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Synthesis of partially methylated tetroses Pierre, Kenneth Jonas 1962

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SYNTHESIS OF PARTIALLY METHYLATED TETROSES by KENNETH JONAS PIERRE B.Sc,  U n i v e r s i t y o f B r i t i s h Columbia, i960  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department of CHEMISTRY  We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e required standard  THE UNIVERSITY OF BRITISH COLUMBIA May,  1962  In presenting  t h i s thesis i n p a r t i a l f u l f i l m e n t of  the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available f o r reference  and study.  I further agree that permission  for extensive copying of t h i s thesis f o r scholarly purposes may granted by the Head of my Department or by his  be  representatives.  It i s understood that copying or publication of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission.  Department of The University of B r i t i s h Columbia, Vancouver 8, Canada. Date  ii  ABSTRACT  i.  I t i s well established that various p a r t i a l l y methylated  tetroses and pentoses occur i n periodate oxidation products  of  methylated polysaccharides. To provide reference compounds as a means of i d e n t i f y i n g some of these sugars 2,3 2,3  di-O-methyi-L-threose,  di-O-methyi-D-erythrose, and 3>U di-O-methyl-L-xylose have been  synthesized. The treose and xylose were synthesized from D-glucoside by benzylidene condensation,  cx>methyi-  methyiation, hydrolysis,  reduction and periodate oxidation, whereas the erythrose was  obtained  from D-mannose by a similar sequence of reactions. The sugars were obtained as syrups and were characterized by the preparation of crystalline derivatives. ii.  Black Spruce hemiceiluioses:By using d i f f e r e n t concentrations of a l k a l i three d i f f e r e n t  hemiceiluioses were extracted from biack spruce h o i o c e i i u i o s e . Of these two fractions, the xyian and the glucomannqn were p u r i f i e d by f r a c t i o n a l p r e c i p i t a t i o n using barium hydroxide  s o l u t i o n and  Fehling's s o l u t i o n . Previous workers have bbserved that the xyian f r a c t i o n was always contaminated by small amounts of galactose. This was also true i n the present instance i f barium hydroxide used f o r p u r i f i c a t i o n , but when t h i s was  alone  was  fojJLowed by two treatments  with Fehling's s o l u t i o n the galactose was completely eliminated. This i s believed to be the f i r s t case of the i s o l a t i o n of such a xyian i n a pure state from the hemiceiluioses of a coniferous wood.  iii  The t h i r d f r a c t i o n - the gaiactogiucomannan was found to be quite d i f f i c u l t t o re-dissolve i n a l k a l i and as a r e s u l t i t s p u r i f i c a t i o n i s not quite complete.  viii  ACKNCWLEDGEMENTS  I w i s h t o e x p r e s s my s i n c e r e s t t h a n k s t o Dr. G.G.S. D u t t o n f o r h i s i n v a l u a b l e guidance and encouragement throughout  t h e c o u r s e o f t h i s work..  I a l s o l i k e t o thank D r . A.M. Unrau f o r i n v a l u a b l e s u g g e s t i o n s a n d f o r t h e use o f h i s s t a n d a r d sugar  curves,  a n d Dr. J.C.J?. Schwarz--.^ f o r an o u t l i n e o f t h e b e n z y l i d e n e condensation.  iv  TABES OF CONTENTS  PART 1 Page HISTORICAL INTRODUCTION  .  .  .  .  .  .  1  .  OUTLINE OF PRESENT RESEARCH  h 12  EXPERIMENTAL A.  S y n t h e s i s o f 2,3 Di-0-methyl-3jD-glucoside . .  .  .  U, 6-Benzylidene-«. - m e t h y l - D - g i u c o s i d e  .  .  ,  .  .  M e t h y l a t i o n o f U , 6 - b e n z y l i d e n e - <*-methyl-Dglucoside . . . . . .  1$ V~>  .  1$  Hydrolysis o f 2,3-di-0-methyI-U,6benzyiidene-<*-methyl-D-glucoside  .  .  .  .  Bromine o x i d a t i o n o f 2,3 d i - O - m e t h y l D-glucose . . . . . . P h e n y i h y d r a z i d e o f 2,3 d i - O - m e t h y l gluconic acid . . . . . R e d u c t i o n o f 2,3 di-O-methyl-D-glucose  .  D-glucitol  .  .  .  .  .  .  .  D-mannoside  . . .  18  18  . .  .  22  di-O-methyi.  .  .  .  .  .  . .  22  . .  .  U , 6 - b e n z y l i d e n e - ot-methyi-D-mannoside M e t h y l a t i o n o f U,6  17  .  B. - S y n t h e s i s o f 2,3-Di-O-methyi-D-erythrose Methyl-ot-D-mannopyranoside  17  di-O-methyi-  l , l i D i - p - n i t r o b e n z o a t e o f 2,3 .  . .  .  2,3 D i - O - m e t h y i - L - t h r e o n o p h e n y l h y d r a z i d e  L-threitoI  17  .  l , U , ! ? , 6 - T e t r a - p - n i t r o b e n z o a t e o f 2,3 d i O-methyl-D-glucitoi . . . . P e r i o d a t e o x i d a t i o n o f 2,3  16  .  . .  .  .  23 23 23  benzylidene-o<-methyl.  .  .  .  .  .  .  2U  V  Page H y d r o l y s i s o f 2,3 d i - U - m e t h y l , U,6h e n z y l i d e n e - o i -methyl-B-mannoside . 2,3-Di-O-methyi mannonophenyihydrazide  . .  Reduction  .  o f 2,3 di-U-methyi-D-mannose  .  . .  .  .  I,h,5,6-Tetra-£-nitrobenzoate o f 2,3 d i - O methyi-D-mannitoI . . . . . P e r i o d a t e o x i d a t i o n o f 2,3 di-O-methyi-Dmannitol . . . .  C.  .' .  .  .  .  2h 2$ 25.  .  .  2:6  .  26  R e d u c t i o n o f 2,3 d i - O - m e t h y i - D - e r y t h r o s e a n d p r e p a r a t i o n o f ±,U-p-nitrobenzoate o f 2,3 d i - O methyi-D-erythritol . . . . . .  .  .  29  2,3 d i - O - m e t h y l e r y t h r o n o p h e n y x h y d r a z i d e .  .  .  29  .  .  31  .  C o n t r o l l e d p e r i o d a t e o x i d a t i o n o f 2,3 d i - O m e t h y l - D - g l u c i t o I a n d s y n t h e s i s o f 3,U-di-0 methyi-L-xylose . . . . . 3,U-Di-0-methyl-L-xyIonoiactone  .  .  .  .  .  3 , U - D i - 0 - m e t h y i - L - x y i i t o i I,2,5-tri-£-nitrobenzoate  .  32  .  33  PART 2 HISTORICAL INTRODUCTION B l a c k Spruce H e m i c e i l u i o s e s EXPERIMENTAL  . .  3h .  .  .  .  .  .  38  .  Delignification  hi o f B l a c k Spruce Wood  .  .  .  .  L i g n i n d e t e r m i n a t i o n o f B l a c k Spruce h o l o - c e l l u l o s e Extraction of Hemiceiluioses .  .  .  I s o l a t i o n of arabino-glucurono-xyian P u r i f i c a t i o n of Glucomannan  .  .  P u r i f i c a t i o n of the Xylan  .  .  Cetyxtriammonium s a l t s o f a c i d i c x y i a n  .  .  .  . .  .  . .  .  .  . .  .  . .  Ul  . .  .  Ul  .  .  1x2  .  .  1x2  .  .  U3  . .  .  UU  .  UU  vi  Page A c e t y l a t i o n of the Xylan.  .  .  .  .  .  P u r i f i c a t i o n o f X y i a n (BSx^) b y Copper Complex.  .  .  .  hh h$  P u r i f i c a t i o n o f Galactogiucomannan  .  .  .  .  .  U5  Q u a n t i t a t i v e A n a l y s i s o f Sugars  .  .  .  .  .  U6  •Degreero"f ^ p o l y m e r i z a t ion.vby formaldehyde e s t i m a t i o n , o f r e duced p o l y s a c c h a r i d e . . . . . . . .Periodate o x i d a t i o n o f X y l a n BIBLIUGBAPHT  .  .  .  .  .  .  .  5U $\x  .  £6  vii  TABLES  TABLE I PERIODATE OXIDATION OF 2 , 3 DI-OMC THTL-D-GLUCITOL . . .  .  TABLE I I PERIODATE OXIDATION OF 2 , 3 DI-O-METHYLD-MA.NNITOL . . . . . TABLE I I I OPTICAL DENSITY OF SUGAR SOLUTIONS TABLE 17 SUGAR RATIOS I N HEMICELLULOSE FRACTIONS . . .  .  I  HISTORICAL INTRODUCTION  Within recent years there has been increasing interest i n the determination of the structure of polysaccharides from natural sources. As a result of these investigations i t has become necessary to develop methods of identifying the units of these polymers by some degradative means. With the introduction of dimethyl sulfate and sodium hydroxide as a methylating agent by Denham and Woodhouse i n 1913 (I, 2 ) , and the subsequent hydrolysis of the methylated polysaccharides, the need for the identification of partially methylated sugars became imminent. Many of these partially methylated sugars are incompletely characterized, some are  known only i n an impure state, while others have never been syn-  thesized ( 1 2 ) . The need for partially methylated sugar derivatives as reference compounds has provided carbohydrate chemists with the problem of synthesising many of these rare sugars i n the laboratory. Extensive work has been done on the hexoses and pentoses but l i t t l e i s known about the tetroses, erythrose and threose. The f i r s t attempt at synthesising these sugars was made by Fischer (3) i n 1887. This was followed by the work of Fenton and Jackson (h) and other groups but with very l i t t l e success until Hockett (5) i n 1935 synthesized D-threose by Ruff's degradation. This he characterized by preparation of the crystalline D-threose triacetate. When Maiaprade (6) discovered the cleavage of 1,2 glycols by periodic acid i n 1928, a new and effective tool became available to synthetic and structural chemists. The mild conditions of the oxidation  2  and the high degree of selectivity of the reagent make i t especially suited for degradation of the sensitive carbohydrate structure. Consequently, cleavage of I, 2 glycols by periodic acid has been extensively u t i l i z e d both for structural studies of simple and complex carbohydrates (7 - i 0 ) and for synthetic purposes ( i i - Ik). The synthesis of some partially methylated tetroses namely, 2-0-methyI-D-threose (12), 2,3-di-O-methyi-D-threose  (13), 2,U-di-0-  methyl-L-erythrose (Ih), and their crystalline derivatives have been achieved by oxidation of well characterized partially methylated sugars or their derivatives by periodic acid. This research was undertaken to synthesise two dimethyl tetroses, namely, 2,3-di-O-methyi-D-erythrose  and 2,3-di-0-methyl-L-  threose and to identify them by preparation of crystalline derivatives. In addition to the above tetroses the synthesis of 3-U-di-Omethyl-L-xylose was achieved by controlled periodate oxidation.  2,3 D i - O - m e t h y l - L - t h r e o s e  ?,3 D i - C - m e t h y l - D - e i y t h r o s e  H  3,U Di-O-methyl-L-xylose  h  OUTLINE OF ^RESENT RESEARCH  In order to synthesize 2,3 di-O-methyl D-erythrose, Dmannose was chosen as the starting material, whereas <*- methyi-Dglucoside, being readily available, was chosen for the 2,3-di-Omethyl-L-threose. The main problem of this synthesis was to selectively block the k and 6 positions of the methyl glycoside leaving the 2 and 3 positions available for methyiation. The path that immediately suggested i t s e l f for blocking the iiidicated positions was to perform a benzyiidene condensation using benzaldehyde. This reagent reacts with the U and 6 positions of methyl glycosides. The oc-methyi-D-mannoside was prepared by the method of Smith and Van Cleve (1$), which i s a modification of the Fischer (i6) method. The crystalline D-mannose was dissolved i n ethylene dichloride containing methanoiic hydrogen chloride, and the mixture refluxed on the steam bath for four hours until a crystalline mass of the methyl glycoside separated from the lower layer. In this method an inert solvent such as ethylene dichloride (l7)  i s added  to cause the methyl glycosides to crystallize directly from the reaction mixture. This avoids neutralization and concentration according to the customary procedure  (16).  The benzyiidene condensation has been extensively used for the preparation of h,6 benzyiidene  -methyi-D-giucoside (VII) (18,  19) using fused zinc chloride as catalyst. In the case of the<*-methyl D-mannoside however, there i s the tendency to form the 2,3j U,6, dibenzyiidene derivative (V(20) under these conditions due to the  5  presence of c i s hydroxyl groups at C ^ )  and C^)«  The reaction  step i s indicated i n the diagram on the following page. This d i f f i c u l t y was overcame however, using a procedure provided by Dr. Schwarz (21). The f i n e l y powdered  cx-methyi-D-  mannoside was dissolved as r a p i d l y as possible i n 98 - 100$ formic a c i d and f r e s h l y d i s t i l l e d benzaldehyde added to the solution. A f t e r f i v e minutes the mixture was poured with s t i r r i n g into l i g h t petroleum ether and water containing potassium carbonate. The benzylidene  U,6  -methyi-D-mannoside (VIII) was obtained from the upper  l a y e r by f i l t r a t i o n . Attempts to prepare the  u -methyl k,6  ethyiidene-D-  mannoside by the method of Honeyman and Morgan gave only the diethylidene compound. This course was not pursued further. With the k and 6 positions blocked, the methylation of the hydroxyl groups at the 2 and 3 positions was  carried out once by  Haworth's methylation technique (22) and three times using the Purdie reagents (23). I t might be noted that attempts at methylating subsequent samples by the Kuhn method (2JU) gave a f u l l y methylated product a f t e r one methylation. A f t e r hydrolysis of the methylated benzylidene derivative, the product, 2,3  di-O-methyi mannose was chromatographically impure,  showing two spots on the paperchromatogram. One of these spots corresponded to 2,3  di-O-methyl mannose and the other to a very f a i n t  trace of a slower running component. The sample was p u r i f i e d by passing through a column of cellulose-hydroceilulose (2J?) using butanone-water azeotrope (26) as developer. The slower running .ponent was not investigated further.  com-  4  Li,6 nenaylidene- «* -methyl D-mannoside  7  The 2,3  di-0-methyI-D-mannose and 2,3  di-O-methyl-D-  glucose were both subjected to sodium borohydride reduction according to the procedure outlined by Smith and h i s co-workers (27). In general the reduction of aldoses by t h i s reagent i s u s u a l l y complete within a few hours. I t was found however, that the reduction of the two above mentioned compounds was not complete within the time expected. Further investigations revealed that according to the work of Pragg and Hough (28) aldoses with a 3-0-substituent undergo reduction much more slowly than other aldoses. They proposed that the reduction of an aldose (e.g. IX) to the h e x i t o l (XI) w i l l be preceded by ring opening of the c y c l i c modification (IX) of the aldose to the aldehydo-form i n the a c y c l i c staggered zig-zag conformation (X). These results are consistent with the view that a bulky subs t i t u e n t at C ^ )  °f t h i s conformation (X) cause the approach of a  borohydride ion to be s t e r i c a l l y hindered. I t was therefore necessary to allow the reduction to go f o r quite long periods. A f t e r reduction the borate complexes were destroyed by treatment of the crude alcohols with methanolic hydrogen chloride followed by passage through cation and anion exchange resins. The  2,3-di-O-raethyl-D-mannitol  c r y s t a l l i z e d on standing but the 2,3-di-O-methyl-D-glucitol could not be induced to c r y s t a l l i z e . The above 2,3-di-O-methyl h e x i t o l s were oxidised using p e r i o d i c acid. The oxidation of these compounds y i e l d s one mole of 2,3-di-0-methyl tetrose, one mole of formic a c i d and one mole of f o r maldehyde. P i l o t scale oxidations were done on the respective h e x i t o l s to f i n d the necessary experimental conditions before the large scale oxidationTwas  attempted.  fx 5  ure  aJ.  9  It was observed that the tetroses were quite volatile and consequently had to be extracted from the aqueous solution by continuous chloroform extraction. The tetroses appeared as clear syrups which could not be induced to crystallize. To identify and characterize these compounds, portions were oxidised with bromine and the acids converted to the crystalline phenylhydrazides which gave satisfactory methoxy and nitrogen analysis. Similar portions of the tetroses were reduced with sodium borohydride and the tetritols converted to the crystalline j>-nitrobenzoates. Following the procedure outlined by Smith (13)  an attempt  was made to perform a controlled periodate oxidation on 2,3  dimethyl  glucitol. The purpose of this study was to determine whether the periodate ion preferentially attacked the 5,6  glycol or the  U,5  glycol. It i s generally accepted that a carbon chain takes up a zig-zag conformation with large substituents i n staggered positions  (12,33, 3k)  • Working with hexitols Schwarz  (35)  has shown that pre-  ferential attack by periodate occurs at diol systems which are represented as trans in the Fischer projection formulae. In effect periodate normally oxidises preferentially a c i s - ot -glycol. The apparent anomaly i s explicable by reference to the diagram  (36).  In view of these arguments, and the views put forward by Buist, Bunton and their co-workers (37)  that periodate cleavage of  diols proceeds through a five membered cyclic intermediate i t was expected that in the 2,3  di-O-mehhyl-D-glucitol preferential attack  would take place at C^yhydroxyl on  to that of  due to the closer proximity of the as compared to that on  Fx j u r e .  H-C-OH  £ I u. c i  to \  H-  Ii  2,3 Di-O-methyl-D-glucitol was consequently subjected to a series of c o n t r o l l e d periodate oxidations using periodate and h e x i t o l i n r a t i o s of 1:1 and 132 at 5"°C. In a l l instances the uptake was quantitative. On completion of the reaction two components with R^ of 0.5h and 0.78  respectively i n solvent A appeared on the chroma-  togram. In the case of 1:1 r a t i o the slower running component was predominant. This experiment indicates that although the attack i s not highly selective i t i s possible to p r e f e r e n t i a l l y oxidise glycols o f certain conformations with periodate. In t h i s way, using a 1:1  ratio of periodate to h e x i t o l , i t  was possible to synthesize 3,U di-O-methyl-L-xyiose which i s the slower running component r e f e r r e d to above. On completion of the consumption of one mole of periodate the reaction mixture was treated with barium carbonate. A f t e r f i l t r a t i o n the aqueous solution was  ex-  tracted with chloroform and the resulting syrup separated on the cellulose-hydrocellulose column to give a c l e a r syrup of 3,h di-0methyl-L-xylo se. In t h i s work, by the selective methylation of certain hydroxyl groups, and by the use of periodic a c i d as an oxidising agent three p a r t i a l l y methylated sugars 2,3 2,3  di-O-methyl-D-erythrose,  di-O-methyl-L-threose and 3)h di-O-methyl-L-xylose have been  synthesised. In addition the preparation of c r y s t a l l i n e 2,3 methyl-D-mannitol was also achieved.  di-O-  ±2  EXPERIMENTAL  Evaporations A l l evaporations were carried out under reduced pressure at a bath temperature not exceeding UO°C. Chromato graphy A l l paper partition chromatography wa's done on Whatman No. 1 chromatography paper by means of the descending method. A l l chromatograms were a i r dried after development and after spraying they were oven dried at HO°C. The solvent systemsused were: A. Butanone-water azeotrope. B. 1-Butanoi-pyridine-water (6:U:3) C. Ethyl acetate : pyridine : water (8:2:1) The spraying reagents used were p-anisidine trichloroacetate (26) and aniline phosphate (29) to detect reducing sugars and ammoniacal silver nitrate to detect non-reducing sugar alcohols. Column chromatography was done on a cellulose-hydrocelxulose ( i : l ) column (2.8x^2 cm), which was kept at a constant temperature (UO°C) by means of a thermostat. Fractions were collected on an automatic fraction collector. Melting Points A l l melting points were taken on a Leitz electrically heated melting point block and are uncojinected. Chemical Analysis. A l l chemical analyses were done by Mrs A. Aidridge of this department.  .13  31}  W  \  Vi''.  H  0  C  H  a  XV  OCH. 3  QCH. •  IT  XiX  D-mannose  Xlil  2,3 Di-O-methyi u,6-benzyiidene-o( -methyl mannoside  I1V  2,3 Di-U-methyl-D-mannose  XV  2,3 Di-C-methyi-D-mannitoi  A.  SYNTHESIS OF 2,3 DI-O-METHYL-L-THREOSE. 1.  Preparation of I+,6 benzyiidene oc -methyl-D-giucoside. Thirty six (36) grams of  -methyl-giucoside was ground with  finely powdered fused zinc chloride (29g), freshly d i s t i l l e d benzaldehyde (105g) added and the mixture shaken for six hours at room temperature. After allowing the solution to stand for twelve hours at 0°C, i t was poured i n a fine stream into ice water (500mi). The benzyiidene compound separated as a white powder which was f i l t e r e d and washed with ice water. The precipitate was shaken with 10% sodium bisulfite solution (five 100 ml portions) to free i t of excess benzaldehyde, the wash liquors being decanted through a Buchner funnel. The benzyiidene:compound i n the funnel was washed with a saturated solution of sodium bicarbonate (300 ml) and ice water (U00 ml) and f i n a l l y with petroleum ether to remove last traces of benzaldehyde. The compound 20 was recrystallized from water. Yield 26 g. m.p. l62-i63°C, £o(fj ^109.2 (C, 1.25  i n CHGIg). Literature values; m.p i63-i6h°C, and £d J + H 1 . 9 ° D  (18). II.  Methylation of U, 6 benzyiidene Crude h,6 benzyiidene  ct -methyl giucoside  oc-methyl giucoside (12.5g) was dis-  solved i n acetone (75ml) at ij.0°C and placed into a three-necked flask equipped with a mechanical stirrer. Dimethyi-sulfate (25ml) and 30% sodium hydroxide (75ml) were added i n ten portions at ten minutes intervals with stirring. After an additional hour's stirring, hot water (lOOml) was added and the mixture heated to 100°C to destroy the excess dimethyl sulfate. The mixture was cooled and the partially methylated product filtered. After drying thoroughly the sample was dissolved i n  16  acetone (25ml) and. methyl iodide (20ml) added. Silver oxide (20gm total) was added over a. period of 2h hours. The methylation was allowed to continue for seventy-two hours after which the excess methyl iodide was recovered by d i s t i l l a t i o n and the insoluble residue was repeatedly extracted with boiling acetone. Evaporation of the extracts gave soft white crystals which gave an indication of hydroxy! groups on the infrared spectrum. The second Furdie methylation was done using methyi iodide as solvent for a similar period of time. An infrared spectrum of a dried sample indicated that there was no free hydroxyl group. Yield 8 . 3 gms m.p. 122-123°C,[«^ + 96.U2°(c, 1.63k in (CH ^CO) 20  Literature values 122-I23°C, H Methoxy content caic 3 0 . 0 0 $ III.  2 0 + +  j  9 97 7..003-° °( C , I.6J4O inCGH^CCj  fd. 2 9 . 8 9 $  Hydrolysis of 2 , 3 di-O-methyl It,6, benzyiidende- Q< -methyl glucoside. The sample ( 8 . 3 g ) was dissolved in IN sulfuric acid ( 1 0 0 m l )  20 * 2 . 9 1 , and the solution heated on the steam bath for a period of twelve hours until the change in optical rotation became constant  ,  2 0  at <*  +  D  1 , 2 5 . The solution was cooled, neutralized with Barium .  carbonate to congo red, centrifuged, and the BaCO^ washed with water until i t gave a negative Moiisch test. The centrifugate and washings were combined and evaporated to:'dryness under reduced pressure. The residue was extracted with boiling chloroform and methanol to y i e l d a syrup of 2 , 3 di-O-methyi-D-glucose,^ Literature v a l u e [ « ^  20  + 81.9-* + U 8 . 3  + 57.£  (C, 2 . 8 I 4 in HgO),  (C, 2 . 8 0 in ^ 0 )  (19).  17  IV.  . Bromine oxidation of 2,3  di-Q-methyl-D-giucose  Seventy (70) mg of 2,3 di-O-methyl-D-glucose was dissolved i n water (10 mi) and bromine added. The oxidation was allowed to continue i n the dark for forty-eight hours at room temperature after which the solution was aerated to free i t of excess bromine. Lead carbonate was added to the solution to precipitate out the bromide ions. The solution was filtered and hydrogen sulfide bubbled through to free i t of lead ions. The solution was centrifuged and the centrifugate treated with activated charcoal to free i t of colloidal sulfur and then evaporated to give a brown syrup of 2,3 di-O-methyl-D-gluconic acid (60mg). The syrup could not be induced to crystallize. V.  Phenyjhydrazide of 2,3 di-O-methyl gluconic acid. UU • i  i•  •  i  i  i  —" •  •  The 2,3 dimethyl gluconic acid (60 mg) was dissolved i n anhydrous ether by .refluxing for three hours. A fine, white precipitate formed at once on addition of freshly d i s t i l l e d phenylhydrazine (ij^mg) and after seven hours refluxing i t was removed by f i l t r a t i o n . Additional material was recovered from the f i l t r a t e by further treatment with phenylhydrazine and concentration of the reaction mixture. The yield was 6lmg (crude). Recrystaliization from ethanoi-ether gave fine short white needles with m.p. I69-I70°C. Literature value l66-l67°C  (l8).  Nitrogen content: Theoretical 8.9$ VI.  Reduction of 2,3  Found. 9.05$.  di-O-methyl-D-glucose.  2,3 Dimethyl glucose (2.3g) was dissolved i n water and sodium borohydride (0.8g)added. The change i n optical rotation was followed to a constant value during a period of twenty-four hours. The reaction  18  was stopped by the addition of acetic acid and the mixture evaporated to dryness i n vacuo. The residue was treated several times with methanolic hydrogen chloride {%) to free i t of borate. The sample was then dissolved i n water and deionized by passing through columns of Amberlite  IR-I20 and Duolite A-lx resins respectively. The aqueous  extract was evaporated to give a clear syrup of 2,3 di-O-methyl glucitol (2.05g).  jVJ^ +11.89° (C=2.U6 i n H o . Literature value 2  [oc]^13.0°f 0.5°(C, 2.$k i n H 0) 2  VII.  (30).  Rf 0.57 i n solvent system B.  l,U,5,6,-Tetra-p-nitrobenzoate of 2,3,di-0-methyi-D-glucitoi. 2,3 Di-O-methyl glucitol (50mg) was dissolved i n anhydrous  pyridine and p-nitrobenzoyl chloride (200mg) added. The reaction mixture was heated on the steam bath for one hour after which a few drops of water were added. After allowing to stand for fifteen minutes the mixture was poured into a saturated solution of sodium bicarbonate (20ml) with stirring. The reaction product,!,Ii,5,6-tetra-p-nitrobenzoate of 2,3. d i - 0 methyl glucitol separated as a gum. The supernatant was decanted and the gum crystallized from methanol. The y i e l d was lk7 mg. m.p. 92-9U°C. Nitrogen content Calc. 6.9556 VIII.  found 7.00$.  Periodate oxidation of 2,3 di-Q-methyl-D-giucitol. 2,3-Di-O-methyI glucitol (Iil8mgj2 m moles) was dissolved i n  d i s t i l l e d water and 0.2M periodic acid (25mij 5m.moles) was added and the volume immediately adjusted to f i f t y mis. with d i s t i l l e d water. The solution was kept i n the dark at room temperature. Aiiquots (1 ml) were withdrawn at intervals (Tabiel) and treated with excess sodium bicarbonate and a measured excess of 0.1N sodium arsenite solution.  19  The solution was allowed to stand for fifteen minutes to complete reduction of the excess periodate. The consumption of periodate was determined by back titration with a standard iodine solution. This procedure was repeated until the periodate uptake became constant. The reaction was then stopped by the addition of barium carbonate to precipitate the periodate and iodate ions. The solution was f i l t e r e d and the 2,3 di-0- methyi-O-threose (210mg) isolated by continuous chloroform extraction over a period of four days as a clear syrup. R  = 0.79 i n solvent A, \oc]*°+  n . 9 ° (C, 2.1 i n CHC1 ).  20  T A B L E  I  PERIODATE OXIDATION OF 2,3-DI-O-METHYL-D:  Time minutes  .  GLUCTTOL  Volume of I ( m l )  — ~ — :  Net Volume of I ( m i )  2  2  ;  I 0  U  Uptake/mole  0  7.79  -  -  15  10.2J4  2.U5  1.520  55  10.36  2.57  1.607  75  11.00  3.21  2.002  90  11.00  3.21  2.002  Concentration of I  rt  solution was 0 . 0 2 U 9 Molar.  2X  30 PERIoDfi-Ts X  f  3-  ox.itATiON mttkyl  -J>-  OF  jludtol  20  3 I 5  o 10  MO  20 TirAC  i>0 //v  |0  /20  22  2,3 Di-O-methyi threonophenylhydrazide.  IX  2,3 Di-O-methyl-L-threose ($Omg) was oxidised with bromine and the product isolated as described for 2,3 di-O-methyl-D-glucose. The 2,3 di-O-methyl threonic.acid (39mg) appeared as a syrup. The acid was dissolved i n anhydrous ether by refluxing on a steam bath. To this solution freshly d i s t i l l e d phenylbydrazine (80mg) was added and the refluxing continued for a further three hours. The solvent was evaporated leaving a brown syrup which was crystallized from ethanolbenezene to give fine, white crystals (26.8mg), m.p. 190-192°C, Calc for C  12  X  R"  l6  0^ N , 2  N 11.02$, OCH^ 2U.09* Found N 11.09$ OCH^ 2U.U$ .  1,U Di-p-nitrobenzoate of 2,3 di-O-methyl-L-threitol. 2,3 Di-O-methyl-L-threose (80mg) was dissolved i n water and  treated with sodium borohydride for twenty-four hours at room temperature. The reaction mixture was worked up i n a similar manner to that outlined for 2,3 di-O-methyl-D-giucitol to y i e l d 2,3 di-O-methyl-Lthreitol (65mg) as a clear syrup which failed to crystallize. The 2,3 di-O-methyl-L-threitol was dissolved i n anhydrous pyridine and p-nitrobenzoyl chloride added. The mixture was heated for thirty minutes on the steam bath. A few drops of water was then added and the mixture ailowed to stand for fifteen minutes after which i t was poured with stirring into a saturated solution of sodium bicarbonate (20ml) to yield a brown gum of the ester which crystallized on treatment with acetone. The sample was dissolved in chloroform and passed through a f l o r i s i l column using chloroform as solvent. The sample was recryst a l l i z e d from acetone-ethanol to give white crystals (2ijmg) 93-9U°C  N calc. 6.2$$j found 7.32$.  m.p.  23  B.  SYNTHESIS OF 2,3-DI-O-METHYL-D-ERYTHROSE. I.  Preparation of methyl- °c -D-mannopyranoside. Crystalline D-mannose (I8g) was added to 3% methanolic  hydrogen chloride (15ml) and ethylene dichloride (30ml). The mixture was refluxed on the steam bath for four hours. During the course of the reaction a two phase liquid system was formed, the lower layer of which turned to a crystalline mass. After cooling, the mixture was f i l t e r e d and washed with a l i t t l e ice-cold methanol followed by ether. The methyl- ot -D-mannoside (7.5g) was re crystallized from 80$ ethanol. m.p. 190-19l°C.[k] J°+75.9 (C, 1.0 i n H 0 ) . Literature value m.p. I9i-192°C 2  W  0 + 7 9 D  '  0  (C  >  1 , 0  ^ \  0 )  U,6 Benzyiidene oc-methyi-D-mannoside.  II.  Finely powdered ot -methyl-D-mannoside (20g) was dissolved as rapidly as possible i n 98-100$ formic acid (iOOmi) and freshly d i s t i l l e d benzaldehyde (lOOml) was immediately added to the solution. After allowing to stand for five- minutes, the mixture was poured with stirring into light petroleum .ether (b.pt 6^-110, 800ml) and water (800ml) containing anhydrous potassium carbonate (275g). Inorganic material separated from the aqueous layer which was discarded. The upper layer was f i l t e r e d and the residue washed with light petroleum. Recrystailization from benzene (hot funnel) gave needles (8.5g), m.p. lii2-lU5°C [ O < " J  2 D  ° +  65.2° (C, 1.8U i n CHCl^). Literature value m.p. HiO-lltf C°  [ot]^ * 6l°(C, 1.8h 21  i n CHGiy (21); also m.p. lh7-lU8°, H^+70.2 0  (C, 0.67 i n CHC1 ) (31).  2!i.  Methylation of k,6 benzylidene- ot -methyl-D-mannoside.  III.  The U>6 benzylidene- ot  -methyi-D-mannoside (£g) was dissolved  i n acetone (U5mi) and placed i n a three necked flask equipped with a mechanical s t i r r e r . To t h i s solution dimethyl sulfate (l^ml) and  30$  sodium hydroxide (hOml) were added i n small portions over an hour. The reaction was allowed to continue f o r a further ninety minutes a f t e r which the excess methyl sulfate was destroyed i n the usual manner. The mixture was cooled and the product extracted with chloroform ( f i v e iOOml portions) to give a brown viscous syrup. The p a r t i a l l y methylated product was further methylated by rurdie's method using methyl iodide (20ml) and s i l v e r oxide (20gm) f o r periods of 32,  U8 and 2U  hours, the product being extracted a f t e r each indicated period with b o i l i n g acetone. A f t e r the l a s t methylation there was no i n d i c a t i o n of the presence of hydroxy! groups i n the i n f r a r e d spectrum. The 2,3  di-O-  methyl U,6 benzylidene- ©c -methyl mannoside (3.28g) could not be i n duced to c r y s t a l l i z e ; [ ] j ° 61.8 ot  2  +  (C,1.22 i n CHCi^jj Literature  20 value  [ot]  D  + 62.7  (C, 1.178  i n CHCi^  (31).  OGH  content,  t h e o r e t i c a l 30.00$ found 30.15$. IY.  Hydrolysis of 2,3 mannoside.  di-O-methyl k,6 benzylidene- QC -methyl  Three grams of the sample was dissolved i n IN HgSO^SOml)  20 1.00 and the mixture heated on the steam bath D f o r f i f t e e n hours u n t i l the o p t i c a l rotation became constant, f i n a l  i n i t i a l rotation  rotation  ot ^° o.295>. Tbe solution was cooled, neutralized with barium D  carbonate to congo red, centrifuged, and evaporated to dryness under reduced pressure. The dry residue was extracted with b o i l i n g chloroform  25  and methanol y i e l d i n g a syrup of  2,3 di-O-methyl mannose (l.2ligm)  which could not be c r y s t a l l i z e d .  = 0.22 i n solvent system A.  22  +  12.6 (C, 1.97 i n EtOH). L i t e r a t u r e value M  D  + 10.6  (C, 1-88 i n EtOH) (31). V.  2,3 Di-O-methyl  mannonophenylhydrazide.  2,3 Di-O-methyl mannose (80mg) was oxidised with bromine at room temperature f o r 72 hours. The bromine was removed by aeration and the solution a f t e r usual treatment yielded a thick syrup which could not be induced to c r y s t a l l i z e . The 2,3 di-O-methyl-mannonic a c i d was dissolved i n ethanol by heating on the steam bath. To t h i s solution freshly d i s t i l l e d phenylhydrazine was added and the mixture allowed to reflux f o r three hours. The solvent was evaporated leaving a brown syrup which was c r y s t a l l i z e d from methanol-ether  m.p,  157°C, L i t . 158°C (38). VI.  Reduction of 2,3 di-O-methyl mannose 2,3 Di-O-methyl mannose ( l . l g ) was dissolved i n water and  (O.Ug) added. The reduction was allowed to continue  sodium borohydride  f o r t h i r t y s i x hours a f t e r which i t was stopped by the addition of a c e t i c a c i d . The mixture was evaporated to dryness and the residue treated several times with methanolic hydrogen chloride to free i t of borate. The residue was dissolved i n water and deionized by passing through columns of Amberlite  IR-120 and Duolite A-k r e s i n respectively.  The aqueous extract was evaporated to give a c l e a r syrup of 2,3 di-0methyi mannitol  (l.03g) which c r y s t a l l i z e d on standing. R e c r y s t a l l i -  zation from M e t h a n o l gave white amorphous c r y s t a l s , m.p. R  f  =  101-103°C  0.57 i n solvent system B. Methoxy content: t h e o r e t i c a l 29.52$  actual  29.96$. [<*]*°± 2° (C, 4.09 i n 1^,0). D  26  VII  l Iy,5 6-Tetra-p-nitrobenzoate of 2,3 mannitol. t  5  S  di-O-methyi-D-  2,3 Di-O-methyl mannitoi (i|2mg) was dissolved i n anhydrous p y r i d i n e and jj-nitrobenzoyi chloride (l60mg) added. The mixture  was  heated on the steam bath f o r one hour a f t e r which a few drops of water were added to hydrolyze the excess £-nitrobenzoyi chloride. The solution was allowed to stand f o r f i f t e e n minutes then i t was poured i n t o a saturated solution of sodium bicarbonate ( 2 0 m l ) .  The  ester separated as a gum which was c r y s t a l l i z e d from acetone m.p. Y i e l d l 0 U . 2 m g . Nitrogen content calc 6.9$$ VIII  Periodate oxidation of 2,3  5>2°Cj  found 6.60%  di-O-methyl-D-mannitoi.  2 , 3 Di-O-methyl mannitoi (]f78mgj 2.32 m.moles) was  dissolved  i n 2$ mis of d i s t i l l e d water and 0.2M periodic a c i d (25mlj 5 ra.moles) added. The solution was kept i n the dark at room temperature. Aliquots (iml) were taken out at i n t e r v a l s and treated with sodium bicarbonate and a measured excess of 0.1N sodium arsenite solution. A f t e r allowing to stand f o r f i f t e e n minutes the periodate uptake was  determined  by back t i t r a t i o n with a standard iodine solution. This procedure  was  repeated u n t i l the periodate uptake became constant. The r e a c t i o n was  stopped by the addition of BaCO^ and the 2,3  di-O-methyl-D-  erythrose extracted from the solution by continuous chloroform extract i o n over a period of f i v e days. system f d . 42.98%.  Yield.  60.98° (C, 2 . 6 4 i n HgO).  306 mg. R  f  0 . 6 7 i n solvent  Methoxy content c a l c . 43-59%  T A B L E  II  PERIODATE OXIDATION OF 2 , 3 DI-O-METHYLD-MA.NNITOL  Time Minutes  Volume of T^mis  o  5.16  Net volume of I m l 2  I0~ Uptake £ e r  m  o  l  15  $.hh  0.28  1.53  h$  5.50  0.3U  1.86  65  5.52  0.36  1.97  120  5.52  O.36  1.97  e  80  29  IX  Reduction of 2,3 di-U-methyx erythrose and preparation of l,U-p-nitrobenzoate of 2,3 di-O-metfayi erythritol.  2,3 M-O-methyi-D-erythrose (x30mg) was treated with sodium borohydride for forty eight hours after which the excess NaBH^ was destroyed by acetic acid and the erythritol treated with methanolic hydrogen ehxoride to destroy the borate compxex. After passing through cation and anion exchange resins respectively the erythritol was extracted from the aqueous solution by chloroform. The 2,3 d i O-methyl-D-erythritol appeared as a syrup vhich could not be induced to crystallize. The sample was dried and dissolved i n anhydrous pyridine. To this solution p_-nitrobenzoyi chloride (200mg) was added and the mixture heated on the steam bath for 30 minutes. The mixture was cooled, a few drops of water added and allowed to stand for fifteen minutes after which i t was decanted into a saturated solution of sodium bicarbonate. The ester precipitated out and was removed by centrifugation. After washing thoroughly with water the ester was reerystaxlized from chloroform-acetone giving xong white needles. M i t l8±-x82°c. N calc 6.25$ fd. 6.12$ OCH^ calc 13.9$.  fd. 18.77$. X  Bromine oxidation of 2,3 di-O-methyl-D-erythrose and preparation of 2,3 di-O-methyl erythronophenylhydrazide. The sample of 2,3 di-methyl-D-eryth rose (220mg) was  oxidised by bromine and the product isolated as previously described. The 2,3 di-O-methyl erythronic acid appeared as a brown syrup. The syrup was dissolved i n ether-ethanoi by refluxing on the steam bath. Freshly d i s t i l l e d phenyhydrazine was added to the solution and the refluxing allowed to continue for a further four hours after which  30  the solvent was evaporated leaving a brown syrup. The syrup was dissolved i n hot ethanol and a few drops of ethyl ether added. The flask was allowed to cool then placed i n the refrigerator overnight during which time short white needles formed (lO.Smg) M.rt. 200-203°C.N calc. 11.02$. fd. 11.09$.  31  CONTROLLED PERIODATE OXIDATION OF 2,3, DI-Q-METHYL-DQLUCITOL AND SYNTHESIS OF 3»k DI-O-METHYL-L-XYLOSE.  C. I.  2,3  Di-O-methyl-D-glucitoI  (8Ljmg) was  dissolved i n cold  water (5°C )and 0.2M p e r i o d i c a c i d (2.5ml) equivalent to 1.2 moles of periodate was  miili-  added. The volume was adjusted to 21 mis and  ~'  the solution kept at 5°C and the periodate uptake determined by the sodium arsenite method. The periodate was consumed q u a n t i t a t i v e l y i n t h i r t y minutes. The solution was t r e a t e d with barium carbonate to prec i p i t a t e the iodate, and a f t e r f i l t r a t i o n the f i l t r a t e was extracted by continuous  chloroform extraction to give a c l e a r syrup. The  syrup  on being chromatographed i n solvent A showed two spots of Rf 0.78  and  0.5U r e s p e c t i v e l y . The oxidation was repeated using the same ratios of periodate and h e x i t o l , and the periodate uptake was 1.13  moles per mole of h e x i -  tol. II.  Oxidation using 1 : 1  molar r a t i o .  The above described oxidation was repeated using a 1:1  molar  r a t i o of periodate and h e x i t o l . The periodate uptake reached a value of 0.96  moles per mole of h e x i t o l i n f i v e minutes and remained at t h i s  value. The product was i s o l a t e d and showed two spots on the chromatogram of Rf  0.5k  and 0.78.  sugar with Rf 0.5k III.  The i n t e n s i t y of the spots indicated that the  was present i n greater quantity.  3,U  Di-O-methyl-L-xylose.  2,3  Di-O-methyl-D-glucitol  with 0.2M p e r i o d i c a c i d (15  (630 mgj  3.0 m.moles) was t r e a t e d  ml; 3.0 m.moles) and the volume adjusted  to 100 mis. The solution was kept i n the dark at 5°0 and the periodate  3%  uptake determined by the a r s e n i t e method using 0.0$ M iodine s o l u t i o n f o r back t i t r a t i o n . The periodate uptake reached a constant value of O.96 moles with no change being observed even on standing overnight. The s o l u t i o n was t r e a t e d w i t h BaCO^ t o p r e c i p i t a t e the i o d a t e i o n s , and a f t e r f i l t r a t i o n the aqueous s o l u t i o n was e x t r a c t e d w i t h chloroform t o give a c l e a r syrup. The syrup showed two spots on the chromatogram as p r e v i o u s l y described. The mixture was separated on the c e l l u l o s e h y d r o c e l l u i o s e column u s i n g solvent A. The f i r s t component was c o l l e c t e d i n tubes 37 to L9 and the second component i n tubes 63 t o 85 the frequency o f change being f i f t e e n minutes. The pure 3,h di-O-methyl-L-xyiose  appeared as a c l e a r  syrup with R^ 0.5U i n Solvent system A . r  7  20  [=<J - 21.9 D  (C, 2.UU i n C C H ^ c o ) . C a l c u l a t e d f o r C  C U7.I3$, H 7.67$, OCH^ 3k.Wo. Found C U6.91$, H IV.  g  ?  0^  8.25$, 00^33.59$  3,l|-Di-0-methyI-L-xyionoiactone. 3,U Di-O-methyl-L-xyiose (Il3mg) was t r e a t e d with bromine  f o r f o r t y - e i g h t (I4.8) hours and the 3,h di-O-methyl-L-xyJuronic a c i d i s o l a t e d as p r e v i o u s l y described. The aqueous s o l u t i o n was placed on the freezedryer overnight during which time a c l u s t e r o f needleshaped c r y s t a l s appeared. The remainder o f the syrup was heated t o 80°c a t a pressure o f 0.1 mm f o r two (2) hours i n a c o l d - f i n g e r t o complete l a c t o n i z a t i o n . On standing overnight the lactone c r y s t a l l i z e d g i v i n g long white needles (UU.2mg). Calculated f o r C o O J F L , , , I P 12  0CH 35.22$, found 35.55$,^]^ 50.9°- 23.8 (72hr KC, 1.035 i n O  3  HgO.).M p t . 68°C.  +  S  33  v  *  3)U D i - O - m e t h y l - L - x y l i t o i i , 2 , 5 - t r i - p - n i t r o - b e n z o a t e 3,U Di-D-methyi-ii-xylose  (120mg) was r e d u c e d b y t r e a t i n g  w i t h sodium b o r o h y d r i d e f o r f o r t y - e i g h t h o u r s a f t e r w h i c h t h e r e duced p r o d u c t was i s o l a t e d as p r e v i o u s l y d e s c r i b e d . The 3 , U - d i - 0 m e t h y l - l - x y l i t o l appeared a s a syrup x-rhich was d r i e d , d i s s o l v e d i n anhydrous p y r i d i n e 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 . The m i x t u r e was h e a t e d on i i i e steam b a t h f o r t h i r t y minutes t h e n a f e w drops o f w a t e r were added. A f t e r a l l o w i n g t o s t a n d f o r twenty m i n u t e s t h e s o l u t i o n was p o u r e d i n t o a s a t u r a t e d s o l u t i o n o f . s o d i u m b i c a r b o n a t e . The e s t e r appeared as a p r e c i p i t a t e which was c o l l e c t e d b y c e n t r i f u g a t i o n . The e s t e r was d i s s o l v e d i n c h l o r o f o r m and p a s s e d t h r o u g h a f l o r i s i l column u s i n g c h l o r o f o r m a s e l u e n t . The e s t e r , on e v a p o r a t i o n  o f t h e c h I o r o f o r m a p p e a r e d as a w h i t e p r e c i p i t a t e j  w h i c h was r e c r y s t a l l i z e d f r o m methanol/water, M p t 8H-86°C. C a l c u l a t e d f o r C_ 0 fl  N H  28 i h 3 25  1U.65$.  N 6.69%, UCH  3  9.88$; f o u n d N 6.83$, OCR" 3  P A R T  2  3U  HISTORICAL INTRODUCTION  The chemistry of the formation and inter-relation of the various components of plant ceii-walx materials i s complex. These materials are probably some of the end products of the metabolism of the ceil-wail protoplasm, but the biochemistry i s not as yet sufficiently well understood to enable one to follow stage by stage the process of the formation of the cell-wall materials. In general terms, the celx-waxi carbohydrate materials can be divided into three classes - "pectin" which i s removed by neutral or acid extraction, "hemiceiluioses" which are removed from the remainder by various strengths of axkaxi, and the residual "cexxuiose". In the woody tissues of higher plants these substances are generally associated with lignin. Simpxe examination shows that each fraction i s a complex mixture, and a c r i t i c a l examination of each of the individual polysaccharides i s necessary, therefore, before one can obtain an exact picture of the amount and distribution of the total polysaccharides. The remainder of this discussion w i l l be concerned with the occurrence, extraction, purification, and elucidation of structures of the various polysaccharides. The most widely distributed hemiceiluioses are those i n which the main constituent i s anhydro D-xylose. The classical studies of 0'Dwyer ( i ) , Anderson (2), and Haworth (3) als well as the more recent studies of numerous other workers have shown that the existence of xylose i s much more universal than heretofore realized.  35  Some hemiceHuioses of this type are composed of xylose only and are classified as true xylans (ii). More frequently however, uronic acids are found i n association with this type of polysaccharide, forming a class of compounds known as xylan polyuronides (5). The pentose L-arabinose i s also often found as an integral part of these materials giving rise to an arabo-glucurono-xylan  of a  branched nature (6,7). In most cases the polyuronides give rise to relatively large amounts of aldobiouronic and aldotriuronie acid upon hydrolysis, and only i n few instances i s the uronic acid isolated in good yields under normal conditions of hydrolysis ( 8 ) . The uronic acid residues that have been isolated from these materials by prolonged and severe hydrolysis are D-galacturonic acid, U-O-methylD-glucuronic acid and D-glucuronic acid ( 9 ) . In addition to the xylan polyuronides associated with wood culiulose there occurs predominantly i n softwoods, a group of polysaccharides that constitute a large percentage of non-ceilulosic carbohydrates. Previously i t has been shown that the hydrolysis of crude hemiceiiulose extracts from certain softwoods led to the isolation of D-mannose (10). Hess and Ludtfce ( l l ) and later Husemann (12) using spruce sulfite pulp and spruce holocellulose, respectively, reported the isolation of a mannan. Unfortunately these workers reported no quantitative data and thus i t was impossible to t e l l whether or not the materials were pure mannans. Timell (13) reported that the presence of mannose residues i n the woods of gynosperms was established early (Ik) but their origin  36  long remained entirely obscure. I t was not until 1952 that Leech (15) isolated a disaccharide containing glucose and mannose from slash pine. This disaccharide was subsequently shown by Anthis (16) to he k-O-p -D-gluropyranosyl-D-mannose, hence substantial evidence was obtained indicating the chemical combination of glucose and mannose. In 1956, Hamilton, Kircher and Thompson (17) reported the isolation of a true, diheteropolymer  glucomannan from western hemlock.  This hemicellulose contained glucose and mannose residues i n a ratio of 1:3 which were linked together by jB(l-U)-gIycosidic bonds (18).  The isolation of oligosaccharides containing both glucose  and mannose units after partial hydrolysis of the glucomannan proved beyond doubt that glucose and mannose were chemically combined i n the polysaccharide. During the last few years many glucomannans have been isolated from the woods of various gynosperms and have been found to contain linear or slightly branched chains of (l-» k)-linked p -D-mannose and j$ -D-glucose residues. Galactose-containing polysaccharides have been isolated i n small quantities from the water soluble extracts of a number of coniferous and deciduous woods.(19) but the structural formulae have yet to be established. Recently, i t has become apparent that some of the galactose units of coniferous woods are associated with a mannose-containing polymer. Adams (20) was able to separate the water-soluble extract of white spruce woodmeai into what appeared to be an arabogalactan and a galactoglucomannan. The isolation and constitution of a galactoglucoma'nnan from slash pine and longleaf pine has recently been reported by Hamilton, Partlow and  37  Thompson (21).  The wood pulp was extracted with $% NaOH and the  portion remaining i n solution after acidification was acetylated and the acetate was extracted with acetone. The acetone-soluble fraction, an deacetylation of the polysaccharide gave rise to a polymer containing galactose glucose and mannose i n the ratio of 1:1:3. An alternative method of isolating the galactoglucomannan from the crude solution was described by Adams (20). He found that Fehling's solution forms an insoluble precipitate with galactoglucomannan. Meier (22) subsequently observed that B a  ++  ions  readily form insoluble complexes with mannans and glucomannans probably by reaction with the cis hydroxy! groups on C  2  and  of  mannose units. These complexes are precipitated from aqueous solutions on the addition of smaii quantities of barium hydroxide solution. Data obtained from periodate oxidation and methylation suggested the presence of a framework of [$ (1-*U)-linked D-glucose and D-mannose residues to which single, terminal side chains of Dgalactose were directly attached by glycosidic (1 - 6) bonds  (21).  Meier (23) i n his work on Norway spruce has reported the isolation of a 6-0--D-galactopyranosyl-D-mannose and an O-^J-Dgalactopyranosyl-(l-> 6 ) - 0 - p -D-mannopyranosyl-(l- k)-D-mannose, >  obtained by partial hydrolysis of a glucomannan. The isolation of these oligosaccharides constitute the f i r s t conclusive evidence that galactose and mannose residues are chemically combined i n wood. These oligosaccharides could have arisen from galactoglucomannan.  38  BLACK SPRUCE HEMICELLULOSES  In recent years there has been increased i n t e r e s t i n the hemicelluloses of plants and plant products. One of the main problems encountered i s the p u r i f i c a t i o n of the various hemicelluloses so as to make the s t r u c t u r a l determination possible. The purpose of t h i s project i s to p u r i f y the hemicelluloses extracted from black spruce (Picea Mariana ( M i l l ) ) by f r a c t i o n a l p r e c i p i tation. Black spruce wood shavings were d e l i g n i f i e d by the method of Wise, Murphy and D'Addieco (2k)  by treatment with sodium c h l o r i t e  a t 70 - 80°C. The resulting h o l o c e l l u l o s e was extracted with 2h% potassium hydroxide by shaking f o r fourteen hours at room temperature. The material was  f i l t e r e d , the residue washed with a l k a l i and  water, and the a l k a l i n e extract immediately  reacted overnight with  sodium borohydride* to minimize ^ degradation. The extract was poured into three volumes of ethanol containing an excess of a c e t i c a c i d giving a hemicellulose f r a c t i o n designated BSx^,  which con-  tained uronic acids, galactose, glucose, mannose, arabinose  and  xylose on hydrolysis. The residue from the potassium hydroxide  ex-  t r a c t i o n was repeatedly washed with large volumes of water and then extracted with 17.5$  sodium hydroxide s o l u t i o n containing k$ boric  a c i d (2k,25). The extract was treated overnight with sodium borohydride and poured into a c i d i f i e d ethanol. The p r e c i p i t a t e d hemic e l l u l o s e , designated BSGM^, i s a glucomannan which contained glucose and mannose with traces of galactose, xylose and arabinose.  39  The mixture extracted with potassium hydroxide was d i s solved i n 10% sodium hydroxide and $% barium hydroxide s o l u t i o n added dropwise to the continuously s t i r r e d s o l u t i o n to give soluble and insoluble portions. The p r e c i p i t a t e i s the galactoglucomannan and i s designated BSGG^, whereas the soluble f r a c t i o n recovered from the supernatant by p r e c i p i t a t i o n i n ethanol i s the xyian port i o n BSXg. The xyian portion (BSXg) was treated twice f u r t h e r with barium hydroxide  s o l u t i o n and the small and decreasing amounts of  p r e c i p i t a t e discarded. Fraction BSx^ s t i l l contained traces of galactose. A c e t y l a t i o n of t h i s hemicellulose by the method of Carson and Maclay (26) was attempted but an acetylated polysaccharide insoluble i n organic solvents such as acetone o r chloroform was not obtained, thereby making separation from the soluble "hexosons" impossible. An attempt was also made to separate the a c i d containi n g xylon from the neutral galactose by f r a c t i o n a l p r e c i p i t a t i o n using cetyltrimel^rlammonium  s a l t (Cetavlon) as a p r e c i p i t a n t (27)  but no p r e c i p i t a t e formed. Neither of these procedures was i n v e s t i gated further. A portion of the xyian sample(BSx^) dissolved i n 5$ sodium hydroxide was treated with an equal volume of f r e s h l y prepared Fehling's s o l u t i o n whereupon the copper complex p r e c i p i t a t e d ( k ) . A l i t t l e acetone was added to quicken the. s e t t l i n g of the p r e c i p i tate which was then c o l l e c t e d by centrifugation. The p r e c i p i t a t e was suspended i n water by vigorous s t i r r i n g , and cold hydrochloric  acid (2N) was then added carefully to decompose the copper complex giving a clear polysaccharide solution. The polysaccharide was precipitated with ethanol to give a white flocculent precipitate BSx£ which s t i l l contained a slight trace of galactose. After a further treatment with Fehling's solution the fraction BSx^ contained traces of copper and was redissolved in $% sodium hydroxide and precipitated again from ethanol to give BSx.. This fraction was 6  chromatographically free of galactose since a colorimetric analysis of an extract from the galactose zone on a paper chromatogram gave a zero reading. The xylose to arabinose ratio was found to be 6 i 3 * l . Other workers have found that the xyian fraction was always contaminated by small amounts of galactose when barium hydroxide alone was used for purification. In this instance however, when the barium hydroxide fractionation was followed by two treatments with Fehling's solution the galactose was completely eliminated. This i s believed to be the f i r s t case of the isolation of such a xylon in.a pure state from the hemiceiluioses of a coniferous wood. The glucomannan (BSGM^) was purified by precipitation with barium hydroxide solution. The precipitate obtained s t i l l contained traces of xylose. This precipitate (BSGMV,) was dissolved i n 10$ sodium hydroxide solution and an equal volume of Fehling's solution added dropwise with stirring. A blue precipitate formed and was collected by centrifugation. After washing with water the precipitate was dissolved in ice cold acid and reprecipitated from ethanol. This fraction (BSGM-) was free of pentoses on hydrolysis.  Ill  EXPERIMENTAL  DELIGNIFICATION OF BLACK SPRUCE WOOD. Black Spruce wood shavings (600g) were placed into a large vessel containing water (9 l i t e r s ) at 70 - 80°C. Glacial acetic acid (120ml) was added followed by sodium chlorite (360g) i n small portions over twenty minutes. The reaction mixture was thoroughly stirred with a mechanical stirrer while steam was passed through to maintain the temperature at 70 - 80°C. This was allowed to continue for four hours with acetic acid and sodium chlorite being added after each hour. The mixture was then allowed to cool, the supernatant siphoned off and the wood shaving thoroughly washed with cold water. The hoioceiluiose was centrifuged, air dried and weighed. This procedure was repeated on an additional 600 grams of a i r dried shaving giving a total weight of 1200 gm. The total yield of a i r dried hoioceiluiose was 980 gms. LIGNIN DETERMINATION OF BLACK SPRUCE HOLOCELLULOSE. Duplicate samples of a i r dried hoioceiluiose were weighed (l.0k7!?g), placed into a beaker and cold (l2-l£°C) 72$ sulphuric acid (l£ml) added with stirring. The mixture was allowed to stand for two hours with frequent stirring, at a temperature of 18 - 20°C (water bath). The material was then added to a one l i t e r Erienmeyer flask, diluted, to a 3% acid concentration by adding $60 ml of d i s t i l l e d water, and boiled for four hours under a reflux condenser. After cooling, the insoluble material was f i l t e r e d into an oven dried, tared filtering crucible, dried and weighed.  U2  The weight of lignin was 0.1l6kg. giving a lignin content of  11.1% (28). EXTRACTION OF HEM CELLULOSES Holocellulose (930g) was extracted with 2lx% potassium hydroxide (12 l i t e r s ) at room temperature for fourteen hours with shaking. The solution was filtered and the residue washed with water to give a total volume of eighteen (18) l i t e r s . The alkaline solution was treated overnight with sodium borohydride (6g) and poured into ethanol (k5 l i t e r s ) containing acetic acid (3 l i t e r s ) . The precipitated hemiceiluiose was recovered by centrifugation, washed i n succession with 80$ ethanol, ethanol and petroleum ether and f i n a l l y dried in vacuo. This fraction was; designated BSx^(xyian). Yield 268g. This amounted to 28$ of the oven dry wood. The residue from the potassium hydroxide extraction was repeatedly washed with large volumes of water and then extracted with 17.5$ sodium hydroxide containing k$ boric acid (i2 l i t e r s ) for lU hours. After f i l t r a t i o n the residue was washed with water to give a total volume of l6 l i t e r s which was reduced overnight and poured into acidified ethanol as before. The precipitate, dried by solvent exchange, amounted to lk8g ar 15.8$ of the oven dried wood and was designated BSGM-^ glucomannan). ISOLATION OF AN AFABINO-GLUCURONO-XYLAN. The potassium hydroxide extract (268g) was dissolved i n 10$ NaOH (5 l i t e r s ) and 5$ barium hydroxide added dropwise to the continuously.stirred solution. The precipitate was the galactoglucomannan fraction (66g) designated BSGG^ and i t was collected  U3  by centrifugation. The c l e a r supernatant was poured into four times i t s volume of ethanol containing  acetic a c i d to give a white  p r e c i p i t a t e of xylan (BSx ) 2  PURIFICATIOM OF GLUCOMA.NNAN. The glucomannan (k£g) BSGM extracted with NaQH/H^BO JL  was  shaken i n 10$ NaOH solution f o r twelve hours. A part of the  sample dissolved. The mixture was centrifuged and the supernatant treated with $£$ Ba.(0E)^ to give a white p r e c i p i t a t e . The residue was  shaken with water (2 l i t e r s ) f o r 12 hours, 20$ NaOH was added  and the solution treated with Ba(OH)^ giving a white p r e c i p i t a t e . These p r e c i p i t a t e s were combined, washed with 5$ NaOH followed by water to give a p r e c i p i t a t e of BSGM . A small sample of BSGMg was hydrolyzed using IN H^SO^. This sample was chromatographed i n s o l vent system Card was  shown to contain a trace of xylose. The sugar  r a t i o of BSGMg was determined by the phenol-sulphuric a c i d method A sample of BSGM^^g) was dissolved i n sodium hydroxide (20$), the solution was then d i l u t e d to £$ i n sodium hydroxide and Fehling's solution (600ml) added dropwise to the A blue gelatinous  s t i r r e d solution.  p r e c i p i t a t e formed which was c o l l e c t e d by c e n t r i -  fugation and washed s i x times with water. The p r e c i p i t a t e was dissolved i n 10$ hydrochloric  acid and poured into four l i t e r s of  ethanol. The white p r e c i p i t a t e (l!?g) was c o l l e c t e d , dried and a small portion hydrolysed. The BSGM^ showed no traces of xylose on the papergram nor by the colorimetric method.  Ilk  PURIFICATION OF THE  XYLAN.  The material BSXg from which the galactoglucomannam BSGtG^ had been separated was subjected to two further treatments with barium hydroxide solutions to give BSx^(92g) which on hydrolys i s s t i l l contained  galactose.  CETYLTRIMETHYLAMMONIUM SALTS OF ACIDIC XYLAN. The xyian (ig) was s o l u t i o n (20$)  dissolved i n water (20ml) and  cetavlon  added slowly. No p r e c i p i t a t e was formed i n d i c a t i n g  that the water insoluble quaternary ammonium s a l t of the a c i d i c xyian was not formed. A portion of the solution was poured into alcohol giving a white p r e c i p i t a t e . This further v e r i f i e d that the complex did not form, since the complex i s insoluble i n alcohol. The cetavlon complex was not investigated further. ACETYLATION OF THE XYLAN (BSx^) The xyian (5g) was added to formamide (60g)  and the mix-  ture shaken f o r 18 hours to e f f e c t complete dispersion. To t h i s mixture pyridine (U3g)  was then added i n four portions over a  period of t h i r t y minutes with constant (30g)  s t i r r i n g . Acetic anhydride  was added i n three portions and the mixture allowed to stand  overnight. The s o l u t i o n was poured into 2% hydrochloric a c i d (600ml) containing crushed i c e . The ester p r e c i p i t a t e d , was washed free of c h l o r i d e . The p r e c i p i t a t e was  f i l t e r e d and  suspended i n ethanol  overnight and dried by solvent exahange using petroleum ether d i e t h y l ether. The acetylated product was both acetone and chloroform, Carson and Maclay (26)  and  found to be soluble i n  an observation opposite to that of  who had found that acetylated xylans were  1  insoluble i n organic solvents and the hexosans soluble, making i t possible to e f f e c t a separation. In view of the circumstances the acetylated xyian was not investigated further. PURIFICATION OF XYLAN (BSx^) BY COPPER COMPLEX. A portion of the sample (30g) was dissolved i n $% NaOH s o l u t i o n (I l i t e r ) and Fehling's solution added dropwise with s t i r r i n g . A blue gelatinous  copper complex p r e c i p i t a t e d out. The  p r e c i p i t a t e was c o l l e c t e d by centrifugation, dissolved i n i c e cold IN hydrochloric  a c i d ( l l i t e r ) to destroy the complex, and poured  i n t o ethanol (U l i t e r s ) whereupon a heavy white p r e c i p i t a t e was formed. A f t e r allowing the p r e c i p i t a t e (22g) to s e t t l e i t was c o l l e c t e d by centrifugation and washed with ethanol. This sample of BSx^  on a c i d hydrolysis showed traces of galactose.  The above proce-  dure was repeated on the BSx^ (22g) to y i e l d BSx^ ( l 5 g ) which contained traces of copper. I t was subsequently dissolved i n ±0% sodium hydroxide (2 l i t e r s ) and p r e c i p i t a t e d again from alcohol-acetic a c i d to give 13 g. of sample (BSx^) which appeared to be free of copper. This l a s t f r a c t i o n was chromatographically free of galactose and a colorimetric analysis o f the extract from the galactose  zone  on a chromatogram showed zero reading. PURIFICATION OF GG An attempt was made to dissolve t h i s sample i n 10% NaOH but only a small f r a c t i o n seemed to d i s s o l v e . The mixture was cent r i f u g e d and the centrifugate treated with $% Ba (OR^ but no prec i p i t a t e formed. The residue was exhaustively  shaken with water and  >  6  most of i t went i n t o solution. To t h i s solution an equal volume of 20$ NaOH was added and Ba(0H)  2  added dropwise as above. A very  small quantity of p r e c i p i t a t e was formed. This was quite inadequate. Consequently the supernatant of both l o t s were d i a i i z e d with acetic a c i d and then evaporated. The galactoglucomannan was subsequently dissolved i n s l i g h t l y a c i d i c s o l u t i o n and then made a l k a l i n e and treated with  5$ Ba(0H)2  upon which a p r e c i p i t a t e was obtained  (3kg).  This sample was dissolved i n 10$ NaOH and the sample treated with Fehling's solution thereby giving a blue p r e c i p i t a t e which was centrifuged, washed, and dissolved i n IN HCI and poured into ethanol. A: portion of the sample, was hydrolyzed and s t i l l contained some xylose. QUANTITATIVE ANALYSIS OF SUGARS Whatman No. I paper (6 x 22") was streaked with the mixture of neutral sugars from the hydrolysate o f the different hemic e l l u i o s e f r a c t i o n s . The chromatograms were developed, along with a blank sheet, i n ethyl acetate pyridine water (8.2:l). The chromatograms were d r i e d and the sugars l o c a t e d by cutting three-quarter inch s t r i p s from e i t h e r side of the paper and spraying them with para-anisidine trichioroacetate spray. The center unsprayed portion of the chromatogram was then cut into sections corresponding t o the l o c a t i o n of the sugar. Each section was transferred to a beaker and eluted with a suitable volume of water (20 or 30 ml) f o r t h i r t y minutes. The solutions, along with the blank, were f i l t e r e d through f i l t e r tubes containing glasswool. A l i q u o t s (2ml) were transferred i n t r i p l i c a t e to matched colorimeter tubes, 80$ aqueous phenol  U7  (lOO liters) was added followed by concentrated HgSO^C^ml) reagent grade by means of a Marklett burette. The solution was allowed to cool and the absorbance read at k°0 mp on a Coleman DU spectrophotometer. The concentration of the sugars was determined from previously prepared standard curves and the moie ratio for the res1  pective polysaccharides determined.  1*8  T A B L E III OPTICAL DENSITY OF SUGAR SOLUTIONS  SAMPLE BSGMg  Xylose  OPTICAL DENSITY -. Arabinose Galactose Glucose Mannose :  :  0.220  0.708  0.221  0.720  0.261*  0.862  0.261*  0.862  0.050  0.050  0.150  0.050  0.055  0.150  0.0L8  o.ol*5  0.152  0.03  oaoi  0.279  0.390  0.03  0.101  0.281  0.390  Trace  BSGM  3  BSGG  2  BSGG  3  B S x  u  BSx^  BSx  6  ;  Trace  0 . O.lUO  0.025  Trace  O.lUO  0.025  Trace  0.590  0.120  0.08  0.570  0.120  0.08  0.500  0.090  0.1*80  0.090  52  TABLE  IV  SUGAR RATIOS IN HEMI CELLULOSE FRACTIONS  Fraction BSGM  2  BSGM  3  BSGG  2  BSGG  3  Xylose  Arabinose  Glucose !  T r a c e  o  x  !  + ( n # e )  0.2  1  Mannose Galactose 3  t  h  3  #  5  ?  <  6  8.0  1  #  5  1.6  U  8.8  1.5  1  SSx^  9.0  1.5  1  BSX6  6.3  1  B S x  S3 *  DETERMINATION OF THE DEGREE OF POLYMERIZATION BY FORMALDEHYDE ESTIMATION OF REDUCED POLYSACCHARIDE. The polymer was dissolved i n 10$ sodium hydroxide solution (l5>ml) and sodium borohydride added (f>Og). The solution was allowed to stand f o r I4.8 hours at room temperature a f t e r which the solution was a c i d i f i e d with acetic acid, cooled and sodium periodate solution (l$ml of 2 M) added. A f t e r every twelve hours a portion of the o x i dation mixture (3ml) was transferred to a large t e s t tube and k0$ p£(OAc)2  S O J L u  ^l°  n  (2ml) added. The solution was mixed properly and a  d i a l y s i s tube, t i e d at one end, containing water ($mi) was placed into the suspension and allowed to equilibrate overnight. The clear solut i o n i n the casing was placed into a • clean .-test tube, chromotropic a c i d reagent ($ml) was added, the suspension centrifuged to remove PbSO^  and the clear solution was decanted i n t o colorimeter  tubes. The  tubes were placed i n a rack i n a b o i l i n g water bath (excluding  light)  f o r t h i r t y minutes. The tubes were cooled i n cold water, the contents mixed and the absorbance read at 570 mjt. D.P. o f Xylan  D.P. of glucomannan lOO - 117  60 - 70.  PERIODATE OXIDATION OF XYLAN. Xylan (i.OOgg) was dissolved i n d i l u t e a l k a l i a f t e r which the solution was made s l i g h t l y a c i d i c , the volume adjusted to 100 mis and a f t e r cooling to $°C 0.2M periodic a c i d solution was added (50mi). The f  periodate uptake was determined a f t e r periods of 2k, k8, 72 and 96 hours, during which time the uptake remained constant at 0.8k9 moles of periodate per sugar residue. To the solution was added barium carbonate to p r e c i p i t a t e the periodate and iodate ions and to the c l e a r f i l t r a t e was added NaBH (I.2g) and the solution l e f t overnight. The solution was  neutralized with acetic acid, evaporated, and borate removed from the poiyoi by several treatments with methanoiic hydrogen chloride.  56  BIBLIOGRAPHY PART 1 1.  W.S. Denham and H. Woodhouse, J. Chem. Soc. 103: 1735. 1913  2.  W.S. Denham and H. Woodhouse, J. Chem. Soc. 2357. 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