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The glucomannans of sitka and black spruces Walker, Roger H. 1971

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THE GLUCOMANNANS OF SITKA AND BLACK SPRUCES  BY  ROGER H. WALKER B.Sc,  The University of B r i t i s h Columbia, 196  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n the Department of  CHEMISTRY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October, 1971  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make I t f r e e l y a v a i l a b l e f o r r e f e r e n c e  and  study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may by h i s r e p r e s e n t a t i v e s .  be granted by  permission.  Department of The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Department or  I t i s understood t h a t copying or p u b l i c a t i o n  of t h i s t h e s i s f o r f i n a n c i a l g a i n written  the Head of my  Columbia  s h a l l not be  allowed without  my  ABSTRACT  A study was made of two glucomannans, one i s o l a t e d by a l k a l i n e borate extraction of Sitka spruce wood and the second from black spruce.  These were methylated by the Hakomori procedure  sodium hydride i n dimethyl sulfoxide. was done to determine  employing  Considerable experimentation  the best conditions f o r methylation and to  demonstrate the u t i l i t y and p r a c t i c a b i l i t y of analysis by these methods.  Some inferences regarding the structure of the glucomannans  are drawn f rom the methylation data.  - iii -  TABLE OF CONTENTS Page  ABSTRACT  i i  TABLE OF CONTENTS  i i i  LIST OF FIGURES  v  LIST OF TABLES ACKNOWLEDGMENTS  V  v  l  1  1  INTRODUCTION  1  DISCUSSION  5  1.  General  5  2.  Isolation  >  3.  Methylation  4.  Hydrolysis . . . .  5.  Analysis  EXPERIMENTAL  1  1  H 16  1  9  1.  General  19  2.  Preliminary treatment of S i t k a spruce  20  3.  Delignification  20  4.  Extraction of h o l o c e l l u l o s e  26  5.  Extraction of glucomannans  27  6.  P u r i f i c a t i o n of glucomannans  27  7.  I s o l a t i o n of black spruce  28  8.  Hydrolysis of glucomannans  28  9.  Preparation of derivatives f o r gas chromatography..  10.  Methylation of Sitka spruce glucomannan  29  - iv Page  11.  Methylation of black spruce glucomannan  33  12.  Hydrolysis of methylated polysaccharides  33  13.  Hydrolysis with formic acid  34  14.  Hydrolysis with s u l f u r i c acid  34  15.  Reduction of methylated sugars  37  16.  Acetylation  37  17.  Paper chromatography  38  18.  Preparative paper chromatography  38  19.  Gas-liquid chromatography  20.  Preparation of methyl glycosides  39  21.  Mass spectrometry  39  22.  Demethylation  44  RESULTS AND INTERPRETATION  .  39  46  I d e n t i f i c a t i o n of methylated sugars  46  Structural s i g n i f i c a n c e of the data  52  BIBLIOGRAPHY  5  7  - v LIST OF FIGURES Figure  Page  1  Types of Polysaccharides  3  2  I s o l a t i o n of Spruce Glucomannans  1  3  P u r i f i c a t i o n of Glucomannans  8  4  Gas Chromatography of Hexitol Acetates  12  5  IR Spectrum of Methylated SSGM  21  6  IR Spectrum of Methylated SSGM  22  7  IR Spectrum of Methylated SSGM  33  8  Gas Chromatography of SSGM Methylated A l d i t o l s  40  9  Gas Chromatography of BSGM Methylated A l d i t o l s  41  Mass Spectrum of Methyl A l d i t o l Acetates  45  10  - vi -  LIST OF TABLES Table  Page 24  1 2  Columns and Conditions f o r Gas Chromatography  25  3  30  4  30  5  35  6  35  7  36  8  Gas Chromatography of Methyl A l d i t o l Acetates  42  9  43  10  48  - v i i-  ACKNOWLEDGMENTS The author wishes to express h i s thanks to the following people whose help and e f f o r t on h i s behalf have been most valuable: Mrs. B.I. Joseleau, Mr. G.D. Jensen and Dr. P.E. Reid who did much of the tedious work involved i n i s o l a t i n g the hemicelluloses and contributed much useful advice.  Mr. P. Borda who performed the  methoxyl analyses and ash determination.  The author also wishes to  p a r t i c u l a r l y thank h i s research director Dr. G.G.S. Dutton who gave advice and guidance throughout the work.  His patience and encouragement  at every stage and h i s a i d i n the preparation of this thesis are s i n c e r e l y appreciated.  - 1 -  INTRODUCTION A large v a r i e t y of polymeric carbohydrate  substances  are i s o l a b l e  from the woody portions of angiosperm and gymnosperm species. hemicellulose has been t r a d i t i o n a l l y applied to such  The term  substances  whether homo- or heteropolymeric i n composition but excluding c e l l u l o s e and starch.  The l i t e r a t u r e appertaining to the general  properties and structures of hemicelluloses i s very extensive and w e l l documented (1-3). be given here.  Therefore only a very b r i e f disucssion w i l l  Three p r i n c i p a l classes of polysaccharides are found  to make up the hemicellulose content of the softwood trees (conifers or gymnosperms).  These are:  glucomannan, galactoglucomannan and  * arabinoglucuronosylxylans. In the spruces which are to be considered here the three types are found i n the approximate proportion of  15%,  5% and 16%, r e s p e c t i v e l y , based on the o r i g i n a l weight of dried wood. A fourth class may  be included, that of the water soluble or water '  These acronyms for the polysaccharides indicate the p r i n c i p a l sugars to be found i n each class. Other sugars may be found i n small amounts, f o r example, glucomannans of softwoods contain a very low percentage of galactose and some arabinoglucuronosylxylans contain 4-0j-methyK)-glucuronic acid. For convenience the abbreviations GM, GGM and xylan have been commonly used to refer to the three types i n this thesis.  - 2 extractable polysaccharides.  They are recovered by d i r e c t water  extraction of the m i l l e d wood (before d e l i g n i f i c a t i o n ) i n a y i e l d of from 1 to 2% and consist mainly of arabinogalactans  (4).  In trees of the hardwood (angiosperm) group the galactoglucomannan class i s generally absent and a roughly corresponding amount of xylose polymers i s observed.  increase i n the  I t i s i n t e r e s t i n g to note  that i n the species Sequoia sempervirens - a tree which i s considered an "evolutionary intermediate" between true angiosperm and gymnosperm types - only a very small amount of galactoglucomannan i s found (5). Any  carbohydrate  polymer, regardless of whether i t consists of a  unique anhydro sugar unit or several may linear. extending  A branched polymer may  be either branched or  have e i t h e r simple one unit side chains  from a l i n e a r backbone or a multiply branched system i n  which no main chain can be defined.  This i s i l l u s t r a t e d i n F i g . 1.  Highly branched systems are quite uncommon i n wood hemicelluloses but are more frequently encountered i n gum  exudates.  With this i n mind i t i s clear that any analysis of a hemicellulose must do more than characterize the type of sugar residues present. The sequence of the d i f f e r e n t monosaccharide u n i t s , the number and type of end groups and the character of the branch points must be demonstrated. The most general  method of obtaining such information i s by  methylation of a l l free hydroxyl functions i n the polymer.  Subsequent  hydrolysis l i b e r a t e s a series of p a r t i a l l y methylated sugars, that have unblocked hydroxyl groups corresponding  to the positions of  - 3 -  Figure 1.  General Types of Polysaccharides  linear  s l i g h t l y branched  ^ complex  - 4 linkage i n the o r i g i n a l polysaccaride.  This method i s a development  of the procedures o r i g i n a l l y used to solve the structures of the c y c l i c glycosides. The e a r l i e s t p r a c t i c a l procedure for methylation of polysaccharides was  that developed by Haworth (6).  been made i n both techniques  Since then many refinements have  of methylation and methods of analysing  the resultant methyl sugars.  A considerable improvement i n the  d e t a i l of analysis i s therefore now t h i s and the methods that may  possible.  The reasons underlying  be employed w i l l be described more  f u l l y i n the discussion. With  this i n mind i t was  decided to undertake an analysis of a  series of softwood hemicelluloses. are working are commercially  Since the woods with which we  important  species considerable work  has already been done on t h e i r hemicelluloses or at least those of closely r e l a t e d species (7-11,60).  Thus i n many cases one has a  considerable amount of information a v a i l a b l e about the gross structure of the polysaccharides to be obtained. directed tox^ard extending  Our work has therefore been  the knowledge of end groups and branch  points present i n the polymer.  As mentioned i n the Abstract, this  thesis describes the i n v e s t i g a t i o n of a n a l y t i c a l and preparative methods, and the results obtained upon the glucomannans from Sitka spruce  (Picea s i t c h e n s i s ) and black spruce  (Picea mariana).  DISCUSSION 1.  General.-  The t r a d i t i o n a l method of analysis of wood  polysaccharides consists f i r s t of i s o l a t i o n and p u r i f i c a t i o n followed by determination of sugar r a t i o s , methylation', analysis of the resultant methylated sugars and, o p t i o n a l l y , periodate degradation. In this study periodate cleavage was not employed as the technique i s better designed  for confirmation of methylation  i n gross structure determinations.  results  Periodate degradation involves  cleavage of any v i c i n a l (unblocked) hydroxyl functions i n the polymer chain.  Analysis of the resultant fragments enables one to  reconstruct the types of linkage and branch points o r i g i n a l l y  present.  The inherant accuracy i s l i m i t e d by over oxidation ( i . e . the amount of periodate consumed becomes greater than that required f o r s t o i c h i o metry with the number of cleavable s i t e s ) , thus r e s t r i c t i n g the degree to which the o r i g i n a l structure can be i n f e r r e d (12,13). 2.  Isolation.-  The i s o l a t i o n and p u r i f i c a t i o n of wood  polysaccharides i s a problem which appears to have no p e r f e c t l y satisfactory solution.  Two basic d i f f i c u l t i e s may be defined.  The  hemicelluloses are very firmly held i n a r i g i d c e l l structure i n the wood i t s e l f  (15,59).  They are, i f not chemically, at least very strongly  - 6 hydrogen bonded to the l i g n i n i n the wood (16). reagents are required to s o l u b i l i z e them. of degradative  Thus very  strong  This raises the p o s s i b i l i t y  elimination (18) or a l k a l i n e peeling reactions  (17)  occurring and c e r t a i n l y any base s e n s i t i v e substituents such as a c e t y l (which some  wood polysaccharides  quickly removed.  are  known to contain)  (19-21) are  Secondly, the problem of separating each hemi-  c e l l u l o s e from the others and e s t a b l i s h i n g i t s p u r i t y i s not  trivial  (14). Chemical methods are generally of l i t t l e use here, as with  the  exception of the separation of a c i d i c polysaccharides by reaction of the carboxyl group, chemical i s not s u f f i c i e n t l y great.  d i f f e r e n t i a t i o n between hemicelluloses Physical methods are therefore generally  employed; p r i n c i p a l l y p r e f e r e n t i a l extraction and p r e c i p i t a t i o n .  A  number of f r a c t i o n a t i o n schemes have been proposed to accomplish the required separation  (11,22,23).  One  applicable i s that of T i m e l l (24).  of the most u s e f u l and generally This method was  used to i s o l a t e  the glucomannans of black and Sitka spruces and i s described i n d e t a i l i n the experimental  section.  The general scheme for the i s o l a t i o n of the polysaccharides  may  be seen i n F i g . 2 and the more d e t a i l e d procedure f o r p u r i f i c a t i o n of the crude glucomannans i s shown i n F i g . 3,  The method of  separation of galactoglucomannan and xylan i n the 24% potassium hydroxide extraction mixture w i l l not be discussed further and i s shown only for c l a r i t y . Upon completing  such an i s o l a t i o n procedure i t would be desirable  i f some means could be employed to rigorously demonstrate the  - 7 Figure 2.  I s o l a t i o n of Glucomannans. m i l l e d wood benzene/ethanol extraction  extracted wood  soluble extractives  chlorite delignification  holocelluloses degraded lignin  insoluble GM + c e l l u l o s e soluble GGM + xylan  17% NaOH 3% borate  insoluble cellulose  soluble  1. alcohol p r e c i p i t a t i o n 2. 50% acetic acid  insoluble  soluble  discard alcohol p r e c i p i t a t i o n  crude glucomannan  Figure 3.  P u r i f i c a t i o n of Glucomannans, crude GM 117% NaOH 3% borate  insoluble  soluble  17% NaOH 3% borate insoluble (GM Res 1)  50% a c e t i c acid soluble  insoluble (discard)  alcohol p r e c i p i t a t i o n GM., 17% NaOH 3% borate insoluble  soluble  50% a c e t i c acid insoluble (GM Res 2)  -  alcohol precipitatioh 2. 17% NaOH 3% borate  1  soluble  insoluble (discard) insoluble  soluble (discard)  50% acetic acid soluble  insoluble (discard)  alcohol precipitation^GM„  - 9 -  p u r i t y and homogeneity of the product.  This, as mentioned before,  i s not e a s i l y accomplished for polymeric carbohydrates and we have been forced to r e l y on a v a r i e t y of methods, none of which are entirely satisfactory.  These are as follows:  gel f i l t r a t i o n  chromatography, sugar analysis, o p t i c a l rotation, and moving boundary electrophoresis. on polysaccharides  The f i r s t of these was attempted i n our laboratory thought to be homogeneous and on deliberate  mixtures of hemicelluloses to G—200 (25).  (GGM's and xylans) on Sephadex from G—25  L i t t l e or no useful separation was achieved.  r a t i o s of constituent  sugars and the o p t i c a l rotation of the polymer  both as free and methylated polysaccharide If r e l a t i v e amounts of constituent successive  The  provide useful clues (1).  sugars remain the same between  p u r i f i c a t i o n s and i n substantial agreement with those  obtained for various  f r a c t i o n s of the methylated polymer a strong  i n d i c a t i o n of homogeneity i s provided.  S i m i l a r l y constant o p t i c a l  r o t a t i o n w i l l indicate e s s e n t i a l homogeneity. experimential  The r e s u l t s i n the  section show that f o r the glucomannans  a high degree of p u r i t y i s achieved.  at l e a s t , quite  I t i s probable that i n a l l  cases, however, complete homogeneity i s not achieved as such highly hydroxylated polymers are strongly co-precipitated and/or c o - s o l u b i l i z e d (26).  An example of t h i s i s the observation  that the residue  obtained  when d i s s o l v i n g the crude glucomannan i n b o r a t e / a l k a l i consists largely of c e l l u l o s e , which would not have been expected to dissolve under the conditions The  of extraction.  technique of moving boundary electrophoresis  ( T i s e l i u s ) has  been used successfully several times to e s t a b l i s h the homogeneity of  - 10 -  polysaccharides (27-29).  I t was attempted with one of our poly-  saccharides and the r e s u l t s were encouraging but not conclusive. Unfortunately, early i n this i n v e s t i g a t i o n the only available T i s e l i u s apparatus f a i l e d and was not replaced.  I t i s l i k e l y , however, that by  using preparative T i s e l i u s techniques small quantities of rigorously p u r i f i e d polysaccharides could be i s o l a t e d .  These would be s u f f i c i e n t  for methylation by the Hakomori procedure and to provide a sample of high q u a l i t y for g a s - l i q u i d chromatographic analysis once the retention times of the sugars to be expected had been established. Sugar r a t i o determinations on the unmethylated polysaccharides were done f i r s t by paper chromatography  to indicate the p r i n c i p a l  sugars present and then by two g a s - l i q u i d chromatographic methods. For gas chromatography  the free sugars were converted to t h e i r  t r i m e t h y l s i l y l ethers i n pyridine (30,31) or reduced to the a l d i t o l s with sodium borohydride and acetylated (32).  Although both methods  give" r e s u l t s i n quite close agreement some d i f f i c u l t y i s encountered i n determining small percentages of galactose.  When s i l y l ethers of  the sugars are prepared mixtures of anomers are formed.  The t o t a l  amount of any sugar i n a mixture i s calculated from the peak s i z e of some anomer which i s resolved completely from peaks contributed by any other sugar.  This implies that s i l y l a t i o n must be done under such  standard conditions that the anomeric r a t i o s remain constant (31). In the case of galactose the peak measured constitutes only about 28% of the t o t a l galactose and therefore a small error i n counting or equilibrium s h i f t may become quite s i g n i f i c a n t . In chromatography  of the a l d i t o l acetates i t i s d i f f i c u l t to  - 11 completely resolve g a l a c t i t o l amounts of the l a t t e r .  from mannitol i n the presence of large  A series of experiments with standard  mixtures showed that i n spite of the d i f f i c u l t i e s of r e s o l u t i o n reasonable accuracy and r e p r o d u c i b i l i t y can be obtained. of g a l a c t i t o l  present may,  i f necessary, be estimated i f i t i s very  small i n proportion to the amount of mannitol present. circumstances  i t may  happen that the g a l a c t i t o l  shoulder on the mannitol peak. reproduced  The amount  Two  Under these  appears only as a  sample chromatograms are  i n F i g . 4.  Considerable experimentation was  done to determine the optimum  conditions f o r gas l i q u i d chromatography of both a l d i t o l acetates and p a r t l y methylated  a l d i t o l acetates.  s o l i d support, percentage  A l l p o s s i b l e parameters of  of l i q u i d phase, flow rate,  initial  * temperature and program rate were v a r i e d . sixteenths inch column was  A four foot by three  used f o r a l d i t o l acetates and an eight  foot by three sixteenths inch one f o r p a r t l y methylated  alditol  acetates. 3.  Methylation.-  The methyl ether f u n c t i o n a l i t y i s one of  the few groups which can be put on the hydroxyl functions of a' polysaccharide and s t i l l survive the rather d r a s t i c conditions required f o r subsequent h y d r o l y s i s . For t h i s reason i t has been Considerable improvement was obtained when using non-acid washed Chromosorb W i n place of Gas Chrom Q as s o l i d support with a 3% coating of ECNSS-M as l i q u i d phase. This modification was suggested by Shaw's work (33) i n which he compared r e s u l t s obtained f o r the g . l . c . of carbohydrate d e r i v a t i v e s on a wide range of l i q u i d phase and s o l i d support systems.  - 12 Figure 4.  Gas Chromatograms of Hexitol Acetates  a l d i t o l acetates of SSGM,  1.  5  xylitol  10  15  20  25  30  35  mii  standard mixture of h e x i t o l acetates  6  12  18  24  30  36  min.  - 13 -  consistently employed for over seventy years and s t i l l forms the basis f o r most polysaccharide analysis. developed i n an attempt methylation.  Numerous methods have been  to improve y i e l d , completeness,  and ease of  Four methods have been commonly employed with  considerable success.  Haworth's (6) and Purdie's (34) methylations  may be considered complementary.  The former employing  aqueous sodium  hydroxide and dimethyl s u l f a t e may be used to p a r t l y methylate  an  unsubstituted polysaccharide insoluble i n organic solvents followed by the l a t t e r using s i l v e r oxide i n refluxing methyl iodide to complete the s u b s t i t u t i o n .  More recently the Kuhn procedure  has been employed using barium hydroxide/barium formamide with methyl iodide.  (35,61)  oxide i n dimethyl  The l a t e s t and most important  development i n methylation i s the Hakomori technique using dimethyl sulfoxide, sodium hydride and methyl iodide (36,37).  I t has to a  great extent revolutionized the procedure of methylation as material may  be brought to a very high degree of s u b s t i t u t i o n i n one  treatment  involving only a few steps.  The y i e l d s obtained are extremely high,  often close to t h e o r e t i c a l .  Thus, much smaller quantities of material  can be used.  The reaction i s carried out by dissolving (or suspending  i f i t i s not soluble) the polysaccharide i n dimethyl sulfoxide.  A  solution of sodium hydride i n dimethyl sulfoxide i s prepared:  NaH  +  0 (l CH„-S-CH 3  0  I The so-called dimsyl ion (I) i s then added and allowed to react to form the alkoxide of the carbohydrate hydroxyl functions.  Methyl  - 14 -  iodide i s then added slowly and with cooling.  Although the reaction  i s straight forward several points have been observed which s i g n i f i c a n t l y improved our r e s u l t s . 1.  The polysaccharide should be i n such a form as to permit maximum solution or at least maximum solvent penetration. Insoluble polysaccharides w i l l require at least two methylations.  2.  Polysaccharide concentration should not exceed 5% as otherwise gel formation may be so intense as to prevent proper mixing.  3.  A i r (oxygen) and moisture should be excluded to a reasonable  4.  degree.  The reaction of dimsyl ion with polysaccharide hydroxyl functions i s not instantaneous and improved r e s u l t s are obtained i f the base i s allowed to react 4-6 hrs with the polysaccharide (the mixture should be s t i r r e d i f i t i s inhomogeneous) before the methyl iodide i s added.  5.  I t was found on analysis of sodium hydride  o i l dispersion  mixtures that had been opened several times that the actual amount of dimsyl ion formed on reaction with dimethyl sulfoxide was only 45% of the expected value.  Thus a check  on the amount of reagent necessary to generate a two-fold excess of dimsyl ion i s advisable. Workup of the methylated polysaccharide i s extremely  easy.  The dimethyl sulfoxide solution i s poured into a large excess of water and dialysed u n t i l a l l dimethyl sulfoxide i s gone.  The solution  - 15 or suspension may  then be e i t h e r e x t r a c t e d w i t h chloroform, f r e e z e  d r i e d or evaporated to dryness under vacuum. Although very high degrees of methyl s u b s t i t u t i o n are obtained by one Hakomori methylation we were unable to o b t a i n complete substitution.  In order to avoid repeated exposure to the r a t h e r  d r a s t i c c o n d i t i o n s of the Hakomori methylation (38) i t was decided to complete the s u b s t i t u t i o n w i t h Purdie's Reagents (methyl i o d i d e , s i l v e r oxide).  This approach was suggested by W a l l e n f e l s  (35) i n h i s work  i n which he r e p o r t s that a combination of methylation techniques i s more e f f e c t i v e than repeated a p p l i c a t i o n of one method. Fractionation  w i t h petroleum ether/chloroform mixtures  c a r r i e d out on the crude methylated p o l y s a c c h a r i d e . degraded and undermethylated  Having  was excluded  m a t e r i a l by t h i s means the l a r g e r p a r t  of the sample was given a Purdie methylation.  The recovered m a t e r i a l  was again f r a c t i o n a t e d and analyzed f o r methoxyl.  The  specific  d e t a i l s of procedures followed f o r each polysaccharide may be seen i n the experimental s e c t i o n .  I t may be noted however, that the methylated  polysaccharides e x t r a c t e d by a 20% chloroform/petroleum  ether mixture  contained a d e t e c t a b l e amount of 2,3-dimethyl x y l i t o l whereas the 15% f r a c t i o n contains none.  In a s i m i l a r a n a l y s i s of ponderosa pine  glucomannan which contained x y l o s e detected by  paper chromatography  before methylation the q u a n t i t y of dimethyl x y l i t o l i n the 20% f r a c t i o n was q u i t e s i g n i f i c a n t but almost n i l i n  the 15% f r a c t i o n .  Thus i t  may be seen that a c e r t a i n s e p a r a t i o n of polysaccharide types does occur during t h i s e x t r a c t i o n . Methylated polysaccharides were examined by IR spectroscopy i n  - 16 c a r b o n t e t r a c h l o r i d e s o l u t i o n t o d e t e r m i n e t h e degree o f s u b s t i t u t i o n ( F i g s . 4-6).  When a p o l y m e r w i t h l i t t l e  o r no a b s o r p t i o n a t  3500 cm ^ was o b t a i n e d d u p l i c a t e m e t h o x y l a n a l y s e s were done b y t h e Z e i s e l method. 4.  Hydrolysis.-  S e v e r a l methods have been used e x t e n s i v e l y f o r  the h y d r o l y s i s o f methylated to  polysaccharides  a c h i e v e c o m p l e t e h y d r o l y s i s w i t h as l i t t l e  degradation as p o s s i b l e .  (39,40).  I t i s important  demethylation o r  We have r o u t i n e l y used two methods b o t h o f  which give acceptable r e s u l t s .  The p o l y s a c c h a r i d e i s f i r s t  solubilized  in  e i t h e r 90% f o r m i c a c i d o r 72% s u l f u r i c a c i d a t 0°C, then r e f l u x e d  in  1 N s u l f u r i c a c i d f o r s i x hours.  o f more v o l a t i l e m e t h y l s u g a r s  To c i r c u m v e n t  t h e n e u t r a l i z e d h y d r o l y s a t e was  extracted with chloroform p r i o r to concentration. e x t r a c t c a n be added t o t h e s u g a r s whole evaporated 5.  possible losses  The c h l o r o f o r m  f r o m t h e aqueous f r a c t i o n and t h e  a t a low temperature.  Analysis.-  When t h e m i x t u r e o f m e t h y l a t e d  sugars  derived  f r o m t h e o r i g i n a l p o l y s a c c h a r i d e i s o b t a i n e d some method o f a n a l y s i s must be s e l e c t e d .  One o f o u r o b j e c t s i n t h i s work was t o a p p l y  L i n d b e r g ' s method o f r e d u c t i o n o f t h e s u g a r s  t o t h e i r a l d i t o l s , gas  chromatography o f t h e a l d i t o l a c e t a t e s and i d e n t i f i c a t i o n by mass spectrometry  t o 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 three d i f f e r e n t hexoses  (38,41-43).  Once one r e d u c e s t h e s u g a r s  to t h e i r a l d i t o l s there i s  o n l y one g a s - l i q u i d chromatograph peak o b t a i n e d f o r each s u g a r . is  This  a d i s t i n c t advantage over procedures employing m e t h y l g l y c o s i d e s  (44,47), s i l y l d e r i v a t i v e s (30,31) o r t h e f r e e s u g a r s , w h e r e i n m i x t u r e s o f anomers a r e o b t a i n e d .  I n a d d i t i o n , t h e mass s p e c t r o m e t r i c  r e s u l t s make i t p o s s i b l e t o d e t e r m i n e  the p a t t e r n o f  methoxyl  s u b s t i t u t i o n on v e r y s m a l l amounts o f sample where t h e p r e p a r a t i o n of a c r y s t a l l i n e d e r i v a t i v e w o u l d be i m p r a c t i c a l . of g a s - l i q u i d chromatographic  From t h e i n f o r m a t i o n  r e t e n t i o n time and f r a g m e n t a t i o n  p a t t e r n s , t h e i d e n t i t y o f t h e a l d i t o l can u s u a l l y be unambiguously confirmed.  I n a d d i t i o n t h e p a r t 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 can  e a s i l y be d e m e t h y l a t e d by t h e method o f Bonner and Bourne (48).  The  f r e e a l d i t o l can t h e n be r e a c e t y l a t e d and i d e n t i f i e d by i t s g a s - l i q u i d chromatography r e t e n t i o n time as f u l l y a c e t y l a t e d h e x i t o l .  The  a l d i t o l a c e t a t e s o f g a l a c t o s e , g l u c o s e and mannose c r y s t a l l i z e r e a d i l y and a m e l t i n g p o i n t may c o l l e c t e d from the g . l . c .  The  be t a k e n i f s u f f i c i e n t sample can be same p r o c e d u r e can be employed t o  g r e a t l y r e d u c e t h e p o s s i b i l i t y t h a t any g a s - l i q u i d peak may  chromatograph  be a c o m b i n a t i o n o f two d i f f e r e n t p a r t l y m e t h y l a t e d  a l d i t o l s having v i r t u a l l y i d e n t i c a l retention times. 2 , 3 , 4 - t r i - O - m e t h y l - D - m a n n i t o l , Rt = 2.48  d i s t i n g u i s h e d by mass s p e c t r o m e t r y .  both g l u c i t o l  sugar  For example,  (38) w o u l d n o t be  f r o m 2 , 3 , 4 - t r i - O - m e t h y l - D - g l u c i t o l , Rt = 2.49 and c o u l d n o t  unmethylated  quite  resolvable be  G a s - l i q u i d chromatography as t h e  a l d i t o l a c e t a t e s w o u l d , however, i n d i c a t e t h e p r e s e n c e o f and m a n n i t o l .  Because o f o u r p a r t i c u l a r i n t e r e s t i n t h e d e t e c t i o n o f end groups we have a l s o done g a s - l i q u i d chromatography on t h e m e t h y l g l y c o s i d e s o f t h e t e t r a m e t h y l s u g a r s o b t a i n e d on h y d r o l y s i s . a p o s i t i v e demonstration of the presence of D-glucose  T h i s method p e r m i t s  2,3,4,6-tetra-O-methyl-  i n t h e p r e s e n c e o f 2,3,4,6-tetra-0^-methyl-p-mannose whereas  t h e s e two a r e e x t r e m e l y d i f f i c u l t t o r e s o l v e as a l d i t o l s and the v e r y  - 18 -  small amounts present make the c o l l e c t i o n of s u f f i c i e n t sample f o r demethylation  quite tedious.  Although the mass spectrum gives a unique fragmentation  pattern  for each d i f f e r e n t p a r t i a l l y methylated h e x i t o l , one complication may a r i s e .  Because the mass spectrometer does not d i s t i n g u i s h  stereochemical d i f f e r e n c e s , there i s loss of information upon reduction. For example, a 2,3-dimethyl and 3,4-dimethyl pentose or a 3,5and 2,4-dimethyl hexose would give the same mass spectrum a f t e r reduction.  In p r a c t i c e confusions of t h i s type r a r e l y a r i s e and can  be resolved by comparison of g a s - l i q u i d chromatography retention times or reduction with borodeuteride which s p e c i f i c a l l y l a b e l s the o r i g i n a l ' ClJ end of the sugar (43) .  - 19 -  EXPERIMENTAL  1.  General.-  To obtain polysaccharides i n an e a s i l y  manipulated  form of highest s o l u b i l i t y the technique of solvent exchange was routinely employed.  A solution of the polysaccharide was poured with constant  s t i r r i n g into four volumes of alcohol containing 5-7% g l a c i a l a c e t i c acid.  The p r e c i p i t a t e so formed was washed once with alcohol/acetic  acid, twice with alcohol, twice with acetone and twice with d i e t h y l ether.  The remaining solvent a f t e r centrifugation and decantation  was removed by heating i n a stream of warm a i r accompanied by constant trituration.  A l l alcohol used f o r these and s i m i l a r p r e c i p i t a t i o n s  was commercial IK formulated from 95% ethanol/5% methanol. Paper chromatography was done at room temperature  on Whatman #1  paper i n 8:2:2 ethyl acetate/pyridine/water f o r twenty-four 4:1:3 butanol/ethanol/water  hours,  f o r twenty-four hours or methyl ethyl  ketone/water azeotrope f o r f i v e hours (49,50).  The r e s u l t s are recorded  i n Table I. Determination of the methoxyl content of methylated polysaccharides was done i n duplicate by the Z e i s e l method and appropriate allowance made f o r any ash found.  The ash was assumed to be inert material as  no uronic acids could be detected i n the hydrolysates of the polysaccharides and i t cannot therefore have arisen from cations  - 20 -  present i n combination with these.  As a preliminary to any methoxyl  determination and as a general monitoring of the progress of methylation IR spectra were routinely run on the methylated polysaccharides i n carbon tetrachloride solution at a concentration of 7-10%.  Sample spectra f o r SSGM's may  be seen i n Figs. 5-7.  They  show that i f care i s taken to exclude traces of moisture an excellent idea may has proceeded.  be obtained of the degree to which s u b s t i t u t i o n  Samples were dried under vacuum at 60°C f o r eight  hours p r i o r to analysis. Gas-liquid chromatography was done on an F & M 720 equipped with a thermal conductivity detector.  Integration of peak area was done  using an Infotronics CRS-100 integrator. parameters are l i s t e d i n Table I I .  Column packings and program  Column packings were prepared by  d i s s o l v i n g the l i q u i d phase i n chloroform or acetone, mixing thoroughly with the s o l i d support and then evaporating the solvent on a rotary evaporator.  The columns were manually packed with v i b r a t i o n and  tamping and conditioned at 100°C and a low flow rate f o r twentyfour hours before use.  A l l rotary evaporations were performed  at  35-40°C i n vacuo except where otherwise noted. Deionizations were done by passing the solutions through a 12 cm column of IR 120 and e l u t i n g with 4 to 5 volumes of d i s t i l l e d water, 2.  Preliminary treatment of Sitka spruce.-  Sitka spruce  sawdust (500 g) was extracted i n a Soxhlet extractor i n two of 250 g with 2000 ml of a 2:1 benzene/ethanol extract was  colourless.  lots  mixture u n t i l the  The extracted sawdust was  then shaken f o r  - 24 -  TABLE I . D e s c e n d i n g P a p e r Chromatography o f M e t h y l a t e d Sugars f r o m SSGM and BSGM MEK/H 0 A z e o t r o p e B l a c k Spruce t  Rf. Obs.  Rel. Int.  Rf. Obs.  .22  .10  .23  -  -  -  -  .04  Spot  #1  S i t k a Spruce  Standards Rf. Lit. 2,3-Me Man 2  '2,6-Me Man 2  Rf. Obs.  Spot  Rf. Obs.  Rel. Int.  .17  0.10  .22  .19  -  -  -  ?  -  -  ?  .05  #1  -  2,6-Me Gluc  .18  -  .28  2,3-Me Gluc  .28  .23  #2  .22  2  #2  .28  #3  .48  3.0  .50  2,3,6-Me Man  .50  .48  #3  .48  3.0  #4  .55  1.0  .56  2,3,6-Me Gluc  .56  .53  U  .54  1.0  #5  .68  .03  .68  2,3,4,6-Me Gal  .68  .67  #5  .66  .02  #6  .78  .10  .78  2,3,4,6-Me^Man  .78  .78  #6  .78  .10  Rg. Lit.  Kg. Obs.  Rg. Obs.  Rel. Int.  #1  .61  0.10  -  - •  -  2  3  3  4  Butanol/Ethanol/Water t  Spot  Obs.  Rel. Int.  Kg. Obs.  Spot*  #1  .60  .1  .60  2,3-Me Man  .54  .62  -  ?  -  -  2,6-Me Man  -  ?  -  -  -  2,6-Me Gluc  .51  n  -  .64  .03  .64  2,3-Me Gluc  .57  .68  #7  .67  #3/4  .84  .84  2,3,6-Me Man and G l u e  .81 .83  .84  #3/4  .85  #5  .88  .03  .89  2,3,4,6-Me Gal  .88  .92  #5  .93  .05  #6  1.00  0.10  1.00  2,3,4,6-Me Man  1.00  1.00  #6  1.00  0.10  t  4.0  2  2  2  2  3  4  4  number i d e n t i f i e s t h e s p o t on t h e p a p e r chromatogram. ' The moving component i s #6.  -  1  The f a s t e s t  .04 4.0  - 25 -  TABLE I I .  Columns and Conditions used  forG.L.C.  Derivative  Column  Packing  Flow Rate  Program  Alditol acetate  4' x 3/16"  3% ECNSS-M 100 ml/min on 60-80 mesh Chrom.W or 100-120 mesh Gas Chrom.Q  Methylated alditol acetate  8' x 3/16"  Persilyl ethers  8' x 1/4" 10% SF 96 on 60-80 mesh Diatoport S  Methyl glycoside  10' x 1/4" 10% ApiezonjV 86 ml/min on 60-80 mesh Diatoport S  175° isotherm.  Separates MeMe^a-mannoside from MeMe^-aglucoside  Methyl glycosides  8' x 1/47 Carbowax 20 M 86 ml/min on 60-80 mesh Diatoport S  140°-210° at 2°/min  Separates MeMe,a-mannoside ana a-glucoside from MeMe^-a-galactoside  Methylated aldose acetate  8' x 3/10" 3% ECNSS-M V / on 86 ml/min 100-120 mesh Gas Chrom Q  a. 170° isotherm, b. 185° isotherm.  The lower temperature isothermal condition was used for separating Me^ acetates  10' at 170 to 190° at 2°/min  c  Chromosorb W (non-silylated) gives longer retention time but improved resolution Column l i f e i s reduced i f used over 195°  86 ml/min  86 ml/min  Comments  180-220° at 2°/min  - 26 -  eight hours with 4 1. of water, allowed to stand eighteen hours and f i l t e r e d .  The residue was washed with 2 x 400 ml water and the  combined f i l t r a t e and washings concentrated to 150 ml and into 600 ml alcohol.  poured  A flocculent p r e c i p i t a t e formed which was  centrifuged o f f , redissolved i n a small volume of water and p r e c i p i t a t e d again.  It was  then dried by solvent exchange to  give Sitka spruce water soluble polysaccharide (SSWSP) i n a y i e l d of 1.63 3.  g or 0.33%. D e l i g n i f i c a t i o n of extracted wood.-  sawdust (200 g) was 80°C.  Water extracted  s t i r r e d with 3 1. of water and brought to  A t o t a l of 331 g of sodium c h l o r i t e and 92 ml of g l a c i a l  a c e t i c acid was  added i n four portions at i n t e r v a l s of one hour  with continuous s t i r r i n g . to reduce foaming.  2-0ctanol was  added dropwise as needed  The extracted sawdust was washed with water  u n t i l the washings were n e u t r a l and then twice with alcohol. y i e l d of 145 g was  obtained.  second l o t of 200 g.  The procedure was  A  repeated on a  The recovered Sitka spruce h o l o c e l l u l o s e  was  142 g or 70% based on the weight of the dry wood. 4.  Extraction of Sitka spruce h o l o c e l l u l o s e . - Holocellulose  (280 g) was  shaken with 2800 ml of 24%  (w/w)  potassium  hydroxide  s o l u t i o n f o r four hours, allowed to stand for ten hours and shaken for a further four hours. 400 ml water.  I t was  then f i l t e r e d and washed twice with  The f i l t r a t e and washings were combined, neutralized  with acetic acid and poured into alcohol.  Drying by solvent  exchange gave the product i n a y i e l d of 59 g equaling 21% of the holocellulose or 14.7% of dry wood.  - 27 5.  Extraction  holocellulose  of Sitka spruce glucomannan.-  The extracted  (above) was washed thoroughly with water and then  shaken for s i x hours with 2000 ml of 17% sodium hydroxide/3% b o r i c acid solution.  I t was then permitted to stand f o r twelve hours,  shaken for two hours and f i l t e r e d .  The residue was washed twice  with 500 ml of water and the combined washings and f i l t r a t e were poured into alcohol/acetic acid. centrifugation  The p r e c i p i t a t e was recovered by  and worked up by solvent  SSGM was 72.8 g.  exchange.  Weight of crude  Ash content was 27%. Corrected weight of SSGM was  53 g or 13% of dry wood. 6.  P u r i f i c a t i o n of Sitka spruce glucomannan.-  Crude SSGM  (45 g) was s t i r r e d f o r twenty-four hours with 500 ml of 17% sodium hydroxide/3% boric acid solution.  As a large portion of the material  appeared to remain undissolved the extraction was repeated with 300 ml fresh solvent  for a further twenty-four hours.  suspensions were centrifuged;  the centrifugate  The combined  i s SSGM Residue 1.  The supernatant was p r e c i p i t a t e d with 500 ml 5% barium hydroxide solution added dropwise with constant s t i r r i n g .  The p r e c i p i t a t e  was washed twice with 25 ml 5% sodium hydroxide s o l u t i o n and then s t i r r e d at 0°C with 150 ml 50% acetic acid. was re-extracted  The insoluble  material  with a further portion of 50 ml of 50% a c e t i c acid.  The combined solutions were precipitated into alcohol/acetic and worked up by solvent  exchange.  acid  Y i e l d of SSGM^ = 23 g or 7% of  dry wood. The SSGM was s t i r r e d with 220 ml 17% sodium hydroxide/3% boric 1  acid f o r twelve hours and centrifuged.  The residue was suspended  - 28 -  i n 100 ml 50% acetic acid and s t i r r e d u n t i l no more material would dissolve.  The mixture was then centrifuged  termed SSGM Residue 2.  and the centrifugate was  The supernatant x^as poured into  acid and recovered by solvent  exchange.  alcohol/acetic  The recovered material was  then dissolved by s t i r r i n g for 4 hrs i n 100 ml 17% sodium hydroxide/ 3% boric acid.  The borate solutions were then combined and  p r e c i p i t a t e d with 200 ml of 5% barium hydroxide. was  dissolved  alcohol/acetic  The p r e c i p i t a t e  i n 150 ml 50% acetic acid, p r e c i p i t a t e d  into  acid and worked up i n the normal manner.  SSGM = 13 g or 4% of dry wood.  This SSGM was used for  2  further methylation studies.  Y i e l d of  2  The complete i s o l a t i o n scheme may be  seen on the flow diagrams, Figs. 2 and 3. 7.  I s o l a t i o n of the black spruce.-  The procedures followed  were e s s e n t i a l l y i d e n t i c a l with those just described for the Sitka spruce.  The black spruce wood was not extracted for water  polysaccharides.  soluble  Comparative y i e l d s and sugar r a t i o s are given i n  Tables I I I and IV. 8.  Hydrolysis of glucomannans.-  The i s o l a t e d polysaccharides  were f i r s t s o l u b i l i z e d by t r i t u r a t i o n i n 72% s u l f u r i c acid at 0°C for about one hour.  The solution was then diluted to 1.0 N and  hydrolyzed i n a sealed tube at 98°C for eight hours. was  neutralized with barium carbonate and centrifuged.  were then extracted with water and centrifuged. was  repeated three times.  The solution The s o l i d s  This procedure  The combined centrifugates  were passed  through a short column of IR 120 and concentrated to a small volume. Paper chromatograms were run on this solution to detect the sugars present.  - 29 -  9.  Preparation of derivatives f o r g a s - l i q u i d  chromatography.-  The t r i m e t h y l s i l y l derivatives were prepared by d i s s o l v i n g 10-20 mg of the sugar mixture, immediately a f t e r concentration just to dryness, i n 2 ml of dry pyridine.  Hexamethyldisilizane (HMDS) (0.5 ml) and  chlorotrimethylsilane (TMS) (0.25 ml) were then added and the mixture allowed to stand f i v e minutes. A l d i t o l acetates were prepared by dissolving a small portion of the sugar mixture i n 15 ml of water and adding a s o l u t i o n containing a one molar excess of sodium borohydride.  After 4-6  hours the excess borohydride was neutralized by the addition of 10% acetic acid.  The solution was then deionized with IR 120 and borate  s a l t s were removed by evaporation to dryness several times with methanol. The a l d i t o l s were dissolved i n 2 ml of a 50:50 mixture of acetic anhydride and pyridine and heated i n a sealed tube at 98°C for 10 minutes.  The acetylation reagents could then be removed on a  vacuum rotary evaporator at 45-50°C as the a l d i t o l acetates are i n s u f f i c i e n t l y v o l a t i l e to be l o s t under these conditions.  The  mixture was then dissolved i n a small volume of chloroform for i n j e c t i o n on the g . l . c . 10. SSGM2  Hakomo r i methylation of Sitka spruce glucomannan.—  The  (1.5 g) was passed through a 200 mesh sieve and dried at 60°  for four hours under vacuum and placed i n a flask f i t t e d with a rubber serum cap.  Dry nitrogen was passed through the flask a f t e r adding  75 ml of dry ( d i s t i l l e d from calcium hydride) DMSO. contents were s t i r r e d  Flask and  (magnetic s t i r r e r ) f o r twenty-four hours.  - 30 -  TABLE I I I .  I s o l a t i o n o f t h e Glucomannans Air dried wood  Oven dried wood  500 g  467 g  Benzene/ ethanol extracted wood  **  Crude GM  Delignified wood  KOH extract  350 g  84 g  71 g  18.3  15.3  73 g  66 g  15.7  14.2  B l a c k Spruce (i)  weight found  ( i i ) percent y i e l d  75  S i t k a Spruce (i)  weight found  500 g  465 g  455 g  *  344 g 74  ( i i ) percent y i e l d  Based on w e i g h t o f oven d r y wood = 100%.  Samples c o n t a i n 18-25% a s h .  TABLE I V . P e r c e n t a g e C o m p o s i t i o n o f I s o l a t e d P o l y s a c c h a r i d e s .  ** Percent Yield  j  Mannose  Glucose  Galactose  Arabinose  Xylose  4.0  1.0  0.1  0.0  Tr  SSGM2  4.0  81.5  16.5  2.0  0.0  Tr  SSGM Res-L  0.5  84.0  SSGM R e s *  0.2  5.0  1.0  BSGM  8.1 4.0  1.0  0.1  0.0  Tr  80.5  18.0  2.0  0.0  Tr  2  2  BSGM *  2.7  BSGM DMSO s o l .  0.1  BSGM H 0 sol.  2.0  BSGMinsol.  0.5  BSGM Res 1  1.0  BSGM Res 2  0.25  2  2  2  D  1 N NaOH  7.3  SSGMi*  [«]  -27°  [«]  D  17% NaOH 3% B o r a t e  -40°  Tr  16.0  -44.5°  -31°  -43°  2  -57°  87.0  13.0  * R a t i o s e s t i m a t e d from paner chromatography.  **  C a l c u l a t e d on an a s h f r e e b a s i s .  - 31 -  Sodium hydride (50% o i l dispersion) was washed free of o i l with dry 30-60° petroleum ether and allowed to react f u l l y with DMSO (15 ml) f i r s t at room temperature f o r two hours then for four hours at 55°. An aliquot of DMSO-dimsyl i o n solution was then removed and t i t r a t e d with 0.1 N HCl a f t e r adding to 25 ml of water containing two drops of phenolphthalein. calculated.  The amount of active sodium hydride was then  Bottles of sodium hydride that had been some time i n use  were found to contain much less than the expected amount of active reagent.  S u f f i c i e n t sodium hydride (1.20 g) to provide a 100%  excess calculated on the basis of three reacting hydroxyl functions per sugar residue was then reacted i n the same manner with 25 ml DMSO.  The dimsyl ion, which formed a clear brown-green s o l u t i o n ,  was then added to the polysaccharide by i n j e c t i n g through the rubber serum cap with a hypodermic  syringe.  There was immediate and  extensive gel formation and the mixture turned a dark brown colour. * After four hours a 100% excess  of methyl iodide was then added  (7.1 g = 3.1 ml) i n increments of 0.25 ml over s i x hours with constant s t i r r i n g . to r i s e above 15°C.  The temperature of the reaction was not allowed The mixture was then l i g h t straw i n colour and  s t i l l s l i g h t l y cloudy i n d i c a t i n g incomplete solution. s t i r r e d for a further 12 hrs  and then diluted with 4 volumes of cold  Based on the reaction:  + Na  _  0  II  CH„-S-CH„ + CH„I  I t was  0 II  • CH„-CH„-S-CH„ + Nal  - 32 -  water and dialyzed against running water for 72 hrs. was  Tbe polysaccharide  then extracted with refluxing chloroform and a methoxyl  analysis was done on the crude extract. The methylation procedure was  OMe  =28.2%.  then repeated on the p a r t l y  methylated polysaccharide a f t e r drying i t under vacuum.  The  polysaccharide was dissolved i n 50 ml DMSO and 1.0 g NaH  (as dimsyl  ion) and 2.5 ml methyl iodide were added.  The  methylated  polysaccharide was recovered as before i n a y i e l d of 1.36 material was  then f r a c t i o n a l l y extracted with  The samples SSGM  for gas-liquid chromatography. 302-308 was  15  The  chloroform/petroleum  308  ether (30-60°) solvents.  g.  368  and SSGM  A portion (165 mg)  remethylated using Purdie's reagents.  2Q  were used  of the f r a c t i o n The sample  dissolved i n 25 ml methyl iodide and 7 g of freshly prepared  was silver  oxide was added at i n t e r v a l s to the r e f l u x i n g solution over a period of seventy-two hours.  The solution was  then centrifuged to remove  the bulk of the s i l v e r s a l t s which were then extracted for twentyfour hours i n a Soxhlet extractor with chloroform.  The solutions  were combined, concentrated to dryness and f r a c t i o n a l l y extracted with chloroform/petroleum ether as f o r the crude methylated polysaccharide.  This procedure e f f e c t i v e l y separates the poly-  saccharide from c o l l o i d a l s i l v e r s a l t s . i n the 15% chloroform  The data f o r the material recovered  solvent i s included i n Table V.  It was  hoped to demonstrate with this more highly methylated polymer that the l a s t peak i n the g a s - l i q u i d chromatograph of methylated sugars derived from sugars present as a result of undermethylation.  was The  gas-liquid chromatography results obtained may be seen i n Table VIII and their s i g n i f i c a n c e i s discussed i n the i n t e r p r e t a t i o n .  - 33 -  11. 1.31  Methylation of the black spruce glucomannan.-  g of BSGM^ was taken for methylation and s t i r r e d for f o r t y -  eight hours at 50° i n 60 ml of dry DMSO. was  Initially  observed  As only a small amount  to d i s s o l v e the insoluble material was removed by  centrifugation.  A portion of the insoluble material was s t i r r e d  with water for twenty-four  hours and a l l samples were dialyzed for  twenty-four hours and recovered by freeze drying. fractions as shown i n Table VI.  This gave three  Methylation was c a r r i e d out on 694 mg  of the water soluble BSGM as described for the SSGM using 1.75 g 2  of NaH (free of o i l ) .  2  The polysaccharide i s o l a t e d a f t e r d i a l y s i s  was  only p a r t i a l l y methylated  NaH  i n 50 ml DMSO.  and was remethylated  using 1.5 g  The material was then dialysed and subjected to  f r a c t i o n a l extraction with petroleum  ether/chloroform a f t e r removal  of water on the rotary evaporator. As the methoxyl value for the combined 10 and 15% extracts was  deemed to be only marginally high enough they were remethylated  using 0.25 g NaH.  L i t t l e g e l formation was seen to occur i n this  instance.  The product was i s o l a t e d i n the customary manner and  analysed.  As the methoxyl value had increased only very s l i g h t l y  this sample was used f o r subsequent analyses. 12.  Hydrolysis of the methylated  polysaccharides.-  of minimizing degradation, demethylation  The importance  and loss of v o l a t i l e  methylated  sugars has been emphasized i n the discussion. Individual  methylated  polysaccharides were hydrolysed by either method as  - 34 -  described below 13.  and l i s t e d i n Table VII.  Hydrolysis with 90% formic acid.-  SSGM  3 0 2  "  3 0 8  (150 mg)"  was placed i n a small round bottomed f l a s k with 10 ml of 90% formic acid and refluxed for one hour.  The formic acid was  then  removed at 40°C on a rotary evaporator and 10 ml of I N was added and refluxed f o r s i x hours.  s u l f u r i c acid  The s u l f u r i c acid was  neutralized with a s l u r r y of barium carbonate.  then  The suspension was  centrifuged and the p r e c i p i t a t e extracted with water and centrifuged. This procedure was repeated three times.  The centrifugates were  deionized with IR 120 and then extracted with chloroform to remove the more highly methylated and, therefore, more v o l a t i l e sugars. The aqueous mixture was then evaporated to dryness at 40°; the chloroform extract 14.  was added and evaporated at 30°C.  Hydrolysis with 72% s u l f u r i c a c i d . -  BSGM^Q (5 mg)  was  mixed with 0.5 ml of 72% s u l f u r i c acid and held at 0°C f o r one hour or u n t i l the polysaccharide was observed to be completely i n solution. six hours.  The s o l u t i o n was then d i l u t e d to 10 ml and refluxed f o r Work up then followed the procedure outlined above.  It  i s u s e f u l , perhaps, to note also that such a 5 mg sample upon reduction and acetylation provided j u s t s u f f i c i e n t material f o r two i n j e c t i o n s on our gas-liquid chromatograph  of s u f f i c i e n t size to  - 35 -  TABLE V.  Fractionation of Methylated SSGM  Solvent (% CHC1 )  Weight  OMe  Ash  OMe*  [a]?f  CHC1  Anal.//  3  5  .021  10  .397  15  .721  43.6  20  .187  39.1  30  .010  302-308 remethylated  .150  44.7  1.78  0.00  44.4  44.7  -25.1°  302-309  -26.6°  302-308  -23.7°  302-368  -23.8°  302-371  TABLE VI. Fractionation of Methylated BSGM Solvent (% CHC1 )  Weight  * OMe  Ash  OMe  43.1  1.6  43.9  [a]^  5  CHC1  3  Anal.//  3  10  .124  -17.4° 302-298  15  .376  -19.4  20  .397  -23.9  25  .031  30  .023  40  .007  302-298 remethylated  .300  43.66  1.11  44.1  302-302  The ash i n these samples may be accounted for i n two ways (see p. 19 ) : either as an inert non-volatile contaminant or as the residue derived from cations o r i g i n a l l y combined with uronic acids. As no traces of uronic acid were found i n hydrolysates of the methylated polysaccharides the former case was assumed.  - 36 -  TABLE VII.  Methods used f o r Hydrolysis of Individual Methylated Polysaccharides  Hydrolysis Method  Polysaccharide  Anal. #  SSGM 15  302-308  Formic acid  SSGM 15  302-371  72% S u l f u r i c  SSGM  302-368  Formic acid  -> H„S0. 1 N 2 4  302-298  Formic acid  -> H.SO. 1 N 2 4  Formic acid  H.SO. I N 2 4  20  BSGM. 10-15 BSGM  a 20  BSGM, 20  72% S u l f u r i c  -> H.SO. 1 N 2 4 »- H_S0. 1 N 2 4  • H.SO. 1 N 2 4  - 37 integrate peaks greater than or equal to about 0.5% of the t o t a l . 15.  Reduction of methylated sugars.-  A l l reductions were  carried out i n aqueous solution with sodium borohydride. mixture of methylated  The  sigars was dissolved i n a small volume of  water and a solution containing a one molar excess of sodium borohydride was added and allowed to stand overnight.  Excess borohydride  was then neutralized by the addition of 10% acetic acid u n t i l evolution of hydrogen  ceased.  The solution was then passed through  a column of IR 120 ion exchange r e s i n and concentrated to dryness. The mixture of reduced sugars and s a l t s was then evaporated three times with methanol to remove b o r i c acid as the v o l a t i l e methyl borate (51). 16.  Acetylation of methylated a l d i t o l s or aldoses.-  The sugars  were removed from the f l a s k by d i s s o l u t i o n three times i n a small volume of a 50:50 mixture of acetic anhydride/pyridine ( t o t a l 8 ml) and placed i n a test tube which was sealed and heated at 98° f o r 15 minutes.  By this method acetylation was found to proceed i n v a r i a b l y  to completion.  The a c e t y l a t i o n was attempted once i n an unsealed  vessel on top of a steam bath but was found to f a i l to go to completion as was shown by continued presence of an OH peak i n the IR spectrum of the product.  A l d i t o l acetates were extracted from the  acetylation mixture by the following procedure.  Extraction three  times i n a separatory funnel with chloroform, extraction twice of the chloroform extract with 1 N HCl, washing once with sodium bicarbonate and once with water.  The chloroform solution was then dried over .  calcium chloride and concentrated to a suitable volume for i n j e c t i o n on the  - 38 -  g a s - l i q u i d chromatograph.  I f t h i s extraction was not performed anomalous  peaks were found i n chromatograms of samples that had remained i n the a c e t y l a t i o n mixture f o r some time. 17.  Paper chromatography.-  Paper chromatography was done both  on free sugars from the unmethylated polysaccharide and on methylated sugars.  The former were run using the eluents 8 : 2 : 2 e t h y l acetate/  pyridine/water or  18:3:1:4  e t h y l acetate/acetic acid/formic acid/water.  The f i r s t solvent i s a basic medium and w i l l resolve glucose, galactose, mannose, arabinose, and xylose i n twenty-four hours.  The second  solvent i s a c i d i c i n nature and i s used to separate a c i d i c sugars. S i m i l a r l y f o r methylated sugars two eluents were r o u t i n e l y employed. Methyl e t h y l ketone/water  azeotrope f o r one front time (approximately  four and one-half hours f o r 7 0 cm paper) or butanol/ethanol/water 4 : 1 : 3 f o r twenty-four hours.  The former was found to be most u s e f u l  when a t t e n t i o n was directed toward the f a s t e r moving tetramethyl hexoses and trimethyl pentoses, the l a t t e r gave superior separation of dimethyl hexoses.  Chromatograms  of free sugars were developed by  dipping i n C I ) s i l v e r n i t r a t e , ( 2 ) sodium hydroxide, ( 3 ) sodium thiosulfate.  Methylated sugars were developed with p-anisidine spray  followed by heating at 1 1 8 ° f o r f i v e minutes. The Rf. and Rg. values observed f o r sugars from polysaccharides, standard samples and recorded l i t e r a t u r e values are l i s t e d i n Table I 18.  Preparative paper chromatography.-  (49,50).  About 1 0 0 mg of the  mixture of methylated polysaccharide hydrolysates of S i t k a spruce and black spruce were streaked on four sheets ( 2 5 x 7 0 cm) of Whatman No. 1 chromatography paper and developed i n methyl e t h y l ketone/water.  A f t e r drying, guide s t r i p s were cut from the sides  - 39 of the papers to determine the p o s i t i o n of the bands.  Four s t r i p s  were cut corresponding to those spots designated 1 and 2, 3 and 4, 5 and 6 i n Table I.  Methylated sugars were removed from the  s t r i p s by eluting with water and then concentrated to dryness at low temperature. 19.  Gas-liquid chromatography.-  Samples were prepared for gas-  l i q u i d chromatography i n s o l u t i o n i n either chloroform or acetone for a l d i t o l acetates, methylated a l d i t o l acetates, methylated sugar acetates and methyl glycosides.  S i l y l derivatives were injected  d i r e c t l y or evaporated to dryness and dissolved i n hexane.  Systems  and conditions used f o r various derivatives are l i s t e d i n Table I I . Sample size was determined by the information desired.  Small samples  were chromatographed to determine r e l a t i v e r a t i o s of major components. Larger sample s i z e was used so that the peaks of minor components appeared at s u f f i c i e n t i n t e n s i t y to activate the i n t e g r a t i o n c i r c u i t s and provide r e l i a b l e measurement.  Sample chromatograms are  reproduced i n Figs. 8 and 9. 20.  Preparation of methyl glycosides.-  Methyl glycosides were  prepared from methyl sugars i s o l a t e d from preparative paper chromatography of methylated BSGM and SSGM hydrolysates.  Sugars were  dissolved i n 25 ml 3% hydrogen chloride/methanol and refluxed f o r eight hours.  Previous work (52,53) had shown that this produced  e s s e n t i a l l y complete e q u i l i b r a t i o n of the isomers formed.  Hydrochloric  acid was neutralized with s i l v e r carbonate and a f t e r f i l t r a t i o n the glycosides were concentrated to dryness at 35°C. 21.  Mass spectroscopy.-  Samples were collected from the gas-  l i q u i d chromatography of methylated a l d i t o l acetates for mass spectro-  Figure 8.  Gas-Liquid Chromatogram of A l d i t o l Acetate of Methylated SSGM  3% ECNSS-M on Chromosorb  W.  10 min at 170° to 190° at 2°/min.  Flow rate = 86 ml/min.  - 42 * TABLE VIII.  Amounts of Methyl A l d i t o l Acetates as Determined by G.L.C.  Polysaccharide  BSGM  #1 Me.M 4  #2 Me.Gal 4  #2' Me X  #3 Me M  //4 Me G  //5 Me M  #6 Me G  #7 Me M  //8 //9 Me G Me?  2.77  1.33  -  76.6  17.3  0.65  tr.  0.80  0-70 t r .  1.6  0.5  0.1  78.0  18.5  0.25  tr.  1.5  0.3  tr.  a  0.45  2.0  1.8  71.6  21.5  0.2  0.3  2.2  0.5  0.4  b  1.15  1.65  2.6  67.8  20.4  1.66  1.04  4.65  0.8  0.7  1.15  1.65  2.6  70.2  20.9  0.2  0.3  2.2  0.5  0.4  a  1.7  0.3  -  76.6  17.2  0.6  0.2  1.5  0.5  0.7  b  3.12  0.92  -  71.5  17.6  1.8  1.15  2.17  0.7  0.9  3.02  0.90  -  75.3  16.8  0.6  0.2  1.5  0.5  0.7  0.72  0.23  •vO.3  67.8  21.4  1.47  1.55  3.7  1.04 1.7  15  BSGM X  BSGM  2 Q  BSGM  2 Q  BSGM  SSGM  1 5  SSGM  1 5  SSGM SSGM  comb.  2Q  15  2()  comb.  2  3  3  2  * E x p r e s s e d i n terms o f mass p e r c e n t  of the t o t a l .  2  2  2  - 43 -  TABLE IX.  Ratios of Methylated Sugars Found i n the Polysaccharides  Methylated polysaccharide  BSGM  2Q  BSGM  2Q  BSGM  15  SSGM  15  SSGM SSGM  15  2()  (%) 1.33  a  3.34  1.65  b  3.32  1.80  3.32  1.70  a  4.45  0.30  b  4.05  0,92  4.45 3.10  comb.  2Q  SSGM  Me4 galactose  4.50  15  BSGM  Ratio of t o t a l methyl mannose/methyl glucoses  comb.  ,  =  (%)  dimethyl sugars  (%)  2,3dimethyl sugars  6  6'  38  51  52  97  (%)  4.13  2.15  1.50  2.80  3.20  2.70  0.92  3.92  2.90  2.00  0.23  0.95  7.76  4.74  100 % Me. sugars-% 2,3-dimethyl  6  Tetra methyl sugars  sugars  100 " % tetramethyl sugars-% t o t a l dimethyl sugars  The 6 value as defined above should i n d i c a t e the degree of p o l y m e r i z a t i o n i n the polymer i f only 2,3-di-0-methy.l hexoses are s t r u c t u r a l l y s i g n i f i c a n t ( i . e . i f 2,6-di-O-methyl hexoses r e s u l t from under- and/or demethylation). I f a l l dimethyl sugars are s i g n i f i c a n t the 6' value should correspond t o the chain length. S i m i l a r l y the percentage of branch p o i n t s i s i n d i c a t e d by the percentage of 2,3-di-0-methyl hexoses o r of a l l dimethyl hexoses.  - 44 -  metric analysis and for demethylation.  Samples were run on either  an A.E.I. MS 902 instrument or a Nuclide 1290 G.  Ionizing voltages  and source temperatures were 70 Ev and 150°C, respectively. r e s u l t s obtained are discussed i n the following section.  The  Proposed  explanations of the f r a c t i o n a t i o n patterns may be found i n the l i t e r a t u r e (42,43,54).  They are not further discussed here as the mass  spectrometry was employed only as a means of q u a l i t a t i v e i d e n t i f i c a t i o n of the methyl a l d i t o l s .  A t y p i c a l s i m p l i f i e d mass spectrum i s shown  i n Figure 10. 22.  Demethylation.-  Samples of methyl a l d i t o l acetates f o r  demethylation were washed from the c a p i l l i a r y tubes i n which they were collected with 2 ml of dichloromethane and cooled i n small f l a s k s to -78°C (dry ice/acetone) (48).  About 1 ml of boron t r i c h l o r i d e  was then added; the mixtures were maintained at -78°C f o r one hour and then allowed to warm to room temperature and l e f t standing for sixteen hours.  They were then evaporated three times with methanol  and acetylated by the procedure previously described.  Demethylation  was generally complete although occasionally small amounts of p a r t i a l l y methylated a l d i t o l acetates could be detected upon subsequent gas chromatography.  Methylated mannitols appeared to be p a r t i c u l a r l y  r e s i s t a n t to complete demethylation.  (150.0)  Figure 10  400 -  300  200 '  100  m/e  43  87  99 101  117  129  161  189  S i m p l i f i e d Mass Spectrum of SSGM peak #6 2,3-di-0_-methyl-D-glucitol tetraacetate.  233  - 46 -  RESULTS AND  INTERPRETATION  The summary of data and i t s i n t e r p r e t a t i o n may be conveniently divided into two parts.  The f i r s t dealing with the establishment  of the i d e n t i t y of methylated a l d i t o l s obtained and the second a discussion of s t r u c t u r a l features of the polysaccharides which may  be i n f e r r e d . For convenience  of reference the peaks observed i n chromatograms  of the methylated a l d i t o l acetates have been designated 1-9.  The  same nine peaks are observed f o r both glucomannans with only the percentages present varying s l i g h t l y . I d e n t i f i c a t i o n of methyl sugars Peak #1.-  This had the mass spectrum of a  2,3,4,6-tetra-0-methyl-  h e x i t o l , showing the following peaks i n high i n t e n s i t y : m/e; 45, 71, 87, 101, 117, 129, 145, 161, and 205.  43,  The peak had the same  retention time as 2,3,4,6-tetra-0-methyl-D-glucitol and -mannitol acetates.  On paper chromatography a spot with the retention time  of 2,3,4,6-tetra-O-methyl-mannose (or glucose) was  observed.  I s o l a t i o n of this and preparation of the methyl glycoside provided the following r e s u l t s . (i)  the glycoside did not co-chromatograph with methyl 2,3,4,6-  tetra-O-methyl-ct-D-galactoside on  Carbowax 20 M and gave a peak  - 47 -  with the same retention times as methyl 2,3,4,6-tetra-C^-methyl-a-Dmannoside or -glucoside (52).  No peak corresponding to methyl  2,3,4,6-tetra-O-methyl-B-p-glucoside  was  observed  ( i f the sugar  was  glucose some of the g-anomer should be formed on methanolysis). (ii)  On Apiezon N the glycoside co-chromatographed with methyl  2,3,4,6-tetra-0-methyl-a-D-mannoside and not with the corresponding ct-D-glucoside. ( i i i ) Additional confirmation was  obtained f o r the BSGM by  g . l . c . of the acetates of the methylated aldoses.  The  relative  retention times observed for some standard samples are reported i n Table X.  In the chromatogram of BSGM methylated sugar acetate peaks  with retention times corresponding to those of 2,3,4,6-tetra-0-methyl-Dmannose and galactose acetates were found. 7  It may  be concluded that peak #1 corresponds to  2,3,4,6-tetra-0-  methyl-D-mannitol and that no tetra-0_-methyl-D-glucitol i s present within the l i m i t of detection (about 1/5 of the amount of tetramethyl mannitol found). Peak ill.-  This has also the mass spectrum expected for a 2,3,4,6-  tetra-O^methyl-D-alditol acetate and the same retention time as 2,3,4,6-tetra-O-methyl-D-galactitol.  On paper chromatography a f a i n t  spot with the retantion time of 2,3,4,6-tetra-£-methyl-D-galactose i s observed. (i)  The gas chromatography of the methyl glycoside showed:  One peak on Carbowax 20 M with the retention times of methyl  2,3,4,6-tetra-0-methyl-a  + 8-D-galactosides and nothing with the  retention times of the corresponding a-D-glucosides of a-D-mannosides. ( i i ) When chromatographed on Apiezon N the glycoside gave two  - 48 -  TABLE X.  Retention Times of Methylated Aldose Acetates  Compound  Ret. time  Ret. time  9.4  11.5  2,3,4,6-tetramethyl galactose acetate  13.4  20.2  2,3,5,6-tetramethyl galactose acetate  15.1  2,3,4,6-tetramethyl mannose acetate  17.9  2,3,4,6-tetramethyl glucose acetate  Conditions:  23.3  Approx. Ratio  .85 1.4  20  Isothermal at 170°C  2,3,4,6-tetramethyl glucose acetate  4.8  5.2  2,3,6-trimethyl glucose acetate  14.8  17.0  1.3  2,3,6-trimethyl mannose acetate  31.2  2,3-dimethyl glucose acetate  34.8  39.7  .3  Conditions:  Isothermal at 185°C  - 49 -  peaks with the retention times of methyl 2,3,4,6-tetra-0-methyl-a and  B  -  D-galactosides. Peak #2 i s therefore 2,3,4,6-tetra-()-methyl-p-galactitol. Peak #3.-  As the largest peak i n the chromatograms this compound  should be 2,3,6-tri-O-methyl-D-mannitol acetate (60)• for i t exhibited the major fragments: m/e; 117, and 233 as expected  The mass spectrum  43,45, 87, 99, 101,  113,  for a 2,3,6-tri-0-methyl-D-alditol acetate.  The retention time on g . l . c . was  the same as that for 2,3,6-tri-O-  methyl-D-mannitol acetate and the Rf. and Rg. values on paper chromatography were the same.  F i n a l l y , upon demethylation  reacetylation only mannitol hexaacetate was  and  found when the product  was gas chromatographed on a column of ECNSS-M.  M.p.  118°C. L i t .  value 123°C. Peak #4.-  As the second major component t h i s peak should be  2,3,6-tri-0^-methyl-p-glucitol acetate. fragments:  m/e;  The mass spectrum, major  43, 45, 87, 99, 101, 113, 117, and 233, i s consistent  only with a 2,3,6-tri-O-methyl-D-hexitol  acetate.  Paper chromatography  i n both solvents showed spots with Rf's exactly corresponding those of authentic 2,3,6-tri-O—methyl-D-glucose.' derived a f t e r demethylation was present.  M.p.  97°C.  Peak #5.m/e;  to  The h e x i t o l acetate  g l u c i t o l with only traces of mannitol  L i t . value 99°C.  The mass spectrum of this peak showed major fragments:  45, 87, 117 and 129 which i s consistent only with a 2,6-di-O-  methyl-D-hexitol  acetate.  The retention time on g . l . c . was  close to  that reported for 2,6-di-O-methyl-D-mannitol acetate (38) (no authentic sample was  available).  Mannitol acetate was  obtained upon gas  liquid  - 50 -  chromatography  of the demethylated and acetylated material.  The  i d e n t i t y may be further substantiated by a process of elimination: 2.3- di-C^-methyl-D-mannitol had been i d e n t i f i e d as peak #7, 2,4- and 3.4- di-O-methyl-D-mannitols  have g . l . c . retention times as reported  by Lindberg (38) that are very much too great and i n addition t h e i r mass spectra are very d i f f e r e n t to that observed.  4,6-Di-O^-  methyl-D-mannitol was eliminated by the absence of intense fragments i n the mass spectrum at m/e;  101, 161 and 261.  2,3,4-, 2,4,6-, and  3,4,6-tri-O-methyl-D-mannitols were eliminated as a l l have reported retention times less than that of 2,3,6-tri-O-methyl-D-glucitol. 2,3,5-Tri-O-methyl-D-mannitol  was eliminated by the absence of any  large amount of the fragment m/e of m/e  101, and the presence of fragments  45, 87, and 129.  Peak #6.-  As may be seen from the figures i n Table VIII only  very small amounts of this peak were present.  The mass spectrum  was e s s e n t i a l l y i d e n t i c a l to that described for peak #5.  The g . l . c .  retention time i s the same as that of 2,6-di-0-methyl-D-glucitol acetate.  After demethylation and reacetylation the presence of  g l u c i t o l was  strongly indicated but not confirmed because of the  very small amount a v a i l a b l e . Peak #7.-  This peak showed the mass spectrum of a 2,3-di-O-  methyl-D-hexitol with major fragments: m/e It co-chromatographed mannitol acetate.  43, 101, 117, and 261.  with a standard sample of  2,3-di-0-methyl-D-  A spot with Rf and Rg corresponding to that of  2,3-di-0-methyl-p-mannose was seen on paper chromatography mannitol hexaacetate was M.p.  119°C.  and  found after demethylation and acetylation.  L i t . value 123°C.  - 51 -  Peak #8.-  The mass spectrum of this peak was very s i m i l a r to  that of peak #7.  Both f i t t e d only the fragmentation pattern  reported for a 2,3-di-O-methyl-D-hexitol although the i n t e n s i t y of peak 101 was rather low and that of 161 was somewhat higher than the values quoted by Lindberg.  The same c h r a c t e r i s t i c s were however  also found when a mass spectrum was made of an authentic sample of 2,3-di-O-methyl-D-mannitol  acetate.  The, differences are presumably  due to changes i n the instrumental parameters used i n obtaining the spectra.  The g . l . c . retention times of the substance was  the same as that of 2,3-di-O-methyl-D-glucitol acetate.  exactly On paper  chromatography a spot s l i g h t l y f a i n t e r than that f o r 2,3-di-()-methylD-mannose was observed with the same retention times as for authentic 2,3-di-0_-methyl-D-glucose. Peak //9.established.  The i d e n t i t y of this peak was not s a t i s f a c t o r i l y  The g . l . c . retention time indicated that i t probably  was-a monomethyl a l d i t o l acetate or a mixture of one or more. mass spectrum was  The  inconclusive as there was at least one s i g n i f i c a n t  fragment for either 2-, 3-, or 6-mono-O-methyl a l d i t o l s which did not appear.  The most probable choice i s a 2-mono-0-methyl derivative as  for this  the only observed discrepancy between the observed  fragmentation pattern and that reported i s the absence of a fragment of m/e  139 (41).  When the material was demethylated and  re-chromatographed  as the a l d i t o l acetate both mannitol and a small amount of g a l a c t i t o l were found.  As i t was thought that the compounds arose from under-  (or de) methylation of the polysaccharide and i t s presence i n very small percentage makes any s t r u c t u r a l significance u n l i k e l y no further  - 52 -  attempt was made to determine i t s i d e n t i t y . S t r u c t u r a l Significance of the data The amounts of i n d i v i d u a l methylated sugars present and their s i g n i f i c a n c e may now be discussed. presented i n Table VIII.  The r e s u l t s obtained are  The peak referred to as 2' has been  i d e n t i f i e d by mass spectrometry etc. as 2,3-di-O-methyl-D-xylitol. It arises from a small amount of contaminant  xylan i n the o r i g i n a l  polysaccharide (16,55,56). When the data were compiled i t was observed that, where comparisons were possible, polysaccharides that had been s o l u b i l i z e d i n 72% s u l f u r i c acid had a higher r a t i o of tetramethyl sugars than those that had been reacted with formic acid.  At the same time  those polysaccharides hydrolyzed i n 72% s u l f u r i c acid also had r e l a t i v e l y higher amounts of dimethyl sugars.  This can be c l e a r l y  seen by r e f e r r i n g to the values recorded f o r SSGM^,. (308) and SSGM^,. (371) the l a t t e r  had  a higher methoxyl value and yet contributed  almost twice as much dimethyl hexose.  I t i s only l o g i c a l to explain  these r e s u l t s by assuming that a small amount of tetramethyl sugar may be l o s t during the r e l a t i v e l y long evaporation time required to remove the formic acid.  S i m i l a r l y i t appears that a s i g n i f i c a n t  increase i n demethylation takes place when 72% s u l f u r i c acid i s used. As no tetramethyl sugars can l o g i c a l l y be present that were not an i n t e g r a l part of the o r i g i n a l polysaccharide the maximum values obtained for them were assumed to be most v a l i d .  S i m i l a r l y no dimethyl sugar  should be l o s t i n work up and minimum values (excess a r i s i n g from demethylation) should be the most r e l i a b l e .  With this i n mind the  - 53 amounts of tetramethyl sugars reported f o r SSGM^Q (which was done only by the formic acid method) are probably a l i t t l e below the actual values. On the whole the amount of s t r u c t u r a l information which may be derived from the r e s u l t s i s disappointingly small.  The.problem  revolves about the combined e f f e c t s of very small amounts of dimethyl sugars  (0.5-1.5%) which may or may not be s i g n i f i c a n t , the problems  of p u r i t y , and of complete methylation. methylation may be dealt with f i r s t .  The problem of under-  Our values of methoxyl content  are w i t h i n .7-1.4% of the t h e o r e t i c a l and were not s i g n i f i c a n t l y raised upon remethylation.  Unfortunately even a three f o l d improvement  to about 0.25% (which i s close to  the l i m i t of r e p r o d u c i b i l i t y of  methoxyl analysis) would s t i l l permit that as much as .55% of a l l possible hydroxyl functions might remain unmethylated (35).  This i s  equivalent to having one and one-half moles of dimethyl sugars f o r every 100 moles of the expected  trimethyl sugar.  C l e a r l y , under such  a l i m i t a t i o n the presence of 0.5% of a p a r t i c u l a r dimethyl sugar cannot be used as a basis f o r s t r u c t u r a l  determination.  The question of p u r i t y of polysaccharides introduces s i m i l a r problems.  I t i s c e r t a i n from the detection of small amounts of  2,3-di-0-methyl-D-xylitol i n some f r a c t i o n s of the methylated glucomannans that a trace of xylan remains i n the polysaccharide after many p u r i f i c a t i o n steps.  The presence of xylan i s not i n  i t s e l f p r e j u d i c i a l to the analysis but i t does i n d i c a t e that one must expect  the presence of some GGM which i n s o l u b i l i t y c h a r a c t e r i s t i c s  i s much more akin to GM than i s xylan.  Having admitted  this p o s s i b i l i t y  one can no longer say with certainty that the tetramethyl g a l a c t i t o l or  galactose found i n the GM i s a c t u a l l y a part of i t or i s derived from a small amount of admixed GGM.  In fact i t i s shown quite c l e a r l y  that the glucomannan used for methylation analysis i s not  completely  homogeneous, even i f the small amount of admixed xylan i s ignored. There i s a marked difference i n the r a t i o s of mannose/glucose determined for  the 15% and 20% chloroform/petroleum  polysaccharides.  ether extracts of methylated  This difference 4.45:1 f o r SSGM^ as compared to  3.10:1 for SSGM2Q i s much greater than any species d i f f e r e n t i a t i o n observed (Table IX).  It i s possible that no true GM or GGM  be defined for these softwood species and that instead a  can  continuous  series of polysaccharides of gradually increasing degree of branching and galactose content may  present a more accurate p i c t u r e .  My  own  opinion, however, i s that the s o l u b i l i t y and p r e c i p i t a t i o n characteri s t i c s of the GM's and GGM's are s u f f i c i e n t l y d i f f e r e n t to i n d i c a t e two d i s t i n c t classes of polysaccharides and that'the GM's  do obtain a  small percentage of galactose and have a low degree of branching 57,58).  (2,  This i s not proven,however but could be by rigorously }  establishing the p u r i t y of the i n i t i a l polysaccharide. made above apply equally to the GM's b r i e f summary may as i n Table IX.  The remarks  of black and Sitka spruces.  A  be made for the s p e c i f i c r e s u l t s obtained f o r each A l l polysaccharides produce 1.5-3,5% 2,3-di-0^-methyl-  D-mannose which indicates that most branching p o s i t i o n of the mannose residue.  occurs through the s i x  A small amount of branching  probably  occurs through the s i x p o s i t i o n of glucose residues as 2,3-di-0methyl-D-glucose i s not l i k e l y to occur as an a r t i f a c t of undermethylation since the s i x p o s i t i o n i s r e l a t i v e l y e a s i l y methylated.  - 55 -  The small amounts of 2,6-di-0-methyl sugars obtained are probably derived from undermethylation. chloroform/petroleum  For the samples extracted i n 15%  ether the amounts of tetramethyl and 2,3-  di-()-methyl sugars suggest a chain length of about f o r t y for BSGM and f i f t y f o r SSGM.  I t i s l i k e l y that some degradation of the  o r i g i n a l polymer has occurred during the methylation (1,62).  or extraction  The r a t i o s of the t o t a l methylated mannose to methylated  glucose 4.45:1.00 for SSGM compares reasonably well to the r a t i o s of about 4.8:1.00 f o r the unmethylated  polysaccharide as does the  amount of 2,3,4,6-tetra-O-methyl-D-galactitol (approximately 1%). The equivalent r e s u l t s for BSGM are mannose/glucose 4.4:1, methylated mannoses/methylated glucoses 4.5:1 and 1.3%  2,3,4,6-tetra-0-methyl-  D-galactose. In conclusion i t may be said that the method of g . l . c . of the a l d i t o l acetates and combined mass spectrometry provides a r e l a t i v e l y rapid and accurate means of analysing the methylated sugars derived from polysaccharides.  I t can be applied to very small quantities  once the i d e n t i t y of components to be expected has been demonstrated. Thus, i n i t i a l analysis could be done on a large, r e l a t i v e l y impure sample and the structure refined by a study on a small amount rigorously p u r i f i e d , perhaps by electrophoretic techniques (28,29).  In the  present case, extremely high degrees of p u r i t y and completeness of methylation would have to be obtained to e s t a b l i s h conclusively what are at most extremely minor s t r u c t u r a l features i n the glucomannans. I f , f o r example, one wishes to prove absolutely the existence of one branch point per 100 sugar residues, the l e v e l of possible  - 56 -  impurity ( i f of a nature which could contribute a species leading to misinterpretation) must be reduced to 0.5% or less and the methoxyl value should be within 0.05% of the t h e o r e t i c a l .  At present the  l a t t e r c r i t e r i o n , even i n view of the improved methylation techniqes cited e a r l i e r does not appear to be a p r a c t i c a l  possibility.  - 57 -  BIBLIOGRAPHY  1.  T . E . T i m e l l , i n "Advances i n C a r b o h y d r a t e C h e m i s t r y " , V o l . 1 9 , p . 2 4 7 , Academic P r e s s , New Y o r k , 1 9 6 4 .  2.  T . E , T i m e l l , i n "Advances i n C a r b o h y d r a t e C h e m i s t r y " , V o l . 2 0 , p . 4 0 9 , Academic P r e s s , New Y o r k , 1 9 6 5 .  3.  G.O. A s p i n a l l , " P o l y s a c c h a r i d e s " ,  4.  G . A . Adams, Can. J .  5.  G . G . S . D u t t o n and J . P .  6.  W.N.  Haworth, J . ' Chem. S o c .  7.  A.R.  Mills  8.  T . E . T i m e l l and A. T y m i n s k i , T a p p i 4 0 , 5 1 9 ( 1 9 5 7 ) .  9.  T . E . T i m e l l and A . T y m i n s k i , J .  Pergamon, 1 9 7 0 .  Chem. 3>6, 7 5 5 ( 1 9 5 8 ) . Joseleau,  unpublished r e s u l t s .  107, 8  and T . E . T i m e l l , Can. J .  (1915). 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