h a r a c t e r -i s t i c of 2,3-di-0-methyl x y l i t o l t r i a c e t a t e as found i n F i g . 18, absent i n F i g s 14 and 16. This peak as d e s c r i b e d before a r i s e s from a l d i t o l s methylated at p o s i t i o n 3 but not at 1 and 2. Hence i t i s e v i d e n t from the above d i s c u s s i o n t h a t assignments and i n t e r p r e t a t i o n s of mass s p e c t r a of d i f f e r e n t p a r t i a l l y methylated a l d i t o l a c e t a t e s permit t h e i r i d e n t i f i c a t i o n . b) Methyl g l y c o s i d e s of p a r t i a l l y methylated sugars: The f r a g m e n t a t i o n p a t t e r n of the methyl g l y c o s i d e of 5,4-di-0-methyl-D-glucose was worked out a c c o r d i n g to the f r a g m e n t a t i o n p a t t e r n proposed by Heyns et a l ( 1 7 ) . The methyl g l y c o s i d e of 5,4-di-O-methyl-D-glucose produces a c h a r a c t e r i s t i c peak at m/e 161. I t s o r i g i n i s i l l u s -t r a t e d i n F i g . 2 1 . Although the mass spectrum of the methyl g l y c o s i d e s of 2,5-di-O-methyl-D-glucose produces a prominent peak at m/e 161, the r e t e n t i o n times of these components on a g a s - l i q u i d chromatogram were not i d e n t i c a l w i t h those of the methyl g l y c o s i d e s of 3,4-di-O-methyl-D-glucose. The mass spectrometry-gas chromatography - 103 -1 0 0 75 8 8 73 -\-> "in n cu **-> c !> OrT 4 5 43 101 .161 50 l ' i ' ' — I 1 — r 100 150 200 m/e Figure 20. Mass spectrum of methyl 3,4-di-O-methyl-glucoside - 104- -CHOH CH 3O SM 3 OH CHQH OH OCH OCH CH 3C + 3 /6 m/e 161 OCH 3OCH 3 m/e 161 OCH OCH 3 m/e 161 F i g u r e 21. Source of the i o n at m/e 161 i n the mass spectrum of methyl 3,4~di-0-methyl- D-glucoside. - 105 -100 8 8 5 0 4 5 0 75 r i • > x 1 0 101 131 145 100 m/e F i g u r e 22, mass spectrum of methyl 2 , $ , 4 - t r i - 0 -methy1-f-D-glucoside C H 3 Q C H O C H C=C H 3 I O C H ^ B 2 m/e 131 J O C H / = O C H . C H 3 0 ^ 3 l B.m/e 17 6 ° C H 3 C H s O C H = C H O C H 3 ] H H , m / e 8 8 A C H 2 O H - O . O C H 3 C H O N 0 0 ^ : 3 O C H . CH„OH 3 6 C H 3 0 - C H = C H - C H - O C H . F. m / e 101 C H . C 2 m/e 145 C H 3 0 C H < H C H 2 K, m/e 8 8 ig 2 3 - . S o u r c e o f t h e ions of methy l 2 3 4 - t r i - o - m e t h y l - ^ D -glucosic le. - 1 0 7 -method i s s u i t a b l e f o r the i d e n t i f i c a t i o n of the methyl g l y c o s i d e of 5,4--di-O-methyl-D-glucose. The f r a g m e n t a t i o n p a t t e r n of the methyl g l y c o s i d e of 2 , 3 , 4 - t r i - O - m e t h y l - D - g l u c o s e i s shown i n F i g . 2 3 . c) S i l y l d e r i v a t i v e s of carbohydrate d e r i v a t i v e s : The mass s p e c t r a l a n a l y s i s of x j ^ l o s y l - g l y c e r o l has not been r e p o r t e d i n the l i t e r a t u r e . 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 x y l o s y l - g l y c e r o l showed a i'I-13 peak of yjo i n t e n s i t y at m/e 5 6 9 . The peak at m/e 389 which i s due t o i-i-(15+90+90) was q u i t e prominent ( 3 / ) . I'he peak at m/e 34-9 i s due to the i o n formed by the l o s s of the s u b s t i t u e n t at C-1 of the x y l o s e moiety of x y l o s y l - g l y c e r o l . d e r i v a t i v e of 5-0-methyl-D-xylose i s i l l u s t r a t e d i n F i g . 26. 1 0 0 - — ! -P •H CO d CD •P H i 50 •H -p 05 i H CD 45 73 5 0 i 1 8 8 101 100 129 147 161 101 2 0 4 217 150 m/e 200 i r o 00 2 5 9 250 Figure 24. Mass. spectrum of t r i m e t h y l s i l y l d e r i v a t i v e of x y l o s y l - g l y c e r o l G O Cr O -Relative I n t e n s i t y , Y<> O O OJ -o 0 1 hi & ro U l ~ o 0 J U) CD o pi cn M f 1 M I - 1 CO o co CO o 1 O o CD O c i -H H-o CD o c f j - ' c r c<! I-1 • CO H-h j O (—1 0 4 H-(D <! _.N ; . J c t O H-<J CD O -o o Ul o~ o -CD CD U l CD CD O O T Ul o Ul 'CD CD Ul UJ 60U -- 110 -45 73 58 75 101 F i g . 2 5 . Mass spectrum of TMS-3-0-methyl-2,4-di-O-TMS-D-xylopyranoside 146 159 131 191 2 1 7 5 0 100 1 5 0 m/e 2 0 0 - 112 -A p a r t i a l s t r u c t u r e of sapote gum: Heteropolysaccharid.es are not s y n t h e s i s e d by a template mechanism and very few enzymes show absolute s p e c i f i c i t y , although they may be very h i g h l y s e l e c t i v e . Exudate gums are s t r u c t u r a l l y more complex than other groups of p l a n t p o l y s a c c h a r i d e s ( 71'- ) • Hence, i t i s r a t h e r d i f f i c u l t to propose a s t r u c t u r e which would not have any l i m i t a t i o n s . According to P a i n t e r ( S 3 ) , the s t r u c t u r e of a h e t e r o p o l y s a c c h a r i d e should be described, i n terms of p r o b a b i l i t y f u n c t i o n s . However, i t i s q u i t e apparent from the present i n v e s t i g a t i o n t h a t sapote gum does hot possess the same s t r u c t u r a l p a t t e r n as o r i g i n a l l y proposed by Smith (7) based on white's data.(4,5,6). L i k e most other p l a n t gums ( 7ZI- ), sapote gum does not have a r e p e a t i n g u n i t s t r u c t u r e . A p o s s i b l e p a r t i a l s t r u c t u r e of sapote gum which may account f o r most of the experimental r e s u l t s i s r e p r e s e n t e d i n P i g . 27 . - 113 -o C - D - G l u A 1 >2XyM A ^ - L - A r a f 1 >2Xyl 3—lAraf-L-c 1 L-Ara-p-1 >2Xyl 1 A R - > 4 - F - D - X y M f2 4 - O - M e - c K - D ' G l u A l * 2 P -D -Xy l 1 ^2 4 - 0 - M e - < - D - G ! u A 1 4 - P - D -Xy 1-1 > 4-P-D-Xy 1-1 >-* 2 A 3 R e* -D-Glu A 1 c<-L -Ara1 1—>2-D~Xyl3—1Araf-L-o<. *4 P-D-XyH t 4 c<-D -GluA1—>2-D-XyH |4 P -D-Xyl1 F i g u r e 2?'. A p o s s i b l e p a r t i a l s t r u c t u r e of sapote gum. GluA r e p r e s e n t s g l u c u r o n i c a c i d and R r e p r e s e n t s x y l o s e c h a i n s . EXPERIMENTAL - 115 -Paper chromatography was c a r r i e d out on v/hatman Nos. 1 and JHFI papers w i t h the f o l l o w i n g s o l v e n t systems ( v / v ) : A. Methyl e t h y l ketone-water azeotrope B. E t h y l a c e t a t e - p y r i d i n e - w a t e r , 8:2:2 C. E t h y l a c e t a t e - a c e t i c a c i d - f o r m i c a c i d - w a t e r , 18:3:1 :4 The descending technique was used f o r paper chroma-tography. Sugars were d e t e c t e d w i t h p - a n i s i d i n e t r i c h l o -r o a c e t a t e . Unless otherwise s t a t e d , a l l evaporations were c a r r i e d out under reduced pressure at a bath temperature of 40°. The m e l t i n g p o i n t s r e p o r t e d are u n c o r r e c t e d and the s p e c i f i c r o t a t i o n s quoted are e q u i l i b r i u m v a l u e s . G a s - l i q u i d chromatography was c a r r i e d out on an P and M 7 2 0 d u a l column instrument f i t t e d w i t h a thermal conduct-i v i t y d e t e c t o r . Helium v/as used as the c a r r i e r gas. Peak areas were ob t a i n e d w i t h the help of an automatic d i g i t a l i n t e g r a t o r . The mass s p e c t r a v/ere recorded on an A.E.I.M.S. 9 mass spectrometer at an i o n i z i n g p o t e n t i a l of 7 0 e.v. The sample of sapote gum which has been used i n the i n v e s t i g a t i o n i s p a r t of the sample o r i g i n a l l y s t u d i e d by Anderson ( 1 ) . - 116 -P u r i f i c a t i o n of the gum: The n a t u r a l gum (25.5 g) c o n s i s t i n g of brown nodules was d i s s o l v e d i n water ( 1 5 0 m l ) , the s o l u t i o n being s t i r r e d c o n t i n u o u s l y . I t went i n t o s o l u t i o n over a p e r i o d of f o u r hours. The s o l u t i o n had a pH of 3*5 •which was a d j u s t e d to pH 2.5 by adding a few drops of cone, h y d r o c h l o r i c a c i d . The s o l u t i o n was immediately t r a n s f e r r e d to a beaker c o n t a i n i n g e t h a n o l (1000 ml) and the p r e c i p i t a t e s were c o l l e c t e d by d e c a n t a t i o n . The p o l y s a c c h a r i d e was .washed w i t h e t h a n o l and d r i e d by treatment w i t h p e t . ether (50°- 60°). The y i e l d of p o l y s a c c h a r i d e was 21.S g. The p u r i f i c a t i o n process was repeated by d i s s o l v i n g the p o l y s a c c h a r i d e (21 g) i n water ( 5 0 0 ml) w i t h constant s t i r r i n g over a p e r i o d of 50 minutes. The s o l u t i o n was t r a n s f e r r e d t o e t h a n o l (1000 ml) and the p r e c i p i t a t e s -were r i n s e d s e v e r a l times w i t h e t h a n o l and p e t . ether ( 5 0 ° - 60°) as b e f o r e . The y i e l d of the d r i e d m a t e r i a l was 1 9 . 2 g. Determination of e q u i v a l e n t weight: A small p o r t i o n of the p o l y s a c c h a r i d e (610 mg) was d i s s o l v e d i n water (10 ml). A s o l u t i o n of sodium hydroxide (0.04 I I , 25 ml) was added to the above s o l u t i o n . o The s o l u t i o n was warmed to 55 f o r 50 minutes. A l i q u o t p o r t i o n s were t i t r a t e d a g a i n s t o x a l i c a c i d (0.008 N) w i t h w i t h p h e n o l p h t h a l e i n as i n d i c a t o r . The e q u i v a l e n t weight was found to be 6 7 6 . - 117 -P r o p i o n a t i o n of sapote P:urn: R o u t i n e l y samples of sapote gum were p r o p i o n a t e d . The procedure d e s c r i b e d i s f o r the amount s t a t e d but the s i z e of the r e a c t i o n c o u l d be a l t e r e d r e a d i l y . A sample of the f i n e l y powdered, p u r i f i e d sapote gum ( 5 g) i n formamide (60 ml) was t r e a t e d w i t h p y r i d i n e ( 4 5 ml) and .propionic anhydride ( 5 0 m l ) . A f t e r storage f o r 2 days, the r e s u l t i n g s o l u t i o n was poured i n t o i c e - c o l d 2 $ h y d r o c h l o r i c a c i d ( 1 l i t r e ) . The p r e c i p i t a t e d m a t e r i a l was f i l t e r e d o f f , washed w i t h i c e - c o l d water, d r i e d i n vacuo, y i e l d 6.2 g. A p o r t i o n of the r e s u l t i n g product (5 g) was r e - t r e a t e d w i t h p y r i d i n e (80 ml) and p r o p i o n i c anhydride ( 1 2 ml) f o r 3 days to y i e l d the p r o p i o n a t e d a c i d (6.8 g ) . F r a c t i o n a t i o n of the p o l y s a c c h a r i d e p r o p i o n a t e : The p o l y s a c c h a r i d e p r o p i o n a t e ( 1 . 7 0 g) was d i s s o l v e d i n c h l o r o f o r m ( 5 0 ml) to g i v e a f a i n t brown s o l u t i o n . Petroleum ether ( 5 0 ° - 6 0 0 ) was added w i t h s t i r r i n g to g i v e p r e c i p i t a t e s , the mixtures being s t i r r e d c o n t i n u o u s l y at the time of each a d d i t i o n . The f r a c t i o n s were removed by c e n t r i f u g a t i c n when d i s t i n c t p r e c i p i t a t e s were formed, washed s e v e r a l times w i t h p e t . ether (30°-6G°). The amount of each f r a c t i o n and i t s o p t i c a l r o t a t i o n are recorded i n Table °J-- 118 -Table 9 F r a c t i o n a l p r e c i p i t a t i o n of sapote gum pr o p i o n a t e i'raction P e t . ether Propionate added ($) p r e c i p i t a t e d 2 r - l 2 ^ (c,1.0 i n CI-IC1-,; m 3 57 80 -41.2° 41 12 -42.4° 3 47 3 -40 . 3 ° P r e p a r a t i o n of DEAE-cellulose column: D i e t h y l a m i n o e t h y l c e l l u l o s e (Whatman powder DE 50, 10 g) was washed a l t e r n a t e l y by 0.5 E h y d r o c h l o r i c a c i d and 0.5 K sodium hydr o x i d e . A f t e r each washing the c e l l u l o s e was allowed to s e t t l e f o r a few minutes a f t e r which the f i n e s and supernatant were decanted. The DE A E - c e l l u l o s e was washed w i t h water before i t was r e s l u r r i e d . The procedure was repeated t h r i c e . The c e l l u l o s e was then d i s p e r s e d i n 0.5'- phosphate b u f f e r , pH 6.1. A st o c k s o l u t i o n of phosphate b u f f e r , pH 6.1, was prepared by d i s s o l v i n g sodium dihydrogen phosphate monohydrate (158 g) i n water (1 1) a n d a d j u s t i n g to pli 6.1 w i t h c o n c e n t r a t e d sodium hydroxide. The d e s i r e d b u f f e r c o n c e n t r a t i o n s were ob t a i n e d by making a p p r o p r i a t e - 119 -d i l u t i o n s of t h i s s o l u t i o n and a d j u s t i n g the pli to 6 . 1 . The column was prepared by adding a t h i n s l u r r y of c e l l u l o s e to a column which c o n t a i n e d , from the bottom, a l a y e r of washed sand about 2 cm. deep. To prevent d i s r u p t i o n of the c e l l u l o s e when sample or b u f f e r was added, the top of the column was covered w i t h a l a y e r of washed sand (2 crn.). P o l y s a c c h a r i d e (60 mg.) was d i s s o l v e d i n 0 . 0 0 5 i'-i-sodium dihydrogen phosphate b u f f e r (pli 6 . 1 , 5 ml) and poured on t o a column of d i e t h y l a m i n o e t h y l c e l l u l o s e (55 cm x 2 .5 cm). The column was e l u t e d w i t h 0 . 0 2 5 I'-— sodium dihydrogen phosphate (500 ml) and a g r a d i e n t of sodium hydroxide (0 . 0 1 - 0 . 5 ^ , 200 m l ) . F r a c t i o n s (ca 10 ml) were c o l l e c t e d and analysed f o r sugar by the p h e n o l - s u l p h u r i c a c i d method (page 121) . P o l y s a c c h a r i d e (57 mg) was e l u t e d i n a s i n g l e band w i t h aqueous s o l u t i o n of sodium hydroxide. The a l k a l i n e s o l u t i o n was d i a l y s e d a g a i n s t running tap water and the cont e n t s of the bag were d e i o n i s e d by passage through A m b e r l i t e IR - 1 2 0(H +) r e s i n . The p o l y s a c c h a r i d e showed a s p e c i f i c r o t a t i o n [c<3 D -6.6 ( c , 1.0 i n water) and e q u i v a l e n t weight of 675 obt a i n e d by t i t r a t i o n as b e f o r e , page 116. - 120 -M o l e c u l a r W e i g h t D e t e r m i n a t i o n o f S a p o t e Gum: S a p o t e gum (1.0 g . ) was d i s s o l v e d i n w a t e r (25 m l . ) and s o d i u m b o r o h y d r i d e (500 mg . ) was added t o i t . The s o l u t i o n was k e p t o v e r n i g h t a t room t e m p e r a t u r e . E x c e s s b o r o h y d r i d e was d e s t r o y e d by t r e a t m e n t w i t h i o n - e x c h a n g e r e s i n I R - 1 2 0 ( H + ) . I t was t h e n d i a l y s e d a g a i n s t r u n n i n g t a p w a t e r f o r 24- h r s . The c o n t e n t s o f t h e b a g were e v a p o r a t e d t o a t h i c k s y r u p (985 mg. ) A p a r t o f t h e m a t e r i a l (4-57 mg. ) was d i s s o l v e d i n m e t a p e r i o d i c a c i d s o l u t i o n ( 0 . 2 M , 5 . 0 m l . ) and k e p t i n t h e d a r k a t room t e m p e r a t u r e . A f t e r 12 h r s . an a l i q u o t ( 2 . 0 m l . ) was r e m o v e d . The p e r i o d a t e was p r e c i p i t a t e d f r o m s o l u t i o n by t h e a d d i t i o n o f s a t u r a t e d l e a d a c e t a t e s o l u t i o n (4- ml . . ) I n t o t h e s o l u t i o n was t h e n i n t r o d u c e d a l e n g t h o f c e l l o p h a n e d i a l y s i s t u b i n g c o n t a i n i n g a vo lume (5 m l . ) o f d i s t i l l e d w a t e r . The s y s t e m was a l l -owed t o s t a n d f o r 12 h r s . a t room t e m p e r a t u r e . D u p l i c a t e a l i q u o t s ( 1 . 0 m l . ) were removed f r o m t h e s o l u t i o n w i t h i n t h e d i a l y s i s b a g , m i x e d w i t h c h r o m o t r o p i c a c i d r e a g e n t ( 5 . 0 m l . ) , and c e n t r i f u g e d t o c l a r i f y . The c l e a r s u p e r -n a t a n t was d e c a n t e d i n t o a m a t c h e d t u b e and h e a t e d , p r o t e c t e d f r o m l i g h t , f o r 30 m i n u t e s a t 1 0 0 ° . A f t e r c o o l i n g t o room t e m p e r a t u r e , t h e a b s o r b a n c e o f c o l o u r p r o d u c e d was d e t e r m i n e d i n a Co leman J u n i o r S p e c t r o p h o t o -m e t e r a t 570 mj* . The c o n c e n t r a t i o n o f f o r m a l d e h y d e was - 1 2 1 -determined by r e f e r e n c e to a standard curve. Formaldehyde p r o d u c t i o n a f t e r 1 2 h was 0.2 mg which corresponded to a number average molecular weight (M n) of sapote gum of 6 9 , 0 0 0 . The value was c a l c u l a t e d on the b a s i s t h a t i n a 1 — 4 l i n k e d x y l a n one mole of formaldehyde was produced per reduced a l d i t o l group. P r e p a r a t i o n of the Chromotropic a c i d reagent: The reagent was prepared by adding a f i l t e r e d s o l u t i o n of naphthalene-4-,5-dihydroxy-2,7-disulphonic a c i d disodium s a l t ( 1 . 0 0 g) i n water ( 1 0 0 ml.) to a s o l u t i o n (4-50 ml.) composed of c o n c e n t r a t e d s u l p h u r i c a c i d ( 3 0 0 ml.) and water ( 1 5 0 ml.) E s t i m a t i o n of sugar by p h e n o l - s u l p h u r i c a c i d method: Sugar s o l u t i o n ( 1 ml) c o n t a i n i n g 1 0 ug to 7 0 ug was t r e a t e d w i t h 2 ml of 5 $ phenol and 5 nil of reagent grade c o n c e n t r a t e d s u l p h u r i c a c i d . The c o l o u r developed almost immediately. A f t e r c o o l i n g to room temperature the absorbances were measured i n a spectrophotometer at 480 mu f o r pentose sugars. A blank was processed i n a s i m i l a r way. A standard curve f o r p h e n o l - s u l p h u r i c a c i d r e a c t i o n of x y l o s e i s shown i n F i g . 28. - 122 -Figure 28. Standard curve f o r phenol-sulphuric acid r e a c t i o n of xylose - 1 2 3 -Determination of formaldehyde:  Lead acetate method The method may be i l l u s t r a t e d by r e f e r e n c e to e r y t h r i t o l . A 2-ral a l i q u o t of standard e r y t h r i t o l s o l u t i o n ( 8 5 * 7 i n 1 0 0 ml of water) was t r e a t e d w i t h 0.2M sodium metaperiodate (2 ml.) f o r 1 3 minutes at room temperature a f t e r which s a t u r a t e d l e a d a c e t a t e ( 1 ml.) was added. The mixture was d i l u t e d to 1 0 0 ml. and c e n t r i f u g e d t o remove l e a d i o d a t e and p e r i o d a t e . Water (2 ml.) was t r e a t e d i n the same way and served as the reagent blank. A l i q u o t s of t h i s s o l u t i o n (or the b l a n k ) c o n t a i n i n g 2 to 8j4 g . formaldehyde ( 0 . 3 ml to 1.0 ml.) were removed to c e n t r i f u g e tubes and d i l u t e d t o 1.0 ml. by a d d i t i o n of water. Chromotropic a c i d ( 1 0 ml. ) was added to each tube, and the s m a l l amount of l e a d sulphate which formed was removed by c e n t r i f u g a t i o n . The l i g h t arnber s o l u t i o n s were then c a r e f u l l y decanted i n t o matched c o l o r i m e t e r tubes. The tubes and contents were heated i n a b o i l i n g water bath f o r 3 0 minutes w i t h e x c l u s i o n of l i g h t . The tubes were promptly c o o l e d i n c o l d water, and the' absorbances were immediately measured at 5 7 0 m|* w i t h a spectrophotometer. Each d e t e r m i n a t i o n was done i n d u p l i c a t e and the average value taken a f t e r c o r r e c t i n g f o r the a p p r o p r i a t e blank experiment. The p l o t of absorbancy a g a i n s t formaldehyde c o n c e n t r a t i o n i s shown i n E i g . 2 9 . Figure 29. Standard curve f o r formaldehyde by the chrbraotropic a c i d method - 125 -T o t a l a c i d h y d r o l y s i s of sapote gum: P u r i f i e d sapote gum (2.00 g) was d i s s o l v e d i n s u l p h u r i c a c i d (1ft, 40 ml) and h y d r o l y s e d over a steam bath f o r a p e r i o d of 6 hours. The brown s o l u t i o n was f i l t e r e d and n e u t r a l i s e d w i t h a s l u r r y of barium carbo-nate. The s o l u t i o n was f i l t e r e d and d e i o n i s e d by passage through c a t i o n exchange and anion exchange r e s i n s s u c c e s s i v e l y . The n e u t r a l components (1.15 g) were examined by paper chromatography i n s o l v e n t system B. Only spots corres p o n d i n g to arabinose and x y l o s e were observed. A p o r t i o n of the m a t e r i a l (20 mg.) was d i s s o l v e d i n anhydrous p y r i d i n e kept over potassium hydroxide p e l l e t s (1 m l . ) . E e x a m e t h y l d i s i l a z a n e (0.4 ml.) and t r i m e t h y l -c h l o r o s i l a n e (0.2 ml) were added to the sugar s o l u t i o n . The mixture was shaken v i g o r o u s l y at room temperature f o r 50 seconds and then allowed t o stand f o r 5 minutes. The mixture was evaporated to dryness u n t i l there was a f a i n t s m e l l of p y r i d i n e . Cyclohexane (4 ml.) was added to the evaporated sample and i t was a g i t a t e d to d i s s o l v e t r i m e t h y l s i l y l e t h e r s . Small amounts of sample were i n j e c t e d i n t o a g a s - l i q u i d chromatography S e p a r a t i o n of sugars was c a r r i e d out on a copper-column (8' x ;•'."), packed w i t h 20/ SP 96 on 60-80 mesh D i a t o p o r t S, i s o t h e r m a l l y at 190° f o r 5 minutes and then programmed at 3° por min. to h o l d at 250°. Only peaks - 126 -corresponding to the t r i n e t h y . l s i l y l d e r i v a t i v e s of arabinose and x y l o s e were observed. The a c i d i c components of the a c i d h y d r o l y z a t e s of sapote gum were i s o l a t e d from the column c o n t a i n i n g anion exchange r e s i n ( D u o l i t e A-4 r e s i n ) by e l u t i n g w i t h 10$ f o r m i c a c i d (10 m l ) . The column was washed w i t h water (500 ml) u n t i l i t gave a n e g a t i v e r i o l i s c h t e s t . The s o l u t i o n was f r e e z e d r i e d (0.80 g ) . The m a t e r i a l was ob t a i n e d as amorphous powder. A s m a l l p o r t i o n of the m a t e r i a l was examined by paper chromato-graphy i n s o l v e n t system C. Pour spots having R X Y i 0 e e 1.16, 0.97, 0.75 and 0.60 were observed. A p o r t i o n of the a c i d i c components (295 nig) was chromatographed on Whatman IIo. papers i n s o l v e n t system C to g i v e f o u r d i f f e r e n t f r a c t i o n s . The f r a c t i o n s were l o c a t e d by s p r a y i n g h a l f - i n c h s t r i p s out from edges of the sheet w i t h p - a n i s i d i n e t r i c h l o r o a c e t a t e reagent. Bands corresponding to ^ y - y ] _ o s e 1«16, 0.97, 0.75 and 0.62 were cut out and e l u t e d w i t h water. F r a c t i o n 1 (31 mg): 4 - 0-Methyl-D-glucuronic a c i d . — The m a t e r i a l h a d F ] " +83.2° (c 1.2 i n water) and i d e n t i c a l chromatographic m o b i l i t y w i t h t h a t of 4-0-methyl-D-g l u c u r o n i c a c i d . F r a c t i o n 2 (115-2): 4 - 0-Nethyl-D-glucopyranosyluronic a c i d (1 2 ) D - x y l o p y r a n o s e . — The compound hadM^f+105° - 1 2 7 -(c 1.0 i n water) and chromatographic m o b i l i t y ^ X y ] _ o s e 0 . 9 7 . F r a c t i o n 5 (26.7 nig): D-Glucuronic a c i d . — I t had £°^2£ +33° ( c , 1.0 i n water) and chromatographic m o b i l i t y R x y l o s e 0 . 7 5 . F r a c t i o n 4 ( 1 0 7 . 0 mg): D-Glucopyranosyluronic a c i d 2J Q ( 1 - - - 2 ) J J - x y l o p y r a n o s e . — I t had IKJJ, +87 (c 1.0 i n water) and chromatographic m o b i l i t y .0.62. Thin l a y e r chromatography of a l d o b i o u r o n i c a c i d s : S i l i c a g e l ( 3 5 g) v/as mixed w i t h gypsum (2.5 s ) i n disodium hydrogen phosphate (0.3H, 1 0 0 m l . ) . The sus-p e n s i o n o b t a i n e d was a p p l i e d to the g l a s s p l a t e s at a t h i c k n e s s of about 0.5 mm. These p l a t e s were allowed to stand f o r 24 hours at room temperature and then used f o r chromatography. The p l a t e s were developed by the ascending t e c h n i q u e . B u t a n o l - e t h a n o l - 0 . 1 K h y d r o c h l o r i c a c i d ( 1 : 1 0 : 5 , v/v) was used as the s o l v e n t system. The p l a t e s were developed i n tanks l i n e d w i t h f i l t e r paper. P l a t e s took 2 hours f o r development. P l a t e s were sprayed w i t h c o n c e n t r a t e d s u l p h u r i c a c i d and heated at 1 5 0 ° . Thin l a y e r chromatographic data of a l d o b i o u r o n i c a c i d s Compounds i-i-, 2-0- (ttf -D-Glucuronosyl) D-xylose 0 . 1 5 2-0- (4-0-he thy l-o(-D-glucuronosyl) D-xylose 0 . 5 8 - 128 -M e t h y l a t i o n of sapote gum p o l y s a c c h a r i d e : The method d e s c r i b e d i s f o r t h i s amount but the • s i z e of the r e a c t i o n c o u l d be a l t e r e d r e a d i l y . 1. P r e p a r a t i o n of m e t h y l s u l p h i n y l anion: Into a dry, 250 ml. three-necked round bottom f l a s k f i t t e d w i t h serum caps and c o n t a i n i n g a magnetic s t i r r -i n g bar was weighed 2.5 g of sodium h y d r i d e , 5 0 $ o i l d i s p e r s i o n . The sodium hydride was washed three times by s t i r r i n g w i t h dry pet., e t h e r (50°-60°) under an atmosphere of dry n i t r o g e n and decanting the wash. A f t e r the t h i r d wash, the r e s i d u a l petroleum ether was evacuated w i t h a vacuum pump through an 18-gauge needle i n s e r t e d i n t o the serum cap. Dimethyl sulphoxide (20 ml. d i s t i l l e d from c a l c i u m hydride under reduced pressure and s t o r e d over d r i e d molecular s i e v e s , was t r a n s f e r r e d i n t o the f l a s k . The mixture was s t i r r e d at 5 0 ° u n t i l the s o l u t i o n became c l e a r , green and e v o l u t i o n of hydrogen gas ceased ( c a . 2 h o u r s ) . 2. Generation of the p o l y s a c c h a r i d e a l k o x i d e : Sapote gum p o l y s a c c h a r i d e was f i r s t passed throug:. a 200-mesh sieve.and d r i e d f o r a p e r i o d of 6 hours at 60° under reduced p r e s s u r e . D r i e d m a t e r i a l (1.24 g) was added to 60 ml. of dry d i m e t h y l sulphoxide i n a 250-ml. three-necked round-bottom, f l a s k c o n t a i n i n g a magnetic bar and f i t t e d w i t h serum caps through which reagents were i n t r o d u c e d and n i t r o g e n gas was passed c o n t i n u o u s l y . The suspension was heated at 60° and s t i r r e d w i t h a magnetic bar u n t i l ( c a . 1 hour) a l l of the p o l y s a c c h -a r i d e d i s s o l v e d to form a pale brown s o l u t i o n . A f t e r c o o l i n g the s o l u t i o n to room temperature, m e t h y l s u l p h i -n y l anion ( 1 5 ml.) was added to the p o l y s a c c h a r i d e s o l u t i o n . The r e s u l t i n g s o l u t i o n became t h i c k and was s t i r r e d f o r 6 hours at room temperature when the mixture appeared homogeneous... 3. M e t h y l a t i o n r e a c t i o n : The p o l y s a c c h a r i d e a l k o x i d e s o l u t i o n was immersed i n an i c e - w a t e r bath and f r e s h l y d i s t i l l e d methyl i o d i d e ( 5 ml.) was added to the s t i r r e d s o l u t i o n at a v e r y slow r a t e such t h a t the temperature d i d not r i s e above 20° (50 min.). W i t h i n a few minutes a f t e r a d d i t i o n of methyl i o d i d e , the s o l u t i o n became c l e a r and v i s c o s i t , y was marked-l y reduced. The mixture was s t i r r e d at room temperature f o an a d d i t i o n a l p e r i o d of 6 hours. Water (60 ml.) was added to the brown c o l o u r e d s o l u t i o n . Immediate p r e c i p i -t a t i o n took p l a c e . The r e a c t i o n mixture was d i a l y s e d o v e r n i g h t vs. running tap water and e x t r a c t e d c o n t i n u o u s l y w i t h c h l o r o f o r m . The chloroform e x t r a c t was d r i e d over anhydrous sodium sulphate and was f i n a l l y evaporated to dryness at 40° under reduced p r e s s u r e . The m e t h y l a t i o n r e a c t i o n was complete as no OH band was found i n the i n f r a -r e d spctrum. The y i e l d of the methylated product was 1.38 - 130 -H y d r o l y s i s of the methylated, sapote gum polysaccharide :: M ethylated p o l y s a c c h a r i d e had fol -48.2° (c 6.2, c h l o r o f o r m ) and methoxyl content 39.3^« A. p o r t i o n of the methylated p o l y s a c c h a r i d e (1.02 g) was d i s s o l v e d i n s u l p h u r i c a c i d (72/, 8 ml) i n a round bottomed f l a s k e x t e r n a l l y c o o l e d w i t h i c e - w a t e r . The s o l u t i o n was kept at room temperature f o r 1 hour to s o l u b i l i z e the p o l y -s a c c h a r i d e . Water was then added to b r i n g the a c i d c o n c e n t r a t i o n t o 8/ and the h y d r o l y s i s mixture was heated at 100° f o r 4 hours, then c o o l e d to room temperature, and. n e u t r a l i s e d w i t h barium carbonate. The p r e c i p i t a t e d barium sulphate was separated by f i l t r a t i o n , s o l i d s being washed s e v e r a l times w i t h water and e t h a n o l . The f i l t r a t e and the e x t r a c t s were combined and evaporated to a syrup at 35° i n vacuo. 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 0-methyl sugars: The mixture of the sugars (996 mg) was d i s s o l v e d i n water passed through a column of A m b e r l i t e IR-120(U +) r e s i n , and the r e s i n was washed w i t h water u n t i l i t gave a n e g a t i v e mo.lisch t e s t . The e f f l u e n t from the c a t i o n exchange r e s i n was passed through a column of D u o l i t e A-4 r e s i n which s e l e c t i v e l y separated the a c i d i c component. The column was washed u n t i l i t gave a n e g a t i v e l - o l i s c h t e s t . The washings were evaporated to a syrup of constant weight (617 mg). - 1 3 1 -The a c i d i c component was i s o l a t e d from the column by e l u t i n g w i t h f o r m i c a c i d ( 1 0 $ , 1 0 ml). Eva p o r a t i o n of the e f f l u e n t f u r n i s h e d the a c i d i c component ( 3 5 5 mg)• The mixture of the methylated n e u t r a l sugars was examined by paper chromatography u s i n g s o l v e n t system A. S e p a r a t i o n of the n e u t r a l components of methylated  n e u t r a l sugars by paper chromatography: A s m a l l amount of methylated n e u t r a l sugars ( 9 6 mg) was r e s o l v e d u s i n g sheets of Whatman ho 1 paper and s o l v e n t A. The q u a n t i t i e s of the components are recorded i n Table 1 0 . Table 1 0 Sep a r a t i o n s of n e u t r a l components of methylated sapote gum by paper chromatography Component Weight, mg. I d e n t i t y 1,2 5 7 . 2 5,4 1 5 . 0 5 1 0 . 0 6 8.5 2,5,5-'Pr i-O-me thy 1-L-arabinose 2,3,4-Tri-O-methy1-D-x y l o s e 2,3,4-Tri-O-mothyl-L-arabinose 2,3-Di-O-methyl-D-x y l o s e D i s a c c h a r i d e V\ o rt o - 0 - m e 11 \ y 1 - D-xy l o s e 7 2 0 . 1 D-Xylose - 132 -C h a r a c t e r i s a t i o n of components: (a) Components 1 and 2: 2 3 o The syrup (37.2 mg) - 27.6 ( c , 1.5 i n methanol) D showed the presence of two o v e r l a p p i n g s p o t s , both having a value c l o s e to 0.80. Comparison w i t h a u t h e n t i c samples i n d i c a t e d t h a t t h i s was c o n s i s t e n t w i t h the behaviour of 2,5 , 5-tri-O-methyl-L—arabinose and 2,3,4-tri-O-methyl-h-xylose. The r o t a t i o n s of these compounds ar e , r e s p e c t i v e l y , -38..5° and +18.5° (84), which show components 1 and 2 t o be a mixture of 2,3,5-t r i - O - m e t h y l - L — a r a b i n o s e and 2,3,4-tri-O-methyl-D-xylose, the molar r a t i o s being 4.3:1. The syrup (37.0 mg) was reduced by an aqueous s o l u t i o n of sodium borohydride (40 mg). The solu t i o n -was kept f o r 18 hours at room temperature. The s o l u t i o n was d e i o n i s e d by treatment w i t h c a t i o n exchange r e s i n ( A m b e r l i t e I.R-120 I i + ) and evaporated to dryness. Borate was removed by the a d d i t i o n of methanol. The r e s i d u e was heated w i t h a mixture of a c e t i c anhydride and p y r i d i n e (1:1, 10 ml) f o r 3 hours at 100°. The r e a c t i o n mixture was evaporated to dryness and d i s s o l v e d i n a s m a l l amount of c h l o r o f o r m (1 m l ) . The mixture was examined on a g a s - l i q u i d chromatogram u s i n g a column (S'xX") c o n t a i n i n g 3$ (w/w) of ECH.33-M on Gas Chrom Q - 135 -(100 -120 mesh) u s i n g a temperature programme 160°-180°. The columns were h e l d i s o t h e r m a l l y at 160° f o r 3 min. and then programmed at 2°/ min to h o l d at 180°. The r e l a t i v e r e t e n t i o n times of 2,5,5-tri-O-methyl-L-a r a b i n i t o l and 2,3,4-tri-O-me t h y l - l ) - x y l i t o l were 1 and 1.14 w i t h a molar r a t i o of 4.5:1 r e s p e c t i v e l y , (b) Components 5 and 4: The mixture ( 1 5 mg) showed the presence of two spots having v a l u e s 0.65 and 0.57. These spots were c h r o m a t o g r a p h i c a l l y i d e n t i c a l w i t h 2 , 5 , 4 - t r i - 0 -methyl-L—arabinose (R^ 0.65) and 2,5-di-O-methyl-D-x y l o s e (R£ 0 . 5 7 ) . A p o r t i o n of the m a t e r i a l (9 mg) was reduced w i t h an aqueous s o l u t i o n of sodium borohydride (100 mg, 10 ml) f o r a p e r i o d of 18 hours. The s o l u t i o n was n e u t r a l i s e d and evaporated. '1'he r e s i d u e was t r e a t e d w i t h methanol to remove b o r i c a c i d . The m a t e r i a l was d i s s o l v e d i n a mixture of a c e t i c a n h y d r i d e : p y r i d i n e (1:1, 10 ml) and heated at 100° f o r 2 hours. The brown s o l u t i o n was evaporated to dryness and d i s s o l v e d i n a s m a l l amount of c h l o r o f o r m (1.0 m l ) . A l i q u o t p o r t i o n s wei^e examined on a g a s - l i q u i d chromatograph u s i n g the same column and c o n d i t i o n s as d e s c r i b e d f o r the s e p a r a t i o n of components 1 and 2. Standard a l d i t o l a c e t a t e s of 2 , 5 , 4 - t r i - 0 - m e t h y l -arabinose and 2 , 3 - d i - 0 - m e t h y l - D - x y l i t o l a c e t a t e s were - 134 -i n j e c t e d under i d e n t i c a l c o n d i t i o n s i n order to i d e n t i f y the components. (c) Component 5:-The m a t e r i a l (10 mg) [og^ -25.3 (c 1.0 i n water) had chromatographic m o b i l i t y R f 0.40 i n s o l v e n t system A . The i d e n t i t y of t h i s component was r e v e a l e d by the f o l l o w i n g e x perimental procedures c. i ) A c i d h y d r o l y s i s A s m a l l p o r t i o n of the m a t e r i a l (3 mg) 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 (1N, 2ml) at 100° f o r 6 hours. The h y d r o l y s a t e was n e u t r a l i s e d w i t h a s l u r r y of barium carbonate, d e i o n i s e d by passage through c a t i o n exchange r e s i n and evaporated to dryness. The m a t e r i a l was examined by paper chromatography u s i n g s o l v e n t system A.. Only spots corresponding to x y l o s e and 2 , 3-cli-O-methyl-xylose were observed. i i ) Reduction and a c i d h y d r o l y s i s :. A p o r t i o n of the m a t e r i a l (3 mg) was reduced w i t h an aqueous s o l u t i o n of sodium borohydride (10 mg, 10 ml) f o r a p e r i o d of 16 hours... D e i o n i s a t i o n and removal of b o r i c a c i d was c a r r i e d out i n the u s u a l way. The r e s i d u e 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 (IM, 2 ml) at 100° f o r 6 hours, n e u t r a l i s e d w i t h a s l u r r y of barium carbonate and d e i o n i s e d as b e f o r e . The m a t e r i a l was examined by paper chromatography u s i n g s o l v e n t system ,A. The compounds were dete c t e d w i t h p - a n i s i d i n e t r i c h l o r o -- 1 5 5 -acetate and s i l v e r n i t r a t e - s o d i u m hydroxide r e a g e n t s . Spots corresponding to x y l i t o l and 2,5-di-G-methyl-D-xylose were observed. i i i ) M e t h y l a t i o n s t u d i e s : The remainder of the m a t e r i a l (4 mg) was d i s s o l v e d i n dry d i m e t h y l s u l p h o x i d e ( 0 . 5 nil) at room temperature, and converted i n t o the sugar a l k o x i d e w i t h f r e s h methyl s u l p h i n y l carbanion ( 0 . 5 m l ) , prepared by the method of . Hakomori ( f o r d e t a i l s c f . page 128 ) at room temperature f o r 5 hours w i t h s t i r r i n g under n i t r o g e n atmosphere. The sugar a l k o x i d e was methylated by the a d d i t i o n of methyl i o d i d e ( 0 - 5 ml) at 2 0 ° , and the methylated product was e x t r a c t e d w i t h c h l o r o f o r m ( 1 0 ml x 3 ) . i'he combined e x t r a c t s were washed s e v e r a l times w i t h water, dehydrated w i t h anhydrous sodium sulphate and evaporated to a syrup. The'methylated product was methanolysed by r e f l u x -i n g i t w i t h 3 $ methanolic hydrogen c h l o r i d e (2 ml) f o r 6 hour's. The methanolysate was n e u t r a l i s e d w i t h s i l v e r carbonate and s u b j e c t e d to g a s - l i q u i d chromatography on a column of 5 $ b u t a n e d i o l s u c c i n a t e i s o t h e r m a l l y at 1 2 0 ° f o r 5 minutes and then programmed at 2° per min. to h o l d at 1 7 0 ° . Two peaks corresponding to c< andj*-methyl-glycosides of 2 , 5 - d i - 0 - m e t h y l - D - x y l o s e were observed. - 136 -d) Component 6 (8.5 mg): The m a t e r i a l was s i l y l a t e d by d i s s o l v i n g i n p y r i d i n e ( 2 ml) and t r e a t i n g w i t h h e x a m e t h y l d i s i l a z a n e ( 0 . 5 ml) and t r i m e t h y l c h l o r o s i l a n e ( 0 . 3 m l ) . The m a t e r i a l was evaporated and d i s s o l v e d i n cyclohexane ( 2 m l ) . A l i q u o t s were i n j e c t e d onto a column of SE-52 ( 8 " x ; ' " ) , the column being h e l d at 1 1 0 ° f o r 3 min. and then programmed at 3 ° per min. to h o l d at 140°. Standard s i l y l a t e d s o l u t i o n of 3-0-methyl-D-x y l o s e was i n j e c t e d under i d e n t i c a l c o n d i t i o n s . S e p a r a t i o n of the n e u t r a l components of methylated  sapote gum by c e l l u l o s e - c o l u m n chromatography: A mixture of methylated n e u t r a l sugars ( 5 0 1 mg) was d i s s o l v e d i n a s m a l l amount of methyl e t h y l ketone-water azeotrope ( 1 . 5 ml) and p l a c e d on a column (40 x 3 cm.) packed w i t h a mixture of h y d r o c e l l u l o s e and c e l l u l o s e ( 1 : 1 ) . The same s o l v e n t was used f o r e l u t i o n and the e f f l u e n t was c o l l e c t e d at 1 3 min. i n t e r v a l s f o r 1 1 5 tubes and then at 3 0 min. i n t e r v a l s f o r the r e s t of the tubes. The d i s t r i b u t i o n of the sugars was determined by p l a c i n g a few drops of the s o l u t i o n of each tube on paper and s p r a y i n g w i t h p - a n i s i d i n e t r i c h l o r o -a c e t a t e . C e r t a i n tubes were co n c e n t r a t e d and examined chromatographica.lly before the contents of the tubes - 137 -Table 11 S e p a r a t i o n of n e u t r a l sugars of methylated sapote gum by c e l l u l o s e - c o l u m n chromatography Tube Number Component Number /eight, mg. I d e n t i t y 22-30 31-50 51-70 71-85 86-100 101-120 140-170 1,2 1 , 2 , 3 4 , 5 5 5 , 6 6 7 15 140 50 25 10 31 97 2 , 3 , 5 - i 1 r i - 0 - m e t h y l - L -arabinose 2 . 5 . 4 - Tri-O-methyl-h-x y l o s e 2 . 5 . 5 - ' 2 r i - 0-methyl-L-arabinose 2 , 3 , 4 - T r i - O - m e t h y l - U -x y l o s e 2 , 3 , 4 - T r i - O - m e t h y 1 - L -arabinose 2 , 5-Di-O-methyl-D-xylose D i s a c c h a r i d e D i s a c c h a r i d e D i s a c c h a r i d e Miono-O-methyl-D-xylose N o n o-0-me thy1-D-xy1o s e D-Xylose T o t a l 483 Recovery 96.6>y - 138 -were c o l l e c t e d , t o g e t h e r . The syrups were r e d i s s o l v e d and evaporated to constant weight. The r e c o v e r y was 96.6$. The r e s u l t s are recorded i n Table 11. C h a r a c t e r i s a t i o n of components i s o l a t e d by c e l l u l o s e - column chromatography: Components 1 and 2: The syrup ( 1 5 2 mg) was d i s s o l v e d i n methanol ( 2 ml.) and r e s e p a r a t e d by c e l l u l o s e column chromatography as before u s i n g methyl e t h y l ketone-water azeotrope as e l u a n t . F r a c t i o n s were c o l l e c t e d at 1 5 min. i n t e r v a l s . The e a r l i e r tubes i . e . , from 2 0 - 2 5 c o n t a i n e d component 1 and the l a t t e r tubes 3 2 - 3 6 c o n t a i n e d component 2 . Component 1 : 2 2 The syrup obtained ( 7 5 mg) had Z^J-Q -38 ( c , 1.0 i n methanol). Examination on a paper chromatogram i n s o l v e n t A r e v e a l e d t h a t the component had i d e n t i c a l chromatographic m o b i l i t y as t h a t of 2 , 5 , 5 - t i ' i - O - m e t h y l -L -arabinose. Component 2: 2 2 The syrup ( 2 5 mg), [oijij +16 ( c , 1.5 i n methanol), was c h r o m a t o g r a p h i c a l l y pure and corresponded w i t h 2 , 3 , 4 — t r i - O - m e t h y l - h - x y l o s e . A p a r t ( 1 0 mg) was methano-l y s e d w i t h methanolic hydrogen c h l o r i d e ( 3 $ , 5 ml) f o r 6 hours. The methanolysecl mateirial was n e u t r a l i s e d w i t h - 139 -s i l v e r carbonate, and the f i l t r a t e evaporated. 'The m a t e r i a l was examined by g a s - l i q u i d chromatography u s i n g a s t a i n l e s s s t e e l column (4'x %") packed w i t h 5$ by weight of bu t a n e - 1 , 4 - d i o l s u c c i n a t e p o l y e s t e r on 80-100 mesh on D i a t o p o r t 8, and the column was run i s o t h e r m a l l y at 120°. Two peaks were observed corresponding to c< and£ methyl g l y c o s i d e s . The components were c o l l e c t e d and the component corresponding t o the -isomer c r y s t a l l i z e d immediately. M.p. and mixed m.p. 49°-50°. Component 3: Contents of the tubes 31-50 (Table 11 ) were separated by paper chromatography. The syrup (29 mg) corr e s p o n d i n g t o 2,3,4-tri-O-methyl-L-arabinose had 23 IK) j) +123° ( c , 1.0 i n w a t e r ) . Demethylation of the component was c a r r i e d out as f o l l o w s : A sample (8 mg) of the syrup was heated f o r 30 min. i n a s e a l e d tube w i t h 48$ hydrobromic a c i d . The tube was c o o l e d i n an acetone-dry i c e m i x t u r e , opened and the contents n e u t r a -l i s e d by passage through an anion-exchange r e s i n ( D u o l i t e A-4) column. The s o l u t i o n was evaporated and the product chromatographed u s i n g s o l v e n t systems A and 3. Components corresponding t o ara b i n o s e , mono-, d i - and t r i - O - m e t h y l -arabinoses were observed. - 140 -Component 4: The contents of the tubes 5 1 - 7 0 (Table 1 1 ) were r e -separated by paper chromatography i n s o l v e n t system A, the paper being developed f o r a p e r i o d of 7 hours. The band corresponding to 2,3-di-O-methyl-h-xylose (18 mg.) v/as cut and e l u t e d w i t h methanol as b e f o r e . A p a r t of the m a t e r i a l ( 3 K g . ) was raethanolysed w i t h methanolic hydrogen c h l o r i d e ( 3 / , 5 ml.) f o r a p e r i o d of 6 hours. The product a f t e r n e u t r a l i s a t i o n w i t h s i l v e r carbonate v/as evaporated t o dryness and examined by g a s - l i q u i d chromatography u s i n g the same column and c o n d i t i o n as d e s c r i b e d f o r component 2 (page 1 3 9 )• Only peaks cor r e s p o n d i n g to < a n d ^ g l y c o s i d e s of 2,3-di-O-methyl-D-x y l o s e s were observed. Component 5 : The m a t e r i a l ( 2 3 mg.) was not examined f u r t h e r as i t had the same chromatographic m o b i l i t y as t h a t of the component 5 d e s c r i b e d on page 134. Component 6: C o n c e n t r a t i o n of the e l u a t e s gave r i s e t o a syrup ( 3 1 mg.) W h i c h had l*J ^ 2 +24.8° ( c , 1.5 i n methanol). On prolonged s t a n d i n g i t c r y s t a l l i z e d , m.p. 9 4 — 9 6 ° Component 7 : C o n c e n t r a t i o n of the contents of tubes 140-170 - 14-1 -y i e l d e d a syrup (97 mg.) which was shown chromatographi-c a l l y to c o n t a i n o n l y x y l o s e . A p a r t of the m a t e r i a l ( 1 2 mg.) was s i l y l a t e d and examined by g a s - l i q u i d chro-matography on a column of 20$ SF-96 on 60-80 mesh D i a t o p o r t 3 (8'x>:"), temperature 1 9 0 ° - 2 2 0 ° at the r a t e of 3° per minute. A n a l y s i s of the a c i d i c f r a c t i o n s from the h y d r o l y s a t e  of the methylated sapote gum: The a c i d i c f r a c t i o n (34-0 mg.) from the h y d r o l y s i s of the methylated p o l y s a c c h a r i d e was r e f l u x e d w i t h methanolic hydrogen c h l o r i d e ( 3 $ , 3 0 ml) f o r 6 hours. The r e s u l t i n g m a t e r i a l a f t e r n e u t r a l i z a t i o n w i t h s i l v e r carbonate and subsequent removal of excess s i l v e r by hydrogen s u l p h i d e , was reduced w i t h l i t h i u m aluminium hydride ( 1 . 0 g) i n t e t r a h y d r o f u r a n ( 3 0 m l ) , the s o l u t i o n b e ing r e f l u x e d f o r 4- hours. A f t e r d e s t r u c t i o n of excess of h y d r i d e by adding d i l u t e aqueous a c e t i c a c i d , the r e a c t i o n mixture was evaporated to dryness. The reduced m a t e r i a l was e x t r a c t e d s e v e r a l times w i t h c h l o r o f o r m . The c h l o r o f o r m e x t r a c t was evaporated t o a syrup ( 3 2 7 mg). The m a t e r i a l 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 ( 1 N , 2 0 ml) at 1 0 0 ° f o r a p e r i o d of 8 hours. The m a t e r i a l was n e u t r a l i s e d w i t h a s l u r r y of barium carbonate, d e i o n i s e d w i t h A m b e r l i t e I R - 1 2 0 ( H + ) r e s i n and concentrated - 14-2 -to a syrup (308 rag). Hydrolysed p r o d u c t s , on chroma-t o g r a p h i c examination on paper i n s o l v e n t system A, were found to have i d e n t i c a l chromatographic m o b i l i t i e s those of 5-0-methyl-D-xylose, 3,4— di-O-methyl-D-glucose and 2 , 3,4—tri-O-methyl-D-glucose. A p a r t of the m a t e r i a l ( 1 0 7 rag) was r e s o l v e d by paper chromatography i n order to o b t a i n 2 ,3,4— tri-O-methyl-D-glucose. By paper chromatography i t v/as not p o s s i b l e t o separate 3-0-methyl D-xylose and 3,4—di-O-methyl-D-glucose. I d e n t i f i c a t i o n of 2,3,4— tri-O-methyl-D-glucose : C o n c e n t r a t i o n of the e l u a t e s corresponding t o Rr G (where g = tetra-O-methyl-D-glucose) 0.64- gave r i s e to 23 a syrup (4-2 rng) which had j><] D +75.3 ( c , 1.1 i n wa t e r ) . A p a r t ( 1 9 mg) of the m a t e r i a l was converted i n t o i t s methyl g l y c o s i d e i n the u s u a l way. The m a t e r i a l v/as then examined on g a s - l i q u i d chromatography u s i n g a column (4-'x/") packed w i t h 5 $ by weight of b u t a n e - 1 , 4 — d i o l s u c c i n a t e p o l y e s t e r on 8 0-100 mesh D i a t o p o r t S w i t h a temperature programme of 1 2 0 ° - 1 7 0 ° at the r a t e of 3 ° per minute. £-Methyl g l y c o s i d e c o l l e c t e d i n a c a p i l l a r y tube c r y s t a l l i z e d a f t e r prolonged s t a n d i n g , m.p., 9 3 ° - 94-- 143 -Reduction of 2 , 5 i 4 - t r i - 0 - m e t h y l - D - g l u c o s e w i t h sodium  borohydride and p e r i o d a t e o x i d a t i o n of the generated 2 , 5 , 4 - t r i - O - m e t h y l - D - g l u c i t o l : The 2 , 3 , 4 - t r i - O - m e t h y l - D - g l u c o s e (20 mg) was d i s s -o l v e d i n water ( 5 0 m l ) , sodium borohydride ( 40 mg) added and r e d u c t i o n a l l o w e d to proceed o v e r n i g h t at room temper-a t u r e . Excess borohydride was destroyed by treatment w i t h A m b e r l i t e I R - 1 2 0 ( H + ) and borate was removed by treatment w i t h methanol. The r e s i d u e was d i s s o l v e d i n 0 . 1 M p e r i o d i c a c i d s o l u t i o n (25 ml) and the r e a c t i o n was c o n t i n u e d f o r a p e r i o d of 24 hours i n the dark. The s o l u t i o n was s a t u r a t e d w i t h sodium sulphate and e x t r a c t e d s e v e r a l times w i t h c h l o r o f o r m ( 5 0 ml a l i q u o t s ) . The combined c h l o r o f o r m e x t r a c t s were d r i e d over anhydr-ous sodium sulphate and evaporated to y i e l d a c o l o u r l e s s syrup (14 mg). Chromatography of the r e s i d u e s u s i n g s o l v e n t A and spray reagent p - a n i s i d i n e t r i c h l o r o a c e t a t e demonstrated t h a t the compound ( 2 , 5 , 4 - t r i - O - m e t h y l - L -x y l o s e ) gave the same c o l o u r w i t h the spray reagent as d i d 2 , 5 , 4 - t r i - O - m e t h y l - D - x y l o s e and the v a l u e s were i d e n t i c a l . &d?-17.5° i n water ( c , 1 . 0 ) ; f o r 2 , 3 , 4 -tr.i-O-methyl-D-xylose l i t ( 8 5 ) , Wp + 18° i n water. - - m -S e p a r a t i o n of reduced a c i d i c methylated sugars by gas-l i o u i d chromatography as t h e i r methyl g l y c o s i d e s :  A p a r t of the m a t e r i a l (90 mg) was converted i n t o methyl g l y c o s i d e s i n the u s u a l way. Methyl g l y c o s i d e s were examined by g a s - l i q u i d chromatography u s i n g the same column and c o n d i t i o n s as d e s c r i b e d on page 135• A u t h e n t i c samples of 2 , 3,4—tri-O-raethyl-D-glucosides and 3-0-methyl - D - x y l o s i d e s were i n j e c t e d i n order to f i n d out d i f f e r e n t components on the chromatogram. Components e l u t i n g out under each peak were c o l l e c t e d i n a c a p i l l a r y tube. I d e n t i f i c a t i o n of 5~0-methyl-D-xylose: The methyl g l y c o s i d e s of 3 -0-methyl-D-xylose c o l l e c t e d i n the c a p i l l a r y tube were 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 2 ml) at 100° f o r 6 hours. The h y d r o l y s a t e a f t e r n e u t r a l i s a t i o n and d e i o n i s a t i o n was examined by paper chromatography i n s o l v e n t A where o n l y a spot corresponding w i t h standard 3 -0-methyl-D-xylose was observed. The m a t e r i a l was s i l y l a t e d and examined by g a s - l i q u i d chromatography u s i n g a column packed w i t h Qjo by weight of SE 52 on 60-80 mesh D i a t o p o r t S. The column was h e l d i s o t h e r m a l l y at 110° f o r 3 minutes and then programmed at 3° per minute t o h o l d at 14-0°. The f l o w r a t e was 75 ml of helium per min. Q;>w0 peaks corresponding to s i l y l d e r i v a t i v e s of 3 -0-methyl-D-xylose were observed. - 145 -I d e n t i f i c a t i o n of 5,4—di-O-methyl-D-glucose; A p a r t of the c o l l e c t e d m a t e r i a l (Peaks 5 and 6 P i g . 5 0 ) was h y d r o l y s e d to generate f r e e sugar i n the u s u a l way. A p o r t i o n of the f r e e sugar was demethylated by h e a t i n g i n a s e a l e d tube w i t h 48/ hydrobromic a c i d f o r 5 0 minutes. A f t e r treatment w i t h D u o l i t e A-4 r e s i n the m a t e r i a l was evaporated and the product chromatographed u s i n g s o l v e n t s A & .3. Components corresponding to gl u c o s e , mono- and di-O-methyl-glucoses were observed. A p a r t of the methyl g l y c o s i d e s was s u b j e c t e d to mass s p e c t r 6 m e t r i c i n v e s t i g a t i o n . The r e s u l t i s d e s c r i b e d on page 1 0 2 . - 146 -(D CO C O Q . to <U J_ L_ O •*-> V <U 0 Q M e t h y l g l y c o s i d e s o f 1 . and 3 . 3 - 0 - M e t h y l - D - x y l o s e 2 and 4 . 2 , 3 , 4 - T r i - O - m e t h y l -I ) - g l u c o s e 5 and 6 . 3 , 4 - M - O - m e t h y l - D -g l u c o s e 10 15 T i me (mjn) 20 25 F i g u r e 3 0 . S e p a r a t i o n o f m e t h y l g l y c o s i d e s o f 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 - 14? -G a s - l i q u i d claromatography of the methylated n e u t r a l  sugars (components 1,2,5,6 and '/ ) ' A p o r t i o n of the methylated n e u t r a l sugars (12 mg) was r e s o l v e d by paper chromatography i n s o l v e n t system A. Since components (same as d e s c r i b e d i n Table 10) 4 and 5 p a r t i a l l y overlapped, components 1,2,3)6 and 7 were i s o l a t e d from paper by e l u t i n g w i t h methanol. The methanol e x t r a c t s were evaporated to a syrup (9 mg) which was reduced w i t h sodium borohydride (10 mg, 10 ml) f o r 16 hours at room temperature. The reduced m a t e r i a l was processed i n the u s u a l way and a c e t y l a t e d w i t h a mixture of a c e t i c a n h y d r i d e : p y r i d i n e (1:1, 5 ^1) at 100° f o r a p e r i o d of 3 hours. The a c e t y l a t e d a l d i t o l s of the methylated sugars were examined by g a s - l i q u i d chromatography u s i n g a column (8' x %") of 3;J 3CK3S-H on Gas Ghrom Q. Separations were made i s o t h e r m a l l y at 160° Tor 3 minutes and then programmed at 2° per min. to h o l d at 180°. One of the s e p a r a t i o n curves i s shown i n P i g . 31. 1. 2 , 3 , 5 - T r i - O - m e t h y l - L - a r a b i n o s e 2 . 2 , 3 , 4 - f r i - O - m e t h y l - D - x y l o s e 3 . 2 , 5 , 4 - T r i - O - m e t h y l - L - a r a b i n o s e 4. 3 - 0 - K e t h y l - D - x y l o s e 5 . h-Xylose 10 —r— 15 — i — 20 2 5 50 F i g u r e t ime (min) 31„ Separation of a l d i t o l a cetates of p a r t i a l l y methylated sugars by g a s - l i q u i d chromatography - 149 -A u t o h y d r o l y s i s of sapote gum: P u r i f i e d sapote gum (20.0 g) i n water (300 ml) was b o i l e d under r e f l u x f o r GO hours. A s m a l l amount of i n s o l u b l e m a t e r i a l was formed. The s o l u t i o n was f i l t e r e d and d i a l y s e d a g a i n s t d i s t i l l e d water (3 l i t r e ) f o r 24 hours. The d i f f u s a t e s were con c e n t r a t e d t o a t h i c k syrup (10.71 g ) . The d i a l y s i s was completed a g a i n s t running tap water f o r 24 hours. The content i n s i d e the bag was f r e e z e d r i e d (8.21 g ) . The f r e e z e - d r i e d m a t e r i a l w i l l be r e f e r r e d t o as the degraded gum. A p o r t i o n of the d i f f u s a t e (3.82 g) was passed through a column of P u o l i t e A-4 r e s i n i n order t o separate n e u t r a l sugars from a c i d i c - sugars. P l u t i o n of the column w i t h water (2 l i t r e ) gave the n e u t r a l components. E l u t i o n w i t h 10$ f o r m i c a c i d (200 ml) gave the a c i d i c f r a c t i o n . Paper chromatography of the n e u t r a l f r a c t i o n i n s o l v e n t 3 f o r a p e r i o d of 24 hours i n d i c a t e d the presence of x y l o s e and a r a b i n o s e . The a c i d i c f r a c t i o n was examined by paper chromatography i n s o l v e n t C. The spots were d e t e c t e d by s p r a y i n g the paper w i t h p - a n i s i d i n e t r i c h l o r o a c e t a t e and h e a t i n g the chromatogram i n an oven at 110° f o r 3 min. The c h a r a c t e r i s t i c c o l o u r f o r the u r o n i c a c i d s was o b t a i n e d . - 1 5 0 -The degraded gum had - 8 ° ( c , 1.0 i n w a t e r ) . The e q u i v a l e n t weight was obtained by d i s s o l v i n g a p o r t i o n of the degraded gum (14-5.5 rag) i n sodium hydroxide ( 1 0 ml, 0 . 1 N ) . A l i q u o t s were withdrawn and t i t r a t e d a g a i n s t M/10 s u l p h u r i c a c i d s o l u t i o n . The e q u i v a l e n t weight was found to be 8 5 0 . M e t h y l a t i o n of the degraded gum: A p o r t i o n of the degraded gum ( 2 . 0 5 g) was methylated a c c o r d i n g to the Kakomori method ( 1 0 ) . Thus the degraded gum 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 d i m e t h y l sulphoxide ( 1 5 0 ml) by s t i r r i n g at 5 0 ° under an atmosphere of dry n i t r o g e n . M e t h y l s u l p h i n y l anion was prepared as before by washing sodium hydride (4-.15 g ) , 5 0 / o i l d i s p e r s i o n , t h r i c e w i t h dry petroleum ether (50° - 60°) and d e c a n t i n g the wash. A f t e r the t h i r d wash, the r e s i d u a l petroleum e t h e r was evacuated w i t h a vacuum pump through an 18-gauge needle i n s e r t e d i n t o the serum cap. Dimethyl sulphoxide ( 5 0 ml) was t r a n s f e r r e d i n t o . t h e f l a s k and the mixture was s t i r r e d at 5 0 ° u n t i l the s o l u t i o n became c l e a r green and e v o l u t i o n of hydrogen gas ceased ( c a . 2 h o u r s ) . The s o l u t i o n of m e t h y l s u l p h i n y l i o n was added to the p o l y s a c c h a r i d e s o l u t i o n and the s t i r r i n g was c o n t i -nued f o r a p e r i o d of 6 hours. To the s o l u t i o n , c o o l e d i n an i c e - w a t e r bath, was added f r e s h l y d i s t i l l e d methyl - 1 5 1 -i o d i d e ( 1 0 ml) during; 4-5 min. W i t h i n a few minutes the s o l u t i o n became c l e a r . . The r e a c t i o n mixture was d i a l y s e a g a i n s t running tap water f o r a p e r i o d of 1 2 hours. I t was then e x t r a c t e d c o n t i n u o u s l y w i t h c h l o r o f o r m , d r i e d over anhydrous sodium sulphate and was f i n a l l y evaporated to dryness at 40° under reduced p r e s s u r e . The c h l o r o f o r m s o l u t i o n showed no h y d r o x y l a b s o r p t i o n i n the i n f r a r e d ( F i g . 5 2 ). The y i e l d of the methylated product was 2 . 2 1 g. H y d r o l y s i s of the Methylated Degraded Gum: A p o r t i o n 0 f the methylated degraded gum ( 1 0 1 mg.) was d i s s o l v e d i n s u l p h u r i c a c i d ( 7 2 / , 1 ml) i n a t e s t tube e x t e r n a l l y c o o l e d w i t h i c e - w a t e r . The s o l u t i o n a f t e r keeping at room temperature was d i l u t e d w i t h water (S ml) and was b o i l e d i n the s e a l e d tube at 1 0 0 ° f o r 4- hours. A f t e r c o o l i n g to room temperature and n e u t r a l i s i n g w i t h a s l u n y of barium carbonate the mixture was c e n t r i f u g e d . . The mixture was f i l t e r e d , a n d the s o l i d s were e x t r a c t e d s e v e r a l times w i t h water and e t h a n o l . The combined e x t r a c t s were evaporated to a syrup. The s e p a r a t i o n of the n e u t r a l and the a c i d i c components was accomplished by passage through c a t i o n and anion exchange r e s i n s . The n e u t r a l e f f l u e n t s were co n c e n t r a t e d and examiner! by paper chromatography u s i n g uinS pepaj3op pGq.axXqq.8iii T O mnjaoods uoTocLiosqe p s j a j i i r r . » g £ oanSx.T • • • n • • • i ; -3.0 4.0 5.0 MICRONS 6.0 i i , , , ] , i . . i . . , . i i > i i i i . i , i . . i t i - 1 5 5 -s o l v e n t system A. Three spots c o r r e s p o n d i n g to 2,5,4-t r i - O - , 2 , 5 , - d i - 0 - and 5-0-methyl-D-xyloses were observed. A p o r t i o n of the n e u t r a l methylated sugars ( 2 5 mg) was d i s s o l v e d i n water (10 ml) and reduced w i t h sodium borohydride (20 mg) f o r a p e r i o d of 24 hours. Excess borohydride was destroyed by t r e a t i n g the s o l u t i o n w i t h cation-exchange r e s i n A m b e r l i t e IE-I20 ( H + ) . Borate was removed i n the u s u a l way by treatment w i t h methanol. The product was a c e t y l a t e d by r e a c t i o n f o r 1 hour- at 100" w i t h ac e t i c a n h y d r i d e - p y r i d i n e (1:1, 10 m l ) . The mixture was evaporated t o dryness and d i s s o l v e d i n c h l o r o f o r m . S e p a r a t i o n of the a c e t y l a t e d a l d i t o l s was c a r r i e d out u s i n g a column ( 8 ' x X " ) c o n t a i n i n g 3$ (w/v;) of ECNSS-H on Gas Chrom 0 (100-120 mesh) u s i n g a temperature programme 145°-180°. The columns were h e l d i s o t h e r m a l l y at 145° f o r 5 minutes and then programmed at 2° per minute t o h o l d at 180°. The i d e n t i f i c a t i o n s of the d i f f e r e n t components on the g a s - l i q u i d chromatogram were c a r r i e d out by comparing the r e t e n t i o n times w i t h those of known a u t h e n t i c samples. - 154 -Reduction ox the methylated, degraded gum: A p o r t i o n of the methylated degraded sapote gum (603 mg) 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 tetrahydr-o— f u r a n (50 ml) and added s l o w l y to a s o l u t i o n of l i t h i u m aluminium h y d r i d e (1.50 g) w i t h anhydrous e t h e r (250 ml) over a steam bath f o r 5 hours.. The r e d u c t i o n was c a r r i e d out under r e f l u x i n g c o n d i t i o n f o r a p e r i o d of 18 hours. Excess hydride was destroyed by the a d d i t i o n of e t h y l acetate.and l a t e r by d i l u t e aqueous a c e t i c a c i d . The m a t e r i a l was e x t r a c t e d w i t h c h l o r o f o r m (5 x 150 ml). I n f r a r e d spectrum showed no a b s o r p t i o n around 1700 cm The amount of m a t e r i a l recovered 'was 560 mg. The i n f r a -r e d spectrum i s shown i n E i g . 55. A p a r t of the reduced, methylated, degraded sapote gum (4-0 mg) was d i s s o l v e d i n s u l p h u r i c a c i d (72/, 0.5 ml) at room temperature over a period, of one hour. I t was then d i l u t e d w i t h water (4- ml) and h y d r o l y s e d at 100°' i n a s e a l e d tube f o r a p e r i o d of 4- hours.. The h y d r o l y s e d m a t e r i a l was processed i n the same way as d e s c r i b e d on page 151. The h y d r o l y s a t e was examined, by paper chromatography u s i n g s o l v e n t A. The c o n c l u s i o n do?awn from t h i s examination was not very c l e a r as the spots of 2,5-d.i-O-methyl-xylose and. 2 , 3 , 4 —tri-C-methyl-glucose almost overlapped w i t h each o t h e r . •U1U..J pupi-vi- -oil pon.t?iiCiTq.oui psonpecr iJ&coqcreo j o rarucj-osds pejreaiuT 4.0 5.0 cc oaiiLJT,.T M I C R O N S 3500 2000 W A V E N U M B E R ( C M 1 ) - 156 -A p a r t of the h y d r o l y s a t e ( 1 5 mg) was d i s s o l v e d i n water ( 1 0 ml) and reduced w i t h sodium borohydride ( 1 5 mg) f o r a p e r i o d of 24 hours. The borohydride reduced m a t e r i a l was processed i n the u s u a l way as d e s c r i b e d on the page 1 5 5 . The product was a c e t y l a t e d by r e a c t i o n w i t h a c e t i c a n h y d r i d e - p y r i d i n e ( 1 : 1 , 1 0 ml). The mixture was evaporated to dryness and d i s s o l v e d i n c h l o r o f o r m . S e p a r a t i o n of the a l d i t o l a c e t a t e s was c a r r i e d out u s i n g the same column and the same programme by the- gas-l i q u i d chromatography as d e s c r i b e d on the page 155» One of the s e p a r a t i o n curves i s shown i n F i g . 34. A c e t a t e s of 1. 2^3,4-Tr ' i -0-methy l -D-xy l i i : o 1 2. 2 , 3 - D i - O - methyl_D-xylito! 3. 2 ; 3 > 4-Tr ' i -0 -methy l_D-g iuc i to l 4. Mono-O-methy l -D-xy l i to l 10 ' J o 25 30 35 4 0 T i m e (min) x'lgure 3'i-. S e p a r a t i o n of methylated a l d i t o l a c e t a t e s from methylted carboxyl-reduced degraded gum - 158 -S p e c t r o p h o t o m e t r y d e t e r m i n a t i o n of p e r i o d a t e uptake: P e r i o d a t e uptake of sapote gum p o l y s a c c h a r i d e was determined by u l t r a v i o l e t spectrophotometry. A s m a l l p o r t i o n of the p o l y s a c c h a r i d e (13.6 mg) was d i s s o l v e d i n sodium metaperiodate s o l u t i o n (0.015 i ' r i , 10 m l ) . A l i q u o t s were withdrav/n at fr e q u e n t i n t e r v a l s and d i l u t e d 250 t i m e s . A blank s o l u t i o n was processed s i m u l t a n e o u s l y . The o p t i c a l d e n s i t i e s of the p o l y s a c c h a r i d e s o l u t i o n and the blank were recorded at 223 mp. The r e s u l t s are recorded i n Table 12. Table 12 P e r i o d a t e uptake of sapote gum p o l y s a c c h a r i d e Time (hour) Moles of 10^ uptake/Moles of sugar 0.5 0.16 1.5 0.25 2.5 0.31 5.0 0.42 19.0 0.59 44.0 0.64 70.0 0.72 - 159 -S m i t h d e g r a d a t i o n o f s a p o t e gum p o l y s a c c h a r i d e : S a p o t e gum p o l y s a c c h a r i d e (5..04 g ) was d i s s o l v e d i n w a t e r (125 m l ) and 0 . 5 h s o d i u m m e t a p e r i o d a t e s o l u t i o n (12'5 m l ) was a d d e d . The s o l u t i o n was a l l o w e d t o s t a n d a t 25° f o r 92 h o u r s . The r e a c t i o n was s t o p p e d a t t h i s p o i n t by a d d i t i o n o f e t h y l e n e g l y c o l (10 m l ) and t h e s o l u t i o n was d i a l y s e d a g a i n s t r u n n i n g t a p w a t e r f o r 24-h o u r s . Sod ium b o r o h y d r i d e ( 2 . 5 g ) was a d d e d , and t h e m i x t u r e k e p t a t room t e m p e r a t u r e , f o r 48 h o u r s . The s o l u t i o n was d i a l y s e d f o r a p e r i o d o f 24 h o u r s , and c o n c e n t r a t e d t o t h i c k s y r u p (4..65 g ) . A'., p o r t i o n o f t h e s y r u p ( 2 . 0 5 g ) was t r e a t e d w i t h N - s u l p h u r i c a c i d (100 m l ) a t room t e m p e r a t u r e f o r 5 h o u r s and n e u t r a l i s e d w i t h a s l u r r y o f b a r i u m c a r b o n a t e . The m i x t u r e was c e n t r i f u g e d and t h e s u p e r n a t a n t was d e c a n t e d . The r e s i d u e was e x t r a c t e d s e v e r a l t i m e s w i t h w a t e r . The c o m b i n e d s o l u t i o n was d e i o n i s e d by p a s s a g e t h r o u g h a c o l u m n o f A m b e r l i t e r e s i n I G - 1 2 0 ( I I + ) and D u o l i t e A - 4 r e s i n . The d e i o n i 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 o a t h i c k s y r u p ( 1 . 4 8 g ) . The a c i d i c componen ts were n o t e x a m i n e d s i n c e an e q u i v a l e n t amount o f i n f o r m a t i o n was o b t a i n e d f r o m t h e p e r i o d a t e o x i d a t i o n o f t h e c a r b o x y l - r e d u c e d p o l y s a c c h a r i d e (page 1 7 7 ) . An a t t e m p t e d s e p a r a t i o n o f a p a r t o f t h e m i x t u r e was c a r r i e d ou t by s h e e t s o f Whatman ho. 5 M i l p a p e r and - 160 -s o l v e n t B. The components were l o c a t e d on paper by t r e a t i n g h a l f i n c h s t r i p s from edges of the sheet w i t h s i l v e r n i t r a t e and sodium hydroxide s o l u t i o n s . Bands corresponding to the d i f f e r e n t components were cut out and e l u t e d w i t h water. A p a r t of the mixture c o n t a i n i n g Smith-degraded, a c i d h y d r o l y s e d p o l y o l s ( 1 5 6 mg) was r e s o l v e d by Dowex 1 X - 2 ( 0 I I ~ ) resin... The n e u t r a l syrup was a p p l i e d to a column of Dowex 1 X - 2 ( 0 H ~ ) r e s i n (26 x 2 cm) and was separated by e l u t i o n w i t h water. The f r a c t i o n s of approximately 5 nil were c o l l e c t e d i n each tube, and the f l o w r a t e was a d j u s t e d to 40 ml per hour. Each f r a c t i o n was co n c e n t r a t e d and then examined by paper chromatography;, f r a c t i o n s of s i m i l a r composition were combined. The data of the i s o l a t e d components of the h y d r o l y s a t e ere summarised i n Table 1 5 . In t o t a l , 1 5 5 rag of m a t e r i a l was e l u t e d from the column (corresponding to 8 5 / r e c o v e r y ) . I d e n t i f i c a t i o n of the separated components: a. Ethylene G l y c o l . A p o r t i o n (20 mg) was d i s s o l v e d i n dry p y r i d i n e (5 ml) and t r e a t e d w i t h p - n i t r o b e n z o y l c h l o r i d e ( 1 9 6 mg) at 9 0 ° f o r 1 hour. The dark brown s o l u t i o n was poured i n t o sodium bi c a r b o n a t e s o l u t i o n , and the product was e x t r a c t e d i n t o c h l o r o f o r m . The ch l o r o f o r m s o l u t i o n was washed w i t h sodium bicarbonate s o l u t i o n and w i t h water . 7 d r i e d over sodium s u l p h a t e , and concent r a t e d to a s o l i d . - 161 -The product was r e c r y s t a l l i s e d from c h l o r o f o r m and pet. e t h e r to give ethylene g l y c o l d i - p - n i t r o b e n z o a t e , m.p. and mixed m.p. 139-141°. b. G l y c e r o l A p o r t i o n ( 2 5 mg) was converted to i t s t r i - p - r i i t r o -benzoate i n the u s u a l way as d e s c r i b e d before by r e a c t i n g w i t h p - n i t r o b e n z o y l c h l o r i d e ( 3 0 0 rag) i n dry p y r i d i n e ( 1 0 ml) at 9 0 ° f o r 1 hour. The product a f t e r r e c r y s t a -l l i z a t i o n from c h l o r o f o r m and p e t . ether gave g l y c e r o l t r i - p - n i t r o b e n z o a t e as y e l l o w c r y s t a l s , m.p. and mixed m.p. 1 8 7 - 1 9 0 ° . c. 2 - 0 - g l y c e r o l - ^ - D - x y l o p y r a n o s i d e The compound had i n water [o(.J 5p-34° (c 1 . 0 ) , ^ x y l o s e i n s o l v e n t B of 0 . 8 5 . H y d r o l y s i s of an a l i q u o t ( 1 0 mg) i n 1 K I-IgSO^ ( 1 0 ml) f o r 6 hours at 1 0 0 ° gave g l y c e r o l and x y l o s e as shown by paper chromatography. To determine the r a t i o of x y l o s e to g l y c e r o l the h y d r o l y s a t e was separated on paper u s i n g s o l v e n t B. The a p p r o p r i a t e areas were e l u t e d , the s o l u t i o n was s u i t a b l y d i l u t e d and a l i q u o t s of the x y l o s e s o l u t i o n were analysed by the p h e n o l - s u l p h u r i c a c i d method (page 1 2 1 ) . The amount of g l y c e r o l present was determined by the chromotropic a c i d method (page 1 2 5 ) . The molar r a t i o between x y l o s e and g l y c e r o l was found to be 1 . 0 : 0 . 9 . The r a t i o between x y l o s e and g l y c e r o l was a l s o determined by g a s - l i q u i d chromatography 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 . Thus a s m a l l p o r t i o n of the Table 1 5 Tube ho Column chromatographic s e p a r a t i o n of Smith-•clep :raded p o l y o l . P ^ x y l o s e Component v/eight (rnp ;) 15-20 Ethylene G l y c o l 26 2.56 21-24 G l y c e r o l 40 - 1.70 2 5 - 5 0 X y l o s y l G l y c e r o l 28 - 5 4 ° 0 . 3 5 3 1 - 5 5 X y l o b i o s y l G l y c e r o l 2 9 -48.2° 0.60 56-40 X y l o b i o s e 5 - 0 . 5 0 41-45 X y l o t r i o s y l G l y c e r o l 1 5 - 5 7 ° 0.52 * Solvent B - 1 6 3 -h y d r o l y s e d m a t e r i a l ( 5 mg) was s i l y l a t e d i n the u s u a l way by d i s s o l v i n g i n p y r i d i n e ( 2 ml) and t r e a t i n g w i t h h e x a m e t b y l d i s i l a z a n e ( 0 . 6 ml) and t r i m e t h y l c h l o r o s i l a n e (0.3 m l ) . The mi x t u r e was i n j e c t e d i n t o a column (8' x packed w i t h 2 0 $ 3 F 96 on 60-80 mesh D i a t o p o r t S. The experiment was c a r r i e d out i s o t h e r m a l l y at 1 1 0 ° f o r 3 min. and then programmed at 3°/min. to h o l d at 2 3 0 ° . The i d e n t i t y of the components was obtained by i n j e c t i n g known a u t h e n t i c samples 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 x y l o s e and g l y c e r o l . From g a s - l i q u i d chromatography a x y l o s e - . g l y c e r o l r a t i o of 0.90:1.00 was c a l c u l a t e d . A p o r t i o n of the x y l o s i d e (4- mg) was s i l y l a t e d i n the u s u a l way and i n j e c t e d i n t o the g a s - l i q u i d chromato-graph onto a column c o n t a i n i n g 2 0 $ 3P 9 6 on 60-80 mesh D i a t o p o r t 3. The s e p a r a t i o n was c a r r i e d out i s o t h e r -m a l l y at 1 5 0 ° f o r 3 min. and then programmed at 2°/min. to h o l d at 2 3 9 ° . The s e p a r a t i o n i s i l l u s t r a t e d i n F i g . 3 3 The compound coming out of the column was c o l l e c t e d i n a narrow g l a s s c a p i l l a r y tube and s u b j e c t e d to mass s p e c t r o m e t r i c i n v e s t i g a t i o n . The r e s u l t i s d e s c r i b e d on page 1 0 7 . The g l y c o s i d e was f u r t h e r c h a r a c t e r i z e d as f o l l o w s : i ) P e r i o d a t e o x i d a t i o n : An a l i q u o t ( 9 mg) was t r e a t e d w i t h a s o l u t i o n of p e r i o d i c a c i d ( 0 . 0 1 h, 1 0 ml) at room temperature i n the dark. A l i q u o t s were w i t h -- 165 -drawn w i t h a s y r i n g e and d i l u t e d 250 times. The absorbances of the r e s u l t i n g s o l u t i o n s were measured i n the spectrophotometer at 223 mp and compared w i t h those of the o r i g i n a l s o l u t i o n of p e r i o d a t e , the t h e o r e t i c a l value being 2.00 moles. A l i q u o t s were withdrawn to d e t e c t the f o r m a t i o n of formaldehyde, but no formaldehyde was formed. Experiments f o r the d e t e c t i o n of formaldehyde were c a r r i e d out e x a c t l i n the same way as d e s c r i b e d on page 123. A p a r t of the dialdehyde (4- mg) was reduced w i t h sodium borohydride (4- mg) f o r a p e r i o d of 24- hours. Excess borohydride was d e s t r o y e d w i t h a cation-exchange r e s i n IR-120(H +) and borate was removed by treatment w i t h methanol. The compound was h y d r o l y s e d w i t h 1ft s u l p h u r i c a c i d (2 ml) on a steam bath f o r 6 hours. A f t e r n e u t r a l i s a t i o n w i t h a s l u r r y of barium carbonate and subsequent d e i o n i s a t i o n w i t h IR-120(H +) the m a t e r i a l was examined on a paper chromatogram u s i n g s o l v e n t B. Only spots corresponding to g l y c e r o l and ethylene g l y c o l were observed. The mixture of ethylene g l y c o l and g l y c e r o l was s i l y l a t e d i n the u s u a l v/ay by d i s s o l v i n g i n p y r i d i n e (1 ml) and t r e a t i n g w i t h h e x a m e t h y l d i s i l a z a n e (0.2 ml) and t r i m e t h y l c h l o r o s i l a n e (0.1 m l ) . The mixture was i n j e c t e d i n t o a g a s - l i q u i d chromatograph, having a - 166 -s t a i n l e s s s t e e l coluran packed w i t h 2 0 $ B'F 96 on 60-80 mesh D i a t o p o r t 8. The experiment was c a r r i e d out i s o t h e r m a l l y at 90° f o r 5 min. and then programmed at 3° per min. to h o l d at 2 0 0 ° . The i d e n t i t y of each component was r e v e a l e d by comparing the r e t e n t i o n times w i t h those of known a u t h e n t i c samples. A r a t i o of 0 . 9 3 : 1.00 was c a l c u l a t e d between g l y c e r o l and ethylene g l y c o l r e s p e c t i v e l y . i i ) m e t h y l a t i o n s t u d i e s : An a l i q u o t (3 mg) was d i s s o l v e d i n dimethylformamide ( 2 m l ) , and t r e a t e d w i t h f r e s h l y prepared s i v e r oxide (0 . 3 g) and methyl i o d i d e (0 . 3 m l ) . The mixture was shaken i n the dark of 4-8 hours. The r e a c t i o n was f o l l o w e d by s p o t t i n g on a t h i n l a y e r p l a t e at frequent I n t e r v a l s . The p l a t e was developed i n the s o l v e n t e t h y l ether:, toluene ( 2 : 1 ) , and the spots were de t e c t e d by s p r a y i n g w i t h 5 0 $ s u l p h u r i c a c i d s o l u t i o n and subsequent h e a t i n g at 1 5 0 ° . The mixture was f i l t e r e d and the f i l t r a t e was evaporated to dryness ( 2 . 5 nig). The methylated x y l o s y l - g l y c e r o l was heated w i t h s u l p h u r i c a c i d (1N, 5.ml) i n a s e a l e d tube at 1 0 0 ° (6 h r s . ) a f t e r which the s o l u t i o n was d i l u t e d to 1 0 ml, n e u t r a l i s e d w i t h barium carbonate, the mixture c e n t r i f a g e d and the i n s o l u b l e s a l t s washed w i t h water (10 m l ) . The combined aqueous s o l u t i o n was e x t r a c t e d w i t h c h l o r o f o r m . The combined c h l o r o f o r m e x t r a c t s were d r i e d w i t h anhydrous - 167 -sodium s u l p h a t e , f i l t e r e d and evaporated to dryness. A sample of the b y d r o l y s a t e was chromatographed on paper u s i n g s o l v e n t A. The chromatogram was sprayed w i t h p - a n i s i d i n e - t r i c h l o r o a c e t a t e and a component observed which corresponded, to 2,3,4— t r i - O - m e t h y l x y l o s e . The h y d r o l y s a t e was r e f l u x e d v/ith methanolic hydrogen c h l o r i d e (3#j 10 ml) f o r 5 hours. A f t e r n e u t r a l i s a t i o n v/ith s i v e r carbonate the s o l u t i o n was evaporated to dryness. A methanolic s o l u t i o n of the m a t e r i a l was i n j e c t e d i n t o a column (4-' x }(") of b u t a n e d i o l s u c c i n a t e (5$) on 80-100 mesh D i a t o p o r t S i s o t h e r m a l l y at 130°. Components having i d e n t i c a l r e t e n t i o n times ofc<and^ methyl g l y c o s i d e s of 2,3,4--tri-0-methyl-D-xyloses were observed. - 168 -d. 2 - 0 - $ - D - x y l o b i o s y l - g l y c e i ' o l : The compound (29 mg) had i n water ft*] D -48.2 (c 1.0 i n w a t e r ) . H y d r o l y s i s of an a l i q u o t (10 mg) i n s u l p h u r i c a c i d (1N, 10 ml) f o r 6 hours at 100° gave g l y c e r o l and x y l o s e on a paper chromatogram i n s o l v e n t B. A' p a r t of x y l o s e and g l y c e r o l was separated from paper w i t h water as e l u a n t . The amount of x y l o s e present was determined by the p h e n o l - s u l p h u r i c a c i d method and the amount of g l y c e r o l by the chromotropic a c i d method ( f o r d e t a i l s see page 1 2 3 ) , the r a t i o between x y l o s e and g ^ c e r o l being 1 .9:1.0. A p o r t i o n of the h y d r o l y s a t e (3 mg) was converted i n t o 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 i n the u s u a l way and examined by g a s - l i q u i d chromatography u s i n g i d e n t i c a l column and o p e r a t i n g c o n d i t i o n as d e s c r i b e d on page 163 . The mole r a t i o of g l y c e r o l and x y l o s e was 1:;1.8 i n good agreement w i t h the c a l c u l a t e d molar r a t i o of 1:2. M e t h y l a t i o n of 2 - 0 - P - B - x y l o b i o s y l g l y c e r o l : A . p o r t i o n of the x y l o b i o s y l g l y c e r o l (5 mg) was shaken w i t h N,IT,-dimethyl formamide (1 m l ) , methyl i o d i d e (0 .5 ral) and s i l v e r oxide (0.3 g) f o r 24 hours at room temperature i n the dark. A f t e r t h a t 0.1 ml of methyl i o d i d e and 0.1 g of s i l v e r oxide were added and shaking was c o n t i n u e d f o r another 24 hours.- The s o l u t i o n was f i l t -ered and the f i l t r a t e was l a t e r evaporated to dryness (4 rag). - 169 -The f u l l y methylated x y l o b i o s y l g l y c e r o l was heated at 100° w i t h s u l p h u r i c a c i d (11?, 2 ml) i n a s e a l e d tube f o r 6 hours. A f t e r n e u t r a l i s a t i o n w i t h a s l u r r y of barium carbonate and subsequent d e i o n i s a t i o n , the e f f l u e n t was evaporated and examined on a paper chromatogram i n s o l v e n t A. Spots c o r r e s p o n d i n g to 2,3-di-O-methyl-D-x y l o s e and 2 , 3,4-tri-O-methyl-D-xylos-e were observed. The m a t e r i a l was d i s s o l v e d i n water.(10 ml) and r e -duced w i t h sodium borohydride ( 2 5 mg) f o r a p e r i o d of 24 hours. Excess borohydride was destroyed by passage through I R - 1 2 0 ( H T ) . and borate was removed by condensation w i t h methanol. The m a t e r i a l was a c e t y l a t e d by treatment w i t h a c e t i c anhydride: p y r i d i n e (1 :.1 , 5 m l ) at 60° f o r 5 hours. A c e t y l a t e d m a t e r i a l was i n j e c t e d i n t o a column (8' x %'")of E0NS3-M'. ( 5 P ) , the temperature of o p e r a t i o n being i s o t h e r m a l f o r 5 min. at 140°, programmed at 5°/min up-to 180°. The peaks were i d e n t i f i e d by i n j e c t i n g known a u t h e n t i c samples of 2 , 5 , 4 - t r i - 0 - m e t h y l and 2 , 5 - d i - 0 -methyl x y l i t o l a c e t a t e s under the same c o n d i t i o n s . i'eriodate o x i d a t i o n of 2 - 0 - f t - D - x y l o b i o s y l g l y c e r o l : A p o r t i o n (8 mg) was t r e a t e d w i t h a s o l u t i o n of sodium metaperiodate (0.01 M, 10 ml) at room temperature i n the dark. A l i q u o t s were withdrawn w i t h a s y r i n g e and d i l u t e d 2 5 0 times. The absorbances of the r e s u l t i n g - 170 -s o l u t i o n s were measured i n the spectrophotometer at 2 2 3 mjm and compared w i t h the blank s o l u t i o n of p e r i o d a t e ( d i t u t e d 250 t i m e s ) . The compound consumed 3 - 1 5 moles of p e r i o d a t e , the t h e o r e t i c a l value being 3.00 moles. The s o l i i t i o n was t e s t e d f o r the presence ox form-aldehyde (see page 1 2 3 ) hut gave a n e g a t i v e t e s t . A p a r t of the dialdehyde ( 3 nig) was reduced w i t h sodium borohydride (5 mg) and subsequently processed i n the u s u a l way. The reduced m a t e r i a l 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 ( 1 N , 2 ml) at 100° f o r 6 hours and n e u t r a l i z e d and d e i o n i s e d i n the u s u a l way. The h y d r o l y s a t e showed spots corresponding to ethylene g l y c o l and g l y c e r o l on a paper chromatogram i n s o l v e n t B. The molar r a t i o between ethylene g l y c o l and g l y c e r o l 1:2.0 was c a l c u l a t e d 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 by g a s - l i q u i d chromatography i n the u s u a l way. X y l o t r i o s y l g l y c e r o l : The compound ( 1 5 mg) had [ "0^-57° (c 1.0 i n w a t e r ) . A p o r t i o n of the m a t e r i a l ( 5 mg) 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 ( 1 h , 1 0 ml) at 100° f o r 6 hours. A f t e r the u s u a l process of n e u r t a l i s a t i o n and d e i o n i s a t i o n , the m a t e r i a l was examined on a paper chromatogram u s i n g s o l v e n t B. Spots corresp o n d i n g to x y l o s e and g l y c e r o l were observed.. A p o r t i o n (2 mg) was s i l y l a t e d and examined by g . l . c . u s i n g the same c o n d i t i o n s as d e s c r i b e d on page 1 6 3 . - 1 7 1 -the molar r a t i o 'between x y l o s e and g l y c e r o l being 1:2.8. A p a r t of the m a t e r i a l was o x i d i s e d with sodium meta-p e r i o d a t e (0.01 I-i, 10 ml) f o r 4-8 hours. moles of p e r i o d a t e uptake being measured s p e c t r o p h o t o m e t r i c a l l y as before was found to be 4.10 ( c a l c u l a t e d 4.0 moles). No formaldehyde was de t e c t e d . Reduction of the p e r i o d a t e o x i d i z e d m a t e r i a l w i t h sodium borohydride and subsequent a c i d h y d r o l y s i s i n the u s u a l way gave ethylene g l y c o l and g l y c e r o l . The molar r a t i o was estimated by g a s - l i q u i d chromatography 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 and was found to be 1:2.8. X y l o b i o s e : The m a t e r i a l (5 mg) 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 (1N, 6 ml) at 100° f o r 6 hours. A f t e r the u s u a l n e u t r a l i s a t i o n and d e i o n i s a t i o n , the m a t e r i a l was examined by paper chromatography u s i n g s o l v e n t system Only a spot corresponding to x y l o s e was observed. - 172 -Carboxyl r e d u c t i o n of sapote gum: The procedure d e s c r i b e d i s f o r 500 mg. of the p o l y s a c c h a r i d e and the s i z e of the r e a c t i o n c o u l d be changed p r o p o r t i o n a t e l y . A p o r t i o n of the p r o p i o n a t e d a c i d (500 mg) was d i s s o l v e d i n t e t r a h y d r o f u r a n (15 m l ) . The s o l u t i o n was added to a c o o l e d (-75°) s o l u t i o n of diazomethane i n d i e t h y l ether (50 m l ) . The mixture was s t i r r e d at -75° f o r 1 hour. The product was p r e c i p i t a t e d by p o u r i n g i n t o 50 ml of l i g h t petroleum ether (b.p. 50°-60°), y i e l d 550 mg. A p o r t i o n of the e s t e r i f i e d m a t e r i a l (4-50 m was d i s s o l v e d i n t e t r a h y d r o f u r a n (50 ml) and t o the r e f l u x i n g s o l u t i o n l i t h i u m borohydride (1 g i n 25 nil of t e t r a h y d r o f u r a n ) was added dropwise d u r i n g 90 min. The r e s u l t i n g s o l u t i o n was r e f l u x e d f o r 18 hours d u r i n g which time a white g e l a t i n o u s p r e c i p i t a t e was formed. The f l a s k was c o o l e d i n i c e and water (25 ml) was added s l o w l y d u r i n g 2 hours to r e a c t w i t h the excess of l i t h i u m b o rohydride. The contents of the f l a s k were d i a l y s e d f o r 24- hours and the p o l y s a c c h a r i d e was recovered by f r e e z e d r y i n g (225 mg). The p o l y s a c c h a r i d e showed a n e g a t i v e t e s t w i t h c a r b a z o l e . A p o r t i o n of the p o l y s a c c h a r i d e (100 mg) 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 (1W, 10 ml) f o r 6 hours at 100°. The h y d r o l y s a t e a f t e r n e u t r a l i s a t i o n w i t h - 173 -barium carbonate and d e i o n i s a t i o n w i t h A m b e r l i t e IR-120(H"r) r e s i n was examined by paper chromatography i n s o l v e n t system B. Spots cox-responding t o L-arabinose, D-xylose, 4 - 0-methyl-D-glucose and D-glucose were observed. The mixture of monosaccharides (75 mg) was reduced i n water (50 ml) w i t h sodium borohydride (100 mg) f o r 12 hours. A f t e r passage through A m b e r l i t e IR-120(H"r) and d i s t i l l a t i o n w i t h methanol the product was a c e t y l a t e d by r e a c t i o n f o r 1 hour at 100° w i t h a c e t i c anhydride-p y r i d i n e (1:1, 10 m l ) . The mixture was evaporated t o dryness and d i s s o l v e d i n ch l o r o f o r m . S e p a r a t i o n of a l d i t o l a c e t a t e s was c a r r i e d out on an P & i'l 720 gas chromatograph w i t h a thermal c o n d u c t i -v i t y d e t e c t o r u s i n g a column (4' x }-,") of 5$ b u t a n e d i o l s u c c i n a t e on 80-100 mesh D i a t o p o r t S. The column was h e l d i s o t h e r m a l l y at 210° f o r 25 min. and then p r o g r a -mmed at 10° per min. to h o l d at 225°. Pour peaks were ob t a i n e d which by comparison w i t h standards ( r e l a t i v e r e t e n t i o n times i n parentheses) were i d e n t i f i e d as corresponding to L - a r a b i n i t o l (0.71), x y l i t o l (1.00), 4 - 0 - m e t h y l - D - g l u c i t o l (1.55) and D - g l u c i t o l (1.75) a c e t a t e s a l l of which were c l e a r l y r e s o l v e d . The r e t e n t i o n time of x y l i t o l p entaacetate was about 21 min. One of the s e p a r a t i o n curves i s shown i n P i g . 38• The s e p a r a t i o n of 4 - 0 - m e t h y l - D - g l u c i t o l pentaacetate A l d i t o l a cetates of 1. L-Arabinose 2. D-Xylose 3 # 4—O-Methyl-D-slucose 4-. D-C-lucose 0 if) C o d. to CD J_ O o (U -*-> (L) P 4 CD 03 CO p ro CO •p I P M H H- P ,o ct p H-O P O O P-4 o P i—» H P CL-<+ H-O ct Co O 4 H P •d P P1 O ro c+ P ro CO cr I - A I y / — r 10 20 30 t ime(min) 40 - 175 -1. 4 - 0 - M e t h y l - i ) -g l u c i t o l pentaacetate 2. G - a l a c t i t o l hexaacetate 4 o 10 12 R e t e n t i o n time, minutes •igure 37. S e p a r a t i o n of a l d i t o l a c e t a t e s of 4 - 0-methri-ll- glue os e and galac to s e by g.1.c. - 176 -and g a l a c t i t o l hexaacetate on the same column operated i s o t h e r m a l l y at 22>° i s shown i n F i g . 37. - 1 7 7 -Spectrophotometric d e t e r m i n a t i o n of p e r i o d a t e uptake  of the carboxyl-reduced p o l y s a c c h a r i d e : P e r i o d a t e uptake of the carboxyl-reduced p o l y s a c c h a r i d e was determined as before by u.v. spectrphotometry. A s m a l l p o r t i o n of the p o l y s a c c h a r i d e ( 1 5 . 2 mg) was d i s s o l v e d i n 0 . 0 1 5 K sodium metaperiodate s o l u t i o n ( 1 0 ml). A l i q u o t s were withdrawn at frequent i n t e r v a l s and d i l u t e d 2 5 0 t i m e s . A blank s o l u t i o n was processed s i m u l t a n e o u s l y . The o p t i c a l d e n s i t i e s of the p o l y s a c c h a r i d e s o l u t i o n and the blank were recorded at 2 2 5 mu. The r e s u l t s are recorded i n Table 14. Table 14 P e r i o d a t e uptake of the carboxyl-reduced p o l y s a c c h a r i d e Time (hour) Moles of p e r i o d a t e / Moles of sugar 0.14 0 . 1 7 0 . 2 1 0 . 3 5 0 . 5 1 0 . 5 S 0 . 6 6 0.5 1.0 2.5 5.0 18.0 48.0 96.0 - 178 -Carboxyl reduced sapote gum (203.2 mg) was d i s s o l v e d i n sodium metaperiodate s o l u t i o n (0.1 H, 25 ml) and the o x i d a t i o n was allowed to proceed at room temperature f o r 98 hours. The r e a c t i o n was stopped by the a d d i t i o n of ethylene g l y c o l (1 ml) and the s o l u t i o n was d i a l y s e d a g a i n s t running tap water o v e r n i g h t . Sodium boro-h y d r i d e (100 mg) was added and the mixture was kept f o r a p e r i o d of 24- hours. The s o l u t i o n was d i a l y s e d f o r a p e r i o d of 24- hours. The s o l u t i o n was then c o n c e n t r a t e d to a syrup (181 .7 mg). A p a r t of the syrup (75.6.mg) 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 (1N, 20 ml) i n s e a l e d tubes f o r a p e r i o d of 8 hours. A f t e r n e u t r a l i s a t i o n w i t h a s l u r r y of barium carbonate and subsequent d e i o n i s a t i o n by passage through c a t i o n and anion exchange r e s i n s , the m a t e r i a l was evaporated to dryness. The m a t e r i a l was spot t e d on a paper chromatogram and developed i n s o l v e n t B. The components were l o c a t e d on the paper by t r e a t i n g the paper w i t h s i l v e r n i t r a t e and the t r i c h l o r o a c e t a t e reagents. Spots corresponding to ethylene g l y c o l , g l y c e r o l , 2 - 0 - m e t h y l - D - e r y t h r i t o l , D-xylose and 4— O-methyl-D-glucose were observed. 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 1. Ethylene g l y c o l . 2. G l y c e r o l 5. Unknown 4-. 2 - 0 - K e t h y l - D - e r y t h r i t o l 5a, 5h, 5c, 5d. D-Xylose -1 ^ « # : # 1 r— 15 3 0 45 6 0 T i m e ( m i n ) F i g u r e 3 8 . S e p a r a t i o n of products of p e r i o d a t e o x i d i z e d p . ' i r h n y v l - r f i d u c e d saoote gum - 180 -A p a r t o f t h e m a t e r i a l ( 2 0 . 3 mg) was s i l y l a t e d i n t h e u s u a l way . G a s - l i q u i d c h r o m a t o g r a p h i c a n a l y s i s was c o n d u c t e d on a s t a i n l e s s s t e e l c o l u m n (8 ' x> ' " ) c o n t a i n i n g SP 96 on 60-80 mesh D i a t o p o r t S , t h e t e m p e r a t u r e programme b e i n g 80°- 240° a t t h e r a t e o f 3 ° p e r m i n u t e w i t h i s o t h e r m a l a t 8 0 ° f o r 5 m i n u t e s . I d e n t i t y o f t h e componen ts was r e v e a l e d by i n j e c t i n g s t a n d a r d s u n d e r i d e n t i c a l c o n d i t i o n . The q u a n t i t a t i v e r a t i o s among t h e d i f f e r e n t componen ts a r e r e c o r d e d i n T a b l e 6. .One o f t h e s e p a r a t i o n c u r v e s i s shown i n P i g . 3 8 . - 181 -A c e t o l y s i s of the Carboxyl-Reduced Sapote Gum: Carboxyl reduced p o l y s a c c h a r i d e (1.10 g) v/as added to formamide (15 ml) at 60° over a p e r i o d of 1 h r . P y r i d i n e (15 ml) was added to i t . A c e t i c anhydride (15 ml) was added to i t dropwise over a p e r i o d of 1 h r . The mixture v/as kept at 60° f o r 5 h r . , and then added drop-wise t o a mixture of i c e - w a t e r (1 l i t r e ) . Dark p r e c i p i t a t e s were formed which were f i l t e r e d and washed s e v e r a l times w i t h i c e - c o l d water. The m a t e r i a l v/as a i r d r i e d at room temperature (1.4-5 g ) . A p o r t i o n of the c a r b o x y l reduced a c e t y l a t e d p o l y -s a c c h a r i d e (712 mg) v/as d i s s o l v e d i n a c e t i c anhydride (24- m l ) , a c e t i c a c i d (24- m l ) , and c o n c e n t r a t e d s u l p h u r i c a c i d (1 ml) i n t h a t order. The s o l u t i o n v/as kept at 4-0° f o r 2}f: h r . The r e a c t i o n was f o l l o w e d by s p o t t i n g on a s i l i c a g e l p l a t e . The r e a c t i o n was stopped by the a d d i t i o n of p y r i d i n e (50 m l ) . The s o l v e n t was evaporated t o dryness at 50° over a r o t a t o r y evaporator. P a r t i a l l y a c e t o l y s e d m a t e r i a l was d i s s o l v e d i n methanol, c o o l e d i n an i c e - w a t e r bath and t r e a t e d w i t h sodium methoxide (1.0 g) to keep the s o l u t i o n a l k a l i n e . The s o l u t i o n was kept over a p e r i o d of 16 hr i n a c o l d room at 5°» I t was then poured i n t o water (500 ml) and t r e a t e d w i t h c a t i o n exchange r e s i n A m b e r l i t e IR -120(H +). The s o l u t i o n was evaporated to a syrup (4-75 a g ) . - 182 -Charc o a l column chromatography ox d e a c e t y l a t e d m a t e r i a l : A c h a r c o a l column was prepared by p l a c i n g a f i l t e r paper (Whatman t:io 1 ) i n a Buchner f u n n e l on which a s l u r r y of c h a r c o a l : c e l i t e ( 1 : 1 , JO g) was s l u r r i e d so as to fox-ra a column 4 cm. h i g h . A c e t o l y s e d m a t e r i a l ( 3 8 0 mg) was d i s s o l v e d i n water ( 1 0 ml) and a p p l i e d to the top of c h a r c o a l column. The column was e l u t e d stepwise as f o l l o w s : water (2 l i t r 5/3 e t h a n o l i n water (2 l i t r e ) , 1 0 / e t h a n o l i n water (2 l i t r > e ) , 1 5 / e t h a n o l i n water (2 l i t r e ) and f i n a l l y 3 / b u t a n o l ( 1 l i t x ^ e ) . . Each f r a c t i o n was conce n t r a t e d and examined by paper chromatogx">aphy i n sovent system B to g i v e d i f f e r e n t o l i g o s a c c h a r i d e s . F r a c t i o n 1 . This f r a c t i o n ( 1 7 5 mg) gave two spots on paper which were i d e n t i c a l w i t h standard arabinose and x y l o s e . F r a c t i o n 2. This f r a c t i o n ( 6 5 mg) was obtained by e l u t i n g c h a r c o a l column w i t h water c o n t a i n i n g 5 / e t h a n o l . The f r a c t i o n had f o u r components^a p a r t of which was separated by f i l t e r sheet chromatography i n s o l v e n t B, the paper being developed twice f o r a t o t a l p e r i o d of 40 hours. Components w i t h d i f f e r e n t chromato-- 183 -g r a p h i c m o b i l i t i e s are quoted as f o l l o w s : P Component Ho. ''xylose 1 1.00 2 0.75 3,4 0.20 Each component was e l u t e d from a paper, f i l t e r e d through a column packed w i t h g l a s s wool and evaporated to dryness. 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 components: Component 1 (7 mg):.. I t had the same chromatographic m o b i l i t y as x y l o s e . On a c i d h y d r o l y s i s i t gave only x y l o s e . Component 2 (8 mg): I t had the same chromatographic m o b i l i t y as a r a b i n o s e . On a c i d h y d r o l y s i s i t gave only a r a b i n o s e . Component 3 (8 mg): I t s o p t i c a l r o t a t i o n was found to be W J ^ + 102.5°, (c 1.0 i n w a t e r ) . A p o r t i o n (4- mg) was d i s s o l v e d i n s u l p h u r i c a c i d (1ft, 5 ml) and h y d r o l y s e d i n a s e a l e d tube f o r 6 hours at 100°. The r e s u l t i n g s o l u t i o n was n e u t r a l i s e d w i t h a s l u r r y of barium carbonate, d e i o n i s e d by passage through ion-exchange r e s i n IR-120(H +) and evaporated to dryness. On a paper chromatogram i t showed spots corresponding to x y l o s e and g l u c o s e . A p a r t of the m a t e r i a l was s i l y l a t e d as before and examined by g . l . c - 184 -M e t h y l a t i o n of a p a r t of the m a t e r i a l was ca r r i e d , out as f o l l o w s : To 2 mg of the o l i g o s a c c h a r i d e i n 0.5 ml of dimethyl!'ormamide were added 0..2 ml of methyl i o d i d e and 0.2 g of s i l v e r o x i d e . The r e a c t i o n mixture was shaken at room temperature f o r 24 hours; Then 0.1 ml of methyl i o d i d e and 0..1 g of s i l v e r oxide were added, and shaking was continued f o r 24 hours. Benzene (10 ml) was added, s a l t s were removed by c e n t r i f u g a t i o n , and the benzene s o l u t i o n was t h r i c e e x t r a c t e d w i t h water, d r i e d over sodium sulphate and evaporated to dryness. The product was methanolysed i n 5$ methanolic hydrogen c h l o r i d e under r e f l u x i n g c o n d i t i o n s f o r 6 hours. A f t e r the removal of the s o l v e n t , the product was d i s s o l v e d i n 0.5 ml of methanol and examined by g a s - l i q u i d chromatography. The s e p a r a t i o n of the methyl g l y c o s i d e s was c a r r i e d out on a 5$ b u t a n e d i o l s u c c i n a t e (4* x %") column i s o t h e r m a l l y at 120° f o r 5 min. and then p r o -grammed at 3° per min. to h o l d at 170°. Component 4 (2 mg):. On a c i d h y d r o l y s i s i t gave x y l o s e and gluc o s e . 25 F r a c t i o n 5 (25 mg), [e<.J0 +109° (c 1.0 i n wat e r ) . On paper chromatographic examination i n s o l v e n t B i t showed R o n l y one spot w i t h chromatographic m o b i l i t y " x y l o s e 0.?'5« The component (9 mg) was i s o l a t e d from paper i n the us u a l -way. The s o l u t i o n was f i l t e r e d through a column - 1 8 5 -c o n t a i n i n g g l a s s wool and evaporated to dryness. A p a r t of the m a t e r i a l ( 2 mg) was d i s s o l v e d i n s u l p h u r i c a c i d (11:, 4- ml) and h y d r o l y s e d at 1 0 0 ° f o r 6 hours. A f t e r n e u t r a l i s a t i o n w i t h a s l u r r y of barium carbonate f o l l o w e d by passage through ion-exchange r e s i n , the material'was evaporated to dryness. On paper chromatographic examination i n s o l v e n t -B, spots corresponding to x y l o s e and 4~C— methyl-D-gl.ucose were observed. The q u a n t i t a t i v e r a t i o between x y l o s e .and 4—0-methyl-13-glucose v/as obtained by d i s s o l v i n g the h y d r o l y s a t e i n water ( 2 ml) and sodium brohydride ( 1 5 rag) was added to i t . The s o l u t i o n was l e f t o v e r n i g h t . A f t e r d e s t r o y i n g excess borohydride w i t h c a t i o n exchange r e s i n by II?-120(H +) and removing borate by d i s t i l l a t i o n w i t h methanol the product v/as a c e t y l a t e d i n a mixture of a c e t i c anhydride i p y r i d i n ( 5 ml, 1:1) at 90° f o r a p e r i o d of 4- hours. The a c e t y l a t e d m a t e r i a l was examined by g a s - l i q u i d chromatography on a column of 5 $ b u t a n e d i o l s u c c i n a t e i s o t h e r m a l l y at 210° f o r 20 min. and then programmed at 1 0 ° per min. to h o l d at 2 2 5 ° . A p a r t of the m a t e r i a l ( 5 mg) was methylated by d i s s o l v i n g i n dimethyIformamide (1 ml) and shaking i n the dark at room temperature w i t h methyl i o d i d e ( 0 . 5 nil) and s i l v e r oxide ( 0 . 5 g) f o r 24- hours. The r e a c t i o n was c o n t i n u e d f u r t h e r by treatment w i t h a d d i t i o n a l s i l v e r - 106 -oxide (0.2 g) and methyl i o d i d e (0.2 ml ) . The methylated m a t e r i a l was methanolysed and examined by g a s - l i q u i d chromatography i n the same way as d e s c r i b e d f o r 'Component 5' of ' F r a c t i o n 2' (page 184). F r a c t i o n 4: The f r a c t i o n (18 mg) on prolonged paper chromatographic examination ( i . e . , two developments, 48 hours) gave r i s e to two spots with .widely separated chromatographic m o b i l i t i e s . The components were i s o l a t e d from the paper 1 i n the u s u a l way by e l u t i n g w i t h water. Component 1: The f a s t e r moving component (8 mg) had fc(]22 + 50° (c 0.5 i n wa t e r ) . A p a r t of the m a t e r i a l (2 mg) 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 (4 ml, 111) at 100° f o r 6 hours. A f t e r n e u t r a l i s a t i o n and d e i o n i s a t i o n , the mixture was s p o t t e d on a paper chromatogram. Only spots of x y l o s e and glucose were observed. The sugar r a t i o was determined 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 , by g a s - l i q u i d chromatography. The method of s i l y l a t i o n and the experimental c o n d i t i o n s on g . l . c . are the same as d e s c r i b e d on page 125. A p a r t of the m a t e r i a l (3 mg) was methylated by u s i n g Ii ,is , d imethylf ormamide (1 m l ) , methyl i o d i d e (0.5 ml) and s i l v e r oxide (0.5 g) and the mixture was shaken i n the dark. A f t e r 24 hours 0.2 ml of methyl i o d i d e and - 187 -0.2 g of s i l v e r oxide were added and shaking was c o n t i n u e d f o r a p e r i o d of 24 hours f u r t h e r . The mixture was f i l t e r e d and the f i l t r a t e v/as e x t r a c t e d w i t h benzene as b e f o r e . The methylated m a t e r i a l was methanolysed w i t h 3 / hydrogen c h l o r i d e f o r a p e r i o d of 6 hours. A f t e r n e u t r a l i s a t i o n w i t h s i l v e r carbonate, f i l t r a t i o n and removal of s o l v e n t , the product was examined by gas-l i q u i d chromatography u s i n g the same, column and c o n d i t i o n as d e s c r i b e d on page 184. Methanolysis of the methylated d e r i v a t i v e gave components w i t h r e t e n t i o n times of methyl g l y c o s i d e s of 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g l u c o s e , 5,4—di-O-methyl-xylose and 2 , 3 - d i - 0 - r n e t h y l - x y l o s e . Component 2: (7 rag) . W ^ + 2 5 ° (c 0.5 i n water) . A p a r t of the component ( 5 mg) was h y d r o l y s e d as before to give x y l o s e and g l u c o s e , the molar 1 r a t i o being determined by g . l . c . 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 . A p a r t of the m a t e r i a l ( 2 mg) was methylated e x a c t l y i n the same way as d e s c r i b e d on page 184 . The methylated m a t e r i a l , v/as methanolysed . i n the u s u a l way and examined by g a s - l i q u i d chromato.graphy. methanolysed products were found to have i d e n t i c a l r e t e n t i o n times as those of methyl g l y c o s i d e s of 2 , 5 , 4 , 6 - t e t r a - 0 - m e t h y l g l u c o s e , 5,4-di-O-methyl x y l o s e and 2 , 3 - d i - 0 - m e t h y l x y l o s e . - 188 -Reducing end of the o l i g o s a c c h a r i d e s : : 2 rag of each of the o l i g o s a c c h a r i d e s were d i s s o l v e d i n water (5 ml) as d e s c r i b e d before and t r e a t e d w i t h sodium borohydride (10 mg) and l e f t at room temperature f o r ?A hours. A f t e r d e s t r u c t i o n of excess hydride w i t h A m b e r l i t e IR-120(H +) the m a t e r i a l was t r e a t e d w i t h methanol to remove b o r a t e . A f t e r e v a p o r a t i o n to dryness, the m a t e r i a l 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 (1N, 2 ml) at 100° f o r 6 hours- A f t e r the u s u a l c l e i o n i s a t i o n , the m a t e r i a l was examined on a paper chromatogram-in s o l v e n t B. The paper chromatogram was developed both by p - a n i s i d i n e t r i c h l o r o a c e t a t e and s i l v e r n i t r a t e - s o d i u m hydroxide to t r a c e a l d i t o l and the r e d u c i n g sugar. - 189 -Methyl g l y c o s i d e s of 1. 2 , 3 , 4 - T r i - 0 - i T i e t h y l - i ) - g l u c o s e 2 . 3,4-D:L - 0-methyl-J)-xylose 3 . 2 , 3 - D i - O - r n e t h y l - i J - x y l o s c 4. 2 , 0 , 4 - T r i - 0 - m e t h y l - l i - r l u c o se 3,4-jJi-O-methyl-J-xylose 5 . 2 , 3 - D i - 0 - m e t h y l - I ) - x y l o s e CD O Q CD ct>d ty A) f-> A) p ct ct H-CO O CO c o CO <D rr c t O CO H, ct {_ 4 B n P Q ' CO ct - H P DJ U O 3 QJ ° V tf O " H P H (D H- CQ l — 1 P< O CD Pi & »d ^ 4 o cq Pi • c M O • c t O CQ • o P 1 5 Methyl g l y c o s i d e s 1 . 2 , 5 , 4-Tri-O-Methyl-D-glucose 2 . j , 4 - h i - 0 - m e t h y l - l ) - x y l o s e 5 . 2 , 3 - D i - 0 - m e t h y l - i ) - x y l o s e 4 . 2 , 5 , 4 —Tri-O-methyl-D-glucose 5,4— Di-O-methyl-D-xylose 5 . 2 , 3 - M - 0 - m e t h y l - i ) - x y l o s e 10 Time (mm) 15 - 191 -P r e p a r a t i o n of the p o l y s a c c h a r i d e amide :. Sapote gum p o l y s a c c h a r i d e was p r o p i o n a t e d , e s t e r i -f i e d w i t h diazomethane as d e s c r i b e d p r e v i o u s l y . A p a r t of the e s t e r i f i e d m a t e r i a l (1..18 g) was d i s s o l v e d i n c h l o r o f o r m (.50 ml) i n a 250 ml round bottomed f l a s k , the l a t t e r being kept i n dry ice-acetone bath. Ammonia gas was bubbled through the s o l u t i o n f o r a p e r i o d of 2 hours. The s o l u t i o n was kept at 5° f o r 24- hours i n the round bottom f l a s k w i t h a mercury t r a p a c t i n g as a stopper. The s o l u t i o n v/as evaporated to dryness.. The f o r m a t i o n of amide v/as f o l l o w e d by i n f r a r e d spectroscopy i n order to d e t e c t the presence of the amide peak ( a b s o r p t i o n at 1700 - 1600 cm ) and.the disappearance of the e s t e r peak ( a b s o r p t i o n at 17^ 5 cm ' ) . The r e a c t i o n had to be repeated f o u r times i n order t o get a q u a n t i t a t i v e f o r m a t i o n of the p o l y s a c c h a r i d e a s i d e . Y i e l d , 592 mg. Cleavage of the p o l y s a c c h a r i d e amide: A s o l u t i o n of the p o l y s a c c h a r i d e amide (203 mg) i n water v/as c o o l e d to 0° and 4/ sodium h y p o c h l o r i t e (10 ml) v/as added w i t h s t i r r i n g . The pK of the s o l u t i o n was a d j u s t e d to 12. A f t e r 1 hour, the s o l u t i o n was heated i n a b o i l i n g water bath f o r 30 min. The c o o l e d s o l u t i o n was a c i d i f i e d to pH 5.0 and l e f t f o r 16 hours at room temperature. The s o l u t i o n was d i a l y s e d - 192 -a g a i n s t d i s t i l l e d water (3 l i t r e ) i n a l a r g e c o n t a i n e r f o r a p e r i o d of 48 hours. The d i f f u s a t e s were evaporated to dryness and were a s s o c i a t e d w i t h some i n o r g a n i c s a l t s . The contents i n s i d e the d i a l y s i s bag were evaporated to dryness. This m a t e r i a l would be r e f e r r e d t o as the •Hofmann degraded p o l y s a c c h a r i d e . Y i e l d 85 mg. M e t h y l a t i o n of the Hofmann degraded p o l y s a c c h a r i d e : A p a r t of the m a t e r i a l (70 mg) was methylated a c c o r d i n g to the procedure of Hakomori. The p o l y s a c c h a r i d e 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 d i m e t h y l s u l f o x i d e (10 ml) by s t i r r i n g at 50° under an atmosphere of dry n i t r o g e n . M e t h y l s u l p h i n y l anion was prepared by t r e a t i n g 200 mg of sodium hydride o i l d i s p e r s i o n (50$), washed s e v e r a l times w i t h pet. e t h e r , w i t h d i m e t h y l sulphoxide (5 ml) at 50° u n t i l the s o l u t i o n became c l e a r , green and e v o l u t i o n of hydrogen gas ceased. The s o l u t i o n of m e t h y l s u l p h i n y l anion was added to the p o l y s a c c h a r i d e s o l u t i o n and s t i r r e d at room temperature f o r a p e r i o d of 6 hours. To the cooled s o l u t i o n was added methyl i o d i d e (2 ml) dropwise over a period of y'i hour. The r e a c t i o n mixture was d i a l y s e d a g a i n s t running tap w>v.ter f o r 12 hours and e x t r a c t e d c o n t i n u o u s l y w i t h c h l o r o f o r m . The c h l o r o f o r m s o l u t i o n was d r i e d over anhydrous sdium s u l p h a t e . The methylated m a t e r i a l d i d - 195 -not show any a b s o r p t i o n due to -OK band i n the i n f r a r e d s p e c t r a . The m a t e r i a l (75 mg) was d i s s o l v e d i n 72/ s u l p h u r i c a c i d (0.6 ml) i n a t e s t tube e x t e r n a l l y c o o l e d w i t h i c e d water. A f t e r one hour water (5 ml) v/as added and the s o l u t i o n was b o i l e d over a p e r i o d of f o u r hours. A f t e r the u s u a l n e u t r a l i s a t i o n , d e i o n i s a t i o n and c o n c e n t r a t i o n the n e u t r a l e f f l u e n t s were examined on a paper chromato-gram u s i n g s o l v e n t system A . Only spots corresponding to 2 , 5,4—tri-O-methyl-xylose, 2,5-d.i-O-methyl-xylose and mono-O-methyl-xylose were obta i n e d . A p a r t (15 mg) was converted i n t o a l d i t o l a c e t a t e s i n the u s u a l way and v/as examined by g . l . c . u s i n g a p a i r of copper columns c o n t a i n i n g ECNSS-M, the temperature programming being 160°- 180° S 3° per min w i t h i s o t h e r m a l at 160° f o r 3 minutes. One of the s e p a r a t i o n curves i s shown i n P i g . 4-1. Gel f i l t r a t i o n of Hofmann d i f f u s a t e s : A column of Sephadex G-10 v/as prepared by a l l o w i n g 60 gms of dry g e l to s w e l l i n water (500 ml) over a p e r i o d of 4- hours. The m a t e r i a l v/as s l u r r i e d i n t o a column (120 cm x 2.5 cm) h a l f f i l l e d w i t h water. The column had a p a c k i n g of g l a s s wool at the bottom w i t h a l a y e r of sand (2 cm) on top of i t . The g e l was allowed to s e t t l e by g r a v i t y . Care was taken so t h a t no a i r bubble was allowed o o s J . X >> sz E i O t± Q i cn C\J cvi X I Q i (U i O o c o CO - 194 -F i g . 41. Separation of a l d i t o l acetates of p a r t i a l l y methylated monosaccharides from methylated Hofmann degraded polysaccharide. CO - 195 -to be trapped i n s i d e the g e l bed. The bed l e n g t h v/as 60 cm . A sponge of 2 cm. h e i g h t v/as put on the top of the g e l bed. The sample of the d i f f u s a t e s from Hofmann degradation v/as a p p l i e d on the top of the column. A f t e r the m a t e r i a l s e t t l e d on the bed s u r f a c e , the column v/as f i l l e d w i t h water and e l u t i o n s t a r t e d . E l u t i o n v/as e f f e c t e d a t a r a t e of 50 ml/hour and 5 ml f r a c t i o n s were c o l l e c t e d by an automatic f r a c t i o n c o l l e c t o r . The e l u t i o n p a t t e r n i s shown i n Fig.4-2. F r a c t i o n s were t e s t e d f o r sugar content by p h e n o l - s u l p h u r i c a c i d method. The o l i g o s a c c h a r i d e v/as almost f r e e of i n o r g a n i c s a l t s . ( 92 mg). A p a r t of the o l i g o s a c c h a r i d e (25 mg) was h y d r o l y s e d o w i t h s u l p h u r i c a c i d (1N, 10 ml) at 100 f o r a p e r i o d of 6 hours. A f t e r n e u t r a l i s a t i o n w i t h a s l u r r y of barium carbonate and d e i o n i s a t i o n by Amberlite IR -120(H +), the s o l u t i o n v/as evaporated to dryness. The m a t e r i a l v/as reduced w i t h an aqueous s o l u t i o n of sodium borohydride (15 ml, 30 mg). The reduced m a t e r i a l a f t e r the d e s t r u c t i o n of excess borohydride and removal of borate by condensation -with methanol v/as a c e t y l a t e d over a steam bath by a mixture of a c e t i c a n h y d r i d e : p y r i d i n e (1:1, 10 m l ) . The a c e t y l a t e d m a t e r i a l was examined on a column of 5$ butane d i o l s u c c i n a t e at 210° i s o t h e r m a l l y . Peaks corresponding to the a c e t a t e s 2 - 0 - m e t h y l - L - x y l i t o l , L-o:rabin.itol and x y l i t o l were observed", the r a t i o between them being 1:2:4-. - 196 -- 197 -Enzymatic degradation of sapote gum: The f o l l o w i n g enzyme p r e p a r a t i o n s , were used f o r the degradation of the p o l y s a c c h a r i d e : ( i ) Commercial p e c t i n a s e ( i i ) Commercial h e m i c e l l u l a s e ( i i i ) Commercial c e l l u l a s e ( i v ) <7<,-L—arabinofuranosidase (a g i f t from Agr. Chem. I n s t . E . T. H. Z u r i c h , S w i t z e r l a n d ) P o l y s a c c h a r i d e (0.5 g) was d i s s o l v e d i n a b u f f e r s o l u t i o n of potassium a c i d p t h a l a t e (pH 4 .0) of enzyme (o.2$, 25 m l ) . I n the case of d i g e s t i o n w i t h enzymes, ( i ) and ( i i ) the s o l u t i o n was put i n s i d e a d i a l y s i n g t u b i n g , h a l f f i l l e d and hung i n s i d e a measuring c y l i n d e r c o n t a i n i n g water (150 m l ) . The r e a c t i o n was c a r r i e d out i n a wooden chamber which v/as maintained at 30°. The water i n s i d e the c y l i n d e r was s t i r r e d c o n t i n u o u s l y w i t h a magnetic s t i r r e r . The water was r e p l a c e d a f t e r every 24 hours. The water e x t r a c t was evaporated to a syrup and s p o t t e d on a paper chromatogram. A blank from a pure gum and enzyme s o l u t i o n was processed i n a s i m i l i a r way. The r e a c t i o n was continued f o r a p e r i o d of 96 hours. - 1 9 8 -In the case of d i g e s t i o n w i t h enzymes ( i i i ) and ( i v ) , the r e a c t i o n mixture was kept i n s i d e a beaker ( i n s t e a d of a d i a l y s i s t u b i n g ) and the r e a c t i o n mixture kept at 3 0 ° f o r 9 6 hours. The enzyme and the p o l y s a c c h a r i d e were p r e c i p i t a t e d w i t h e t h a n o l . The c l e a r supernatant was con c e n t r a t e d and examined by paper chromatography. The r e s u l t s of the enzymatic degradation of sapote gum are l i s t e d below. enzyme P e c t i n a s e H e m i c e l l u l a s e C e l l u l a s e e<-L-arabinofurano-s i d a s e Sugar r e l e a s e d x y l o s e (+), arabinose(+) xylose(+) x y l o s e (+) arabinose (++++) - 1 9 9 -References 1. E. Anderson and H.C.Ledbetter, J . Am. Pharm. Assoc., 4 0 , 623 ( 1 9 5 1 ) . 2 . m.C. Denises, Compt. rend., 150, 5 2 9 ( 1 9 1 0 ) ; Chem. ;.bst., 4 , 1 4 4 5 . 5 . T. 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