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Periodate oxidation products of some partially methylated hexoses and hexitols Gibney, Kelly Blair 1967

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PERIODATE OXIDATION PRODUCTS OF SOME PARTIALLY METHYLATED HEXOSES AND HEXITOLS by KELLY BLAIR GIBNEY B.Sc, The University of B r i t i s h Columbia, 1963 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Chemistry We accept t h i s t h e s i s as conforming to the required standard. The Uni v e r s i t y of B r i t i s h Columbia A p r i l , 1967 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I ag r ee t h a t t he 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 s t u d y , I f u r t h e r ag ree 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 c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f 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 s h a l l no t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depa r tment o f Ch&HVllkft' The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada ABSTRACT Chairman: Professor G.G.S. Dutton Periodate Oxidation Products of Some Partially Methylated Hexoses and Hexitols Complete periodate oxidation of 2,3-di-O-methyl-D-glucitol and 2,3-di-O-methyl-D-mannitol produced 2,3-di-O-methyl-L-threose and 2,3-di-0-methyl-Q-erythrose respectively. Reduction with sodium borohydride produced the corresponding t e t r i t o l s , 2,3-di-O-methyl-L-threitol and 2,3-di-0-methyl-erythritol. Incomplete periodate oxidation of the two hexitols lead to the formation of large amounts of the respective pentoses, 3,4-di-0-methyl-L-xylose and 3,4-di-O-methyl-D-arabinose. Attempts to prepare 4-0-methyl-D-threose from methyl 6-0-methyl-ag-g-galactofuranoside and 2-0-methyl-D-erythrose from methyl 4-0-methyl-a-D-mannopyranoside by periodate oxidation were only pa r t i a l l y successful. Although the oxidation proceeded smoothly, the cleavage of the oxidized product could not be readily effected with mineral acid, methanolysis or mercaptolysis without extensive degradation taking place. Reduction of the aldehydes generated by periodate oxidation allowed the facile preparation of the corresponding t e t r i t o l s . A scheme to correlate the original aldehyde structure to the reduced t e t r i t o l i s proposed. The structure of an unknown component in the hydrolysis of methyl 2,3-di-0-methyl-a-D-glucopyranoside in the presence of 2,3-di-0-methyl-D-glucitol was shown to be 1,4-anhydro-2,3-di-0-methyl-D-glucitol. The paper chromatographic characteristics of the prepared tetroses, t e t r i t o l s and pentoses in the two most commonly used solvent systems have been recorded. - i i i -TABLE OF CONTENTS Page No. INTRODUCTION 1 HISTORICAL 4 METHODS OF SYNTHESIS . 7 2,3-Di-O-methyl-L-threose 7 2,3-Di-O-methyl-Q-erythrose 11 4-O-Methyl-D-threose 13 2-O-Methyl-D-erythrose 17 DISCUSSION • • 19 D-l Unknown in the hydrolysis of methyl 2,3-di-O-methyl-a-D-glucopyranoside 19 Proton Magentic Resonance 20 Mass Spectra 23 D-2 A solution to hydrolysis problems 29 D-3 Review and Discussion of Periodate Oxidation 33 Formyl Ester 41 Thin Layer Chromatography for Monitoring Carbohydrate reactions . . . . . . . . . . . . . 43 EXPERIMENTAL 46 Gas Liquid Chromatography 46 Silylation of Samples for Gas Chromatography 46 Periodate Oxidations 46 Synthesis of 2,3-di-0-methyl-L-threose 47 Methyl 4,6-benzylidene-a-D-glucoside . . 47 Methyl 2,3-di-O-methyl-a-D-glucopyranoside 48 2,3-Di-O-methyl-D-glucitol . 48 - iv -TABLE OF CONTENTS (cont'd) Page No 2,3-Di-0-methyl-l,4,5,6-tetra-0-£-nitrobenzoyl-D-glucitol 48 2,3-Di-O-methyl-L-threose . . . 49 2,3-Di-0-methyl-L-threitol 49 2.3- Di-0-raethyl-l,4-di-0-p_-nitrobenzoyl-L-threitol . . . . 50 1.4- Anhydro-2,3-di-O-methyl-D-glucitol 50 1,4-Anhydro-2,3-di-0-methyl-5,6-di-0-p_-nitrobenzoyl-D-glucitol 51 Synthesis of 2,3-di-0-methyl-D-erythrose . . . 51 Methyl a-D-mannopyranoside 51 Methyl 4,6-0-benzylidene-a-D-mannopyranoside . 52 Methyl 4,6-0-benzylidene-2,3-di-0-methyl-a-D-mannopyranoside 52 2,3-Di-O-methyl-D-mannitol 52 2,3-Di-O-methyl-D-erythrose . 52 2,3-Di-O-methylerythritol 53 2,3-Di-0-methyl-l,4-di -0-p_-nitrobenzoylerythritol 53 Synthesis of 2-O-methyl-D-erythritol . . . . 53 Methyl 2,3-isopropylidene-a-D-mannoside 53 Methyl 4-0-methyl-a-D-mannopyranoside . . . . . . . . . 54 2-O-Methyl-D-erythritol . . . 54 2-0-Methyl-l,3,4-tri-0-£-nitrobenzoyl-D-erythritol . . . 55 Synthesis of 4-O-methyl-D-threose ' 55 6-O-Methyl-D-galactose 55 Methyl 6-0-methyl-a-D-galactofuranoside . . . . . . . . . 55 Methyl 6-0-methyl-B-D-galactofuranoside 55 Methyl 6-O-methyl-a-D-galactopyranoside 55 Methyl 6-0-methyl-2, 3,5-tri -0-p_-nitrobenzoyl-a-D-galacto- 55 furanoside . . . . . . . . . . . . - V -TABLE OF CONTENTS (cont'd) Page No. Methyl 6-0-methyl-2,3,S-tri -0-p_-nitrobenzoyl-6-D-galactofuranoside 56 4-O-Methyl-D-threose-di-methylacetal 5^ 4-O-Methyl-D-threose . 57 4-O-Methyl-D-threitol . 57 4-0-Methyl-l,2,3-tri-0-p_-nitrobenzoyl-D-threitol . . . 57 . , . 57 Controlled Periodate Oxidations 3,4-Di-O-methyl-L-xylose. 57 Methyl 3,4-di-O-methyl-cx-L-xyloside 58 3,4-Di-O-methyl-D-arabinose 58 3,4-Di-O-methyl-D-arabonamide 58 Bibliography • 59 - v i -LIST OF TABLES Table 1. Molecular Ion Peaks for 5,6-di-0-acetyl-l,4-anhydro-2,5-di-O-methyl-D-glucitol . . . . . . 24 Table 2. Molecular Ion Peaks for 1,4:3,6-dianhydro-2,5-di-0-methyl-D-glucitol • 27 Table 3. Molar Response Factors for some Sample Compounds . . 31 Table 4. Paper Chromatographic Characteristics of Known and New Tetroses, Pentoses and Tetritols 45 LIST OF FIGURES Figure No. Figure 1. Reduction of three periodate oxidation products to one Tetr i t o l 3 Figure 2. T.L.C. on S i l i c a Gel, developed butanone-water azeotrope, of methyl 2,3-di-O-methyl-a-D-glucopyranoside hydrolysate 7 Figure 3. Steric hinderance of substituent at C-3 to borohydride reduction 9 Figure 4. Synthesis of 2,3-di-O-methyl-L-threose 10 Figure 5. Synthesis of 2,3-di-O-methyl-D-erythrose . . . . . . 12 Figure 6. Synthesis of 6-0-methyl-D-galactosides 15 Figure 7. Modes of periodate oxidation of 6-0-methyl-D-galactosides 15 Figure 8. Synthesis of 4-0-methyl-D-threose 16 Figure 9. Synthesis of 2-0-methyl-D-erythritol . . . . . . . . . 18 Figure 10. Envelope conformation of gluco configuration 21 Figure 11. P.M.R. spectrum of 5,6-di-0-acetyl-l,4-anhydro-2,3-di-0-methyl-Q-glucitol 22 Figure 12. Mass spectrum of 5,6,-di-0-acetyl-l,4-anhydro-2,3-di-O-methyl-D-glucitol . . . . . . . . . . . . 25 - v i i -LIST OF FIGURES (Cont'd) Figure No. Page No. Figure 13. Source of m/e 58 peak 26 Figure 14. Source of m/e 60 peak 26 Figure 15. Mass spectrum of l,4;3,6-dianhydro-2,5-di-0-methyl-D-glucitol 28 Figure 16. Characteristic fragments from labelled isomeric t e t r i t o l s 30 Figure 17. Periodate oxidation, of adjacent hydroxyl groups . 33 Figure 18. Formation of formic acid . . . 33 Figure 19. Periodate oxidation of a primary hydroxyl . . . 34 Figure 20. Course of periodate oxidation 34 Figure 21. Positions of periodate oxidation in various hexitols 36 Figure 22. Periodate oxidation of 2,3-di-O-methyl-Q-glucitol 38 Figure 23. Periodate oxidation of 2,3-di-O-methyl-Q-mannitol 39 Figure 24. Inhibition of periodate oxidation 40 Figure 25. Periodate oxidation of a malondialdehyde . . . . 40 Figure 26. Formation of elemental iodine on periodate oxidation 40 Figure 27. Course of periodate oxidation of ketoses . . . 41 Figure 28. Periodate oxidation showing formate ester formation 42 Figure 29. P.M.R. spectrum of formyl and aldehyde hydrogens. 43 - v i i i -ACKNOWLEDGEMENTS The author wishes to express his sincere thanks to Dr. G.G.S. Dutton for his guidance and support during this investigation. A special thanks is due Dr. P.E. Reid, who during the period of Dr. Dutton's sabbatical year guided this research. The counsel of Dr. K.N. Slessor is also gratefully acknowledged as is that of Dr. L.D. Hall. Thanks.are due Mr. J. Haywood-Farmer for a sample of l,4:3,6-dianhydroglucitol. The author also wishes to thank his wife for her patience and support during the course of this work. INTRODUCTION One of the most important methods for the determination of the structure of polysaccharides involves the preparation of the polysaccharide methyl ether and subsequent identification of the methylated monosaccharides resulting from i t s hydrolysis. These methylated sugars are often present in extremely complex mixtures and partition chromatography is the preferred method for the separation of the components. Although many advances have been made in chromatography (1), i t is not possible in every case to separate completely a l l the components nor to identify readily some of the sugars which are resolved (2). Many techniques have been employed to identify these sugars or sugar mixtures: paper electrophoresis (3), determination of hexose or pentose structure (4), demethylation (5), complete methylation (6), optical rotation (7), formation of an osazone to test for substitution at C-2 (8), furanoside formation (9), oxidation to the lactone and subsequent examination of hydrolysis rate, or IR spectra indicating lactone ring size (10), formation of the t r i t y l ether at C-6 (11), and periodate oxidation of the sugar or sugar alcohol (12,13,14). Periodate oxidation has been employed in both the structural deter-minations of synthetic methylated sugars and in the identification of partially methylated sugars obtained from hydrolysed methylated polysaccharides. Bell, for example, employed periodate oxidation in the determination of the structure of such sugars as 2,6-di-O-methyl-D-galactose (15) and 3,4-di-0-methyl-D-galactose (16). 1 . Lemieux and Bauer (17) used periodate oxidation as a means of identi-fication for mono-O-methylglucoses. Alkaline hydrolysis of the periodate - 2 -oxidation product of any mono-O-methyl glucopyranose yields a substance which can be positively distinguished by paper chromatography from the products formed under the same conditions from the other mono-O-methylglucopyranoses. Extension of the method to di and tri-O-methyl glucoses was discussed. The value of the procedure was illustrated by characterization of the O-methyl glucoses derived from a water-soluble O-methylcellulose. The value of the product obtained from the oxidation of 4-0-methyl-D-glucOpyranose was of the order expected for 2-0-methyl-D-erythrose. The reducing substance formed from 2,3,6-tri-O-methyl-D-glucitol was assumed to be 2,3-di-O-methyl-L-threose. Stephen (18) in the structural investigation of V i r g i l i a oroboides gum obtained a chromatographically fast running spot on periodate oxidation of 2,3,6-tri-O-methyl-D-galactitol, 2,3-di-O-methyl-D-galactitol, 2,3-di-0-methyl-L-arabinitol and 2, 3-di-O-methyl-D-glucitol assumed to correspond in each case to 2,3-di-O-methyl-L-threose. Dutton and Unrau in structural studies of mesquite gum (19) and a number of synthetic polysaccharides: glucan (20), xylan (21), arabinan (22), rhamman (23), galactan (24) and mannan (25) have employed periodate oxidation extensively as a tool in the identification of the components of complex mixtures of methylated sugars and the alcohols derived from them. In the glucan study,these periodate oxidations produced reducing fragments believed to be partially methylated tetroses. In subsequent papers, reduction of the reducing fragments lead to the formation of a considerable number of methylated t e t r i t o l s which were subsequently analysed by gas liquid chromato-graphy (G.L.C.). A common factor in the work of Lemieux and Bauer (17), Stephen (18) and Dutton and Unrau was the production of methylated tetrose or t e t r i t o l fragments resulting from the periodate oxidation of mixtures of methylated sugars or their derived alcohols. Identification of the unreduced periodate oxidation products i s desirable since the product provides more information than does the derived p o l y o l . For example, reduction of the reducing fr a g -ments below y i e l d the same t e t r i t o l : K C O C H O -H -H C H O H C H O H-+OCH--OCH: H-C H 2 O H H-C H O •OCH. H- -OCH. C H O * N a B K H-H-C H 2 O H O C H 3 O C H 3 C H 2 O H Figure 1. Reduction of three periodate oxidation products to one t e t r i t o l . The studies mentioned above would have been f a c i l i t a t e d by the e x i s t ence of a well characterized series of reference compounds. This laboratory has been engaged i n a programme to synthesize these reference compounds. The four isomeric 2,4-di-0-methyl.tetroses were synthesized by Slessor and Dutton (26) and i n a continuation of t h i s programme t h i s thesis reports the synthesis of 2,3-di-O-methyl-L-threose, 2,3-di-O-methyl-D-erythrose, 2,3-di-O-methyl-L-threitol, 2,3-di-0-methylerythritol, 4-0-methyl-D-threose, 4-0-methyl-D-threitol, 2-0-methyl-D-erythritol and the two pentoses 3,4-di-0-methyl-L-xylose and 3,4-di-O-methyl-D-arabinose. - 4 -HISTORICAL The generic name tetrose refers to the four carbon straight chain aldoses. There are three possible approaches to the synthesis of tetrose derivatives: 1. Lengthening the carbon chain of a lower sugar. 2. Fission of the carbon chain of a higher sugar. 3. Reaction of the tetroses themselves. The K i l i a n i and Fischer (27) cyanohydrin synthesis is based on the formation of cyanohydrins (28) from aldehydes and ketones with hydrogen cyanide. Fischer (29) showed that the a-hydroxy acids formed on hydrolysis of the cyano-hydrins could be ut i l i z e d for the preparation of higher aldoses by reduction of their derived aldonic acid lactones. More recently, the nitromethane synthesis has been used to lengthen sugar chains. The addition of nitromethane to aldehydes yields nitroalcohols which can be decomposed in acid solution to yield hydroxyaldehydes. This method, f i r s t applied to sugars by Pictet (30), was thoroughly investiaged by Sowden and Fischer (31) as a general method for lengthening the carbon chain of aldoses. The reaction of diazomethane with aldonic acid chlorides results (on decomposition) in the formation of ketoses with one more carbon atom (32). Enzymatic action has also been ut i l i z e d to lengthen carbon chains of sugars (33,34). There are three inherent problems in applying chain lengthening by nitromethane and Kiliani-Fischer syntheses to the formation of tetrose derivatives.Firstly, a new asymmetric centre is introduced alpha to the aldehyde, resulting in an epimeric mixture which must be separated. Secondly, the nature of the synthesis makes impossible the formation of a derivative sub-stituted at C-2 unless further reactions are carried out. The third problem is the d i f f i c u l t y of working with glyceraldehyde. The use of diazomethane is not applicable since aldoses are desired. The use of enzymes would be involved since specific enzymes would be d i f f i c u l t to obtain and they might be expected to reject the compounds i f derivatives - 5 -were prepared before chain lengthening. There are several methods for shortening the chain length of mono-saccharides. The classical Wohl (35), Ruff (36) and Weerman (37) degrada-tions which eliminate hydrogen cyanide, carbon dioxide and sodium isocyanate respectively, result in the reduction of the chain length by one carbon atom. More recently, i t was reported that aldose dithioacetals may be suitable starting materials for the preparation of lower aldoses (38,39). Oxidation of the dithioacetals with monoperphthalic acid yields the disulphone which, in an alkaline medium,loses bis-(ethanesulphonyl)methane yielding an aldose with one less carbon atom. Probably the most useful method is degradation through glycol cleavage. Cleavage between two adjacent hydroxyls by periodate (40), or the less frequently used lead tetracetate (41), results in the formation of two aldehydes. The use of these reagents has been recently reviewed (42,43). The inherent problem with these degradative methods as a synthetic tool is the necessity of preparing a suitable derivative. The classical cleavages that eliminate the anomeric carbon require that there be no sub-stituent at C-2. The glycol cleaving agents require blockage of a l l vicinal hydroxyls not to be cleaved in the reaction. Although u t i l i z a t i o n of the tetroses as the starting material for preparation of pa r t i a l l y methylated derivatives is a feasible approach, l i t t l e is known about tetrose derivatives and the tetroses needed would normally have to be prepared by the conventional means of oxidative cleavage of higher sugars. There have been few reports of the synthesis of methylated tetroses and,except for one report, a l l procedures have employed periodate oxidation. In the f i r s t recorded synthesis of a methylated tetrose, Gatzi and Reichstein (44) methylated 1,2-0-isopropylidene-L-threose. Mild hydrolysis yielded 3- 0-methyl-L-threose. However, the physical constants of the compound were not reported. Smith et al. (45, 46) in periodate oxidation studies of some partially methylated sugars, had occasion to synthesize two partially methyl-ated tetroses. Periodate oxidation of 3-0-methyl-g-xylose and 2,3-di-0-methyl-D-arabinitol yielded 2-0-methyl-Q-threose and 2,3-di-O-methyl-Q-threose respectively. Richards (47) oxidized methyl 6-O-methyl-D-galactofuranosides with sodium metaperiodate. The dialdehyde formed was hydrolysed to yield 4- 0-methyl-D-threose. The derivative prepared to characterize the compound was the phenylosazone. This derivative destroys the optically active centre at C-2 .and hence is not a characteristic derivative of 4-0-methyl-D-threose. This compound has been resynthesised in order to prepare a characteristic derivative. Dutton and Slessor (26) in their synthesis of the four isomeric 2,4-di-0-methyl tetroses employed periodate oxidation of methylated sugar alcohols. 2,4-Di-O-methyl-D- and L-erythroses were prepared from 4,6-di-0-methyl-D-glucitol and 3,5-di-0-methy1-L-arabinitol respectively. The 2,4-di-O-methyl-D- and L-threoses v/ere prepared from 3,5-di-O-methyl-D-xylitol and the mixed alcohols produced on reduction of 1,4,6-tri-O-methyl-L-sorbose. The method chosen to synthesize the partially methylated tetroses reported in this study was the periodate oxidation of suitably substituted hexoses and hexitols. The reasons for this choice were:-(a) the quantitative yields resulting from periodate oxidation (48) (b) the substrates required are known carbohydrates or readily obtained from known carbohydrates (c) periodate oxidation has been extensively studied in relation to i t s use in carbohydrate chemistry and the conditions and side reactions are well characterized. METHODS OF SYNTHESIS 2,3-Di-O-methyl-L-threose. The synthesis of 2,3-di-O-methyl-L-threose was achieved by periodate oxidation of 2,3-di-O-methyl-D-glucitol. The glucitol was prepared by the sodium borohydride reduction of 2,3-di-O-methyl-D-glucose. and Scott (49). The excess benzaldehyde present after the formation of methyl 4,6-0-benzylidene-a-D-glucoside was removed conveniently by steam d i s t i l l a t i o n . Addition of an excess of potassium carbonate to the reaction mixture to neutralize the zinc chloride and any benzoic acid formed by oxidation of benzaldehyde prevents hydrolysis of the acetal during steam d i s t i l l a t i o n . The hydrolysis of methyl 2,3-di-O-methyl-a-D-glucopyranoside to 2,3-di-O-methyl-D-glucose was extremely d i f f i c u l t and required prolonged heating with 2N. sulfuric acid. The reaction required thirty-six hours to reach completion. Thin layer chromatography (T.L.C.) of the hydrolysate, Figure 2, revealed a component running chromatographically faster than the starting material and components running slower than the hydrolysis product. 2,3-Di-O-methyl-D-glucose was synthesized by the method of Irvine Solvent front 2,3-Di-O-methyl-D-glucose Methyl 2,3-di-O-methyl -oc-D-glucopyranoside Unknown (demethyl-ation?) Figure 2. T.L.C. on s i l i c a gel, developed with butanone-water azeotrope, of methyl 2,3-di-O-methyl-ct-D-gluco-pyranoside hydrolysate. - 8 -Due to the complexity of the hydrolysis problem i t was only investigated in part. It is important to note, however, that further study of this problem should be undertaken. Hydrolysis of methylated polysaccharides and sugars has been extensively studied. One of the assumptions that must be made in polysaccharide investigations is that l i t t l e or no demethylation occurs when methylated polysaccharides are hydrolysed. Demethylation and degradation have been observed, however, and various hydrolysis schemes have been presented in order to minimize these occurrences. Hydrochloric acid, both aqueous (50) and methanolic, causes considerably more demethylation than sulfuric acid. Freudenberg and Boppel (51) found that when 2,3,6-tri-O-methyl-D-glucose was treated with concentrated hydrochloric acid at +5° considerable demethylation to a mixture of di-O-methyl-D-glucoses occurred. Smith et al (52) reported the demethylation of 1,4-di-0-methyl-erythritol to the extent of 1.3 % when refluxed for 18 hours with 3 % methanolic hydrogen chloride. Treatment with aqueous formic acid (53) is reported to result in less degradation than a mixture of acetic and hydrochloric acids. Croon and Lindberg (54) report the use of 72 % sulfuric acid is superior to both methanolysis and formolysis, causing only about 5 % degradation and demethyl-ation of less than 0.5 %. Methyl 2,3-di-O-methyl-a-D-glucopyranoside would appear to provide a good model compound for a systematic investigation of degradation and demethylation since the hydrolysis product is found in the hydrolysis of methylated glucomannans and amylopectin. The starting material and demethylation products are readily synthesized f a c i l i t a t i n g the detailed study of the demethylation and degradation products. The reduction of 2,3-di-O-methyl-D-glucose required prolonged treatment with sodium borohydride (24 h.). This finding is in agreement with the resistance reported in the reduction of 3-substituted aldoses (55) with potassium borohydride. The effect is attributed to steric hinderance of the borohydride ion as i t approaches the 1,3 system. - 9 -C(4) Figure 3. S t e r i c hinderance of substituent at C-3 to borohydride reduction. Two periodate oxidations were c a r r i e d out with 2,3-di-O-methyl-D-g l u c i t o l , an oxidation with one mole of periodate and an oxidation with excess periodate. The products from the one mole oxidation are 3,4-di-O-methyl-L-xylose and 2,3-di-O-methyl-L-threose. The oxidation with excess periodate y i e l d s 2,3-di-O-methyl-L-threose. The products from these oxidations are discussed more f u l l y i n section D-3. - 10 -)HgOH ZnCl H O V H I / O C H , 0 C H O H 0 >H / O C H . •H M e l A g 2 0 H O C H , H O x?CH 3 / 6 C H 3 mi ld >CH, -3> :H 2 OH H C W C H 3 VQCH, >CH 3 :H 2 OH H' > /|H,OH H O V C H 3 -r >CH. N a B H 4 ChUOH --OCH, hLCO--~ O H --OH C H 2 O H IQ7 C H O — O C H -H L C O — C H O H NaBK 9> C H 2 O H — O C H o K C O — t h L O H Figure 4. Synthesis of 2,3-di-O-methyl-L-threose. - 11 -2,3-pi-O-methyl-D-erythrose The synthesis of 2,3-di-O-methyl-D-erythrose was achieved by periodate oxidation of 2,3-di-O-methyl-D-mannitol. The mannitol was prepared by boro-hydride reduction of 2,3-di-O-methyl-D-mannose, see Figure 5. Methyl a-D-mannopyranoside was prepared by the method of Smith and Van Cleve.(56)'which is a modification of the Fischer method (57). The benzylidene condensation using benzaldehyde and fused zinc chloride has been used extensively for the preparation of methyl 4,6-0-benzylidene-a-Q-glucoside (58,59). The use of these reagents to prepare the corresponding methyl a-D-mannoside derivative is complicated by the cis configuration of the hydroxyls at C-2. and C-3 and the major product formed is methyl 2,3;4,6-di-0-benzylidene-a-D-mannoside (60). This d i f f i c u l t y was overcome by using a procedure pro-vided by Schwarz (61). The methyl a-D-mannopyranoside was ground to a fine powder and dissolved as rapidly as possible in 98-100 % formic acid. Benz-aldehyde was added and after five minutes the reaction mixture was poured into a solution of potassium carbonate. Steam d i s t i l l a t i o n was again used to remove the excess benzaldehyde and the methyl 4,6-0-benzylidene-a-D-mannoside was extracted from the resulting aqueous phase with chloroform. The methylation product, methyl 2,3-di-0-methyl-4,6-0-benzylidene-a-D-mannoside on hydrolysis yields 2,3-di-O-methyl-D-mannose. Reduction of the 2,3-di-O-methyl-g-mannose with sodium borohydride yielded crystalline 2,3,di-0-methyl-D-mannitol. Two periodate oxidations were carried out with the 2,3-di-O-methyl-D-mannitol in a similar manner to the 2,3-di-O-methyl-D-glucitol. Oxidation with one mole of peridate yields 3,4-di-O-methyl-Q-arabinose and 2,3-di-O-methyl-D-erythrose. Oxidation with excess periodate yields 2,3-di-O-methyl-D-erythrose. The oxidations and their products w i l l be discussed in section D-3. . . - 12 : H 2 O H Q :H 2 OH H6\9H HQ/ ' ' M e O H / H + H6VH H C ^ > C H . ZnC I , <|)CHb H C Q 2 H <J)CHO O C H , + &\OH HO/OCH, OCH< 3 H H Q / i c H A g 2 o D C H H-O C H mi ld H H 2 OH > I C H 3 H 3C ' O C H , HgOH — C "> K£H 3H 3C^H,OH H( N a B H 4 C H ? O H HCO— K £ 0 — — O H --OH C H 2 O H C H O — O C H . — O C H 3 C H 2 O H NaBH , CHpOH - - O C H , - - O C H 3 CKpH Figure 5. Synthesis of 2,3-di-O-methyl-D-erythrose. - 13 -4-O-Methyl-D-threose. The synthesis of 4-0-methyl-D-threose was accomplished by the method of Richards (47) employing minor modifications. 6-0-Methyl-D-glactose was prepared by methylating 1,2:3,4-di-0-iso-propylidene-D-galactose. Mild acid hydrolysis yielded 6-0-methyl-D-galactose (62). Methyl 6-0-methyl-Q-galactofuranosides were prepared by reaction of 6-O^methyl-D-galactose with 0.0125 % methanolic hydrogen chloride (63). The methyl glycosides were separated on a cellulose-hydrocellulose column to yield pure methyl 6-0-methyl-a-D-galactofuranoside, methyl 6-0-methyl-6-g-galactofuranoside and methyl 6-0-methyl-a-D-galactopyranoside. The glycosides were characterized by their proton magnetic resonance spectra (64) and optical rotations. The p_-nitrobenzoate derivatives were prepared from the furanosides. The methyl 6-0-methyl-a-D-galactopyranoside crystal-lized and a mixed melting point with an authentic sample was undepressed. The periodate oxidations to prepare 4-0-methyl-D-threose were per-formed on mixed galactofuranosides rather than on pure anomers. As a result, large samples of methyl glycosides could be applied to the column for chromatography. The periodate (0.0076M) consumption for the mixed furanosides was 0.91 moles after 53 hours. Although this is a much longer time than indicated by Richards (47) i t is in agreement with the findings of Hudson (65). Richards'shorter reaction time may reflect a certain amount of methyl 6-0-methyl-D-galactopyranoside. In an attempt to reduce the extensive decomposition reported (47) on hydrolysis of the dialdehyde formed from the periodate oxidation of methyl 6-0-methyl-a,B-D-galactofuranosides, a methanolysis was employed. Decomposition was s t i l l encountered,however,and the product was isolated chromatographically as the dimethyl acetal of 4-0-methyl-D-threose. Acid 14 -hydrolysis yielded 4-0-methyl-D-threose. However attempts to prepare the 2,4-dinitrophenylhydrazone were unsuccessful and the compound was characterized as the p_-nitrobenzoate of the derived polyol. - 15 -H 2 OH H C l C H 3 O H Figure 6. Synthesis of 6-0-methyl-D-galactosides C H , + H 2 O C H 3 -4-OH OH C H 2 O C H 3 2 I O ; H,OH -+OH OH C H 2 O C H 3 H 2 O C H 3 H H< 'OCH< 11 O IO4 -f-OH C H 2 O C H 3 H + O H C H 2 O C H 3 Figure 7. Modes of periodate oxidation of 6-0-methyl-D-galactosides. - 16 -104 -f-OH C H 2 O C H 3 ~S„ o t H 2 O C H 3 C H , H 3 C O H 2 H(OH) C H 2 O C H 3 A T Y P I C A L RING F O R M M e O H H 3 C O v / O C H 3 \ - H H O + — O H CH2OCH3 H" N a B H 4 C H O H O — — O H t H 2 O C H 3 N a B K -3> . C H 2 O H H O — - - O H £ H 2 O C H 3 Figure 8. Synthesis of 4-0-methyl-D-threose. - 17 -2-0-Methyl-D-erythrose. The synthesis of 2-0-methyl-D-erythrose was accomplished by periodate oxidation of methyl 4-0-methyl-a-D-mannoside. Methyl 4-0-methyl-a-D-mannoside was synthesized by the procedure of Smith (66). Attempts in this laboratory and another (67) to synthesize methyl 2,3-O-isopropylidene-a-D-mannopyranoside by the method of Ault, Haworth and Hirst (68) were unsuccessful. A modification of the benzylidene condensation provided by.Schwarz (61) was devised in a successful attempt to, synthesize this compound. Methyl a-D-mannoside was dissolved in 98-100 % formic acid and acetone was added to the reaction mixture. The formic acid was neutra-lized after several days of reaction by pouring into a saturated solution of potassium carbonate. A mixture of methyl 2,3-0-isopropylidene-a-D-.mannoside and methyl 2,3: 4,6-di-O-isopropylidene-a-D-mannoside was extracted from the aqueous phase with chloroform. Extraction of the mixture with light petroleum (B.p. 30-60°) removed the diisopropylidene derivative from the monoisopropylidene derivative. During the Kuhn (69) methylation of methyl 6-0-trityl-2,3-O-isopro-pylidene-a-D-mannoside, T.L.C. monitoring revealed detritylation in the prolonged treatment. Column chromatography was employed, therefore, in order to obtain a pure sample of methyl 4-0-methyl-a-D-mannoside. Periodate oxidation of 4-0-methyl-a-D-mannoside produced a dialdehyde which was extensively degraded in a l l hydrolysis attempts. The best results were obtained when formation of the dithioacetal was attempted. However, i n s u f f i -cient material was obtained.and characterization was not possible. The dialdehyde was reduced and hydrolysed to give 2-0-methyl-D-erythritol. - 18 -: H 2 OH o C H 3 C C H ^ H C 0 2 H 4>3CCI H2OCCt) : pyridine CH 3I C H 3 A g 2 0 H 3 C O : H 2 O H H-H , C O ^ H H V/OCH. IO4 : H 3 O H H 3C6\ CH H C / O C H 3 H C I II II o 1. N a B H 4 2. H + C H 2 O H - - O C H 3 - O H C H 2 O H * HC (SR ) 2 HSR - -OCH3 , -> _ _ 0 H + HoOH C H 2 O H H s C O ^ H H c / O C H 3 (SR)p (SR)o C d C 0 3 + HgO ^ R =CH2<|> C H O - - O C H 3 — O H C H 2 O H Figure 9. Synthesis of 2 - 0-methyl-D-erythritol, - 19 -D-1. Unknown in the hydrolysis of methyl 2,3-di-O-methyl-a-D-glucopyranoside. In the preparation of 2,3-di-O-methyl-D-erythrose, the presence of small amounts of methyl 2,3-di-O-methyl-a-D-glucopyranoside i n the2,3-di-O-methyl-D-glucitol did not i n t e r f e r e with the periodate oxidation because of i t s i n a b i l i t y to be oxidized. The larger value of the tetrose allowed i t s easy removal from the trace of glycoside. However, during the oxidation of 2,3-di-O-methyl-D-glucitol with one mole of periodate to form 3,4-di-O-methyl-L-xylose i t was necessary that no trace of methyl 2,3-di-O-methyl a-D-glucopyranoside remained, as the R^'s of the 3,4-di-O-methyl-L-xylose and the methyl 2,3-di-O-methyl-a-D-glucopyranoside are very s i m i l a r i n solvent A. The 2,3-di-O-methyl-D-glucose however, moves with nearly the same R^ as i t s methyl glycoside and i n order to c l e a r l y see that the hydrolysis had taken place the sample was reduced a f t e r hydrolysis and the slower of the p o l y o l allowed a c l e a r look at any remaining glycoside. Resubjecting a sample which had been treated i n the above manner to further hydrolysis, r e s u l t e d i n a sample which contained a unique, chromatographically fast-moving component which was not the f u r f u r a l type of compound seen i n previous hydrolyses of methyl 2,3-di-O-methyl-a-D-gluco-pyranoside. Reduction of the 2,3-di-O-methyl-D-glucose with sodium boro-hydride produced no change i n the unknown components R^ i n d i c a t i n g that there was no free hemiacetal function i n the molecule. Due to the large amount of t h i s substance ( 20 % ) , i t was decided to determine i t s structure. I s o l a t i o n was achieved by subjecting the aqueous sample of 2,3-di-O-methyl-D - g l u c i t o l to continuous chloroform extraction. The chloroform s o l u t i o n on evaporation y i e l d e d a r e l a t i v e l y pure f r a c t i o n of the unknown compound. - 20 -Proton magnetic resonance (60 mc.p.s.) studies on this product as i n i t i a l l y isolated, provided l i t t l e information because of the environmental similarity of the protons. It was not possible however,to see a proton resonance at low f i e l d which is characteristic of the anomeric hydrogen (70). This would appear to indicate that no acetal functionality existed in the. unknown component. Addition of trifluoroacetic acid shifted the hydroxyl resonances to lower f i e l d but the spectrum was not appreciably improved. Further purification of the unknown compound was achieved by acetylation and subsequent gas liquid chromatography. Acetylation was chosen for several reasons:-(a) the influence an acetate has on the position of resonance of of the hydrogen joined to the carbon whose hydroxyl has been acetylated (71); (b) the acetylation mixture of acetic anhydride and pyridine can be injected directly onto the column of the gas chromatograph without work-up of the reaction (72); (c) the acetate blocking group can be readily removed to yield the starting material (73) and .(d) a large body of information regarding the mass spectra of sugar acetates is available (74). Proton Magnetic Resonance (p.m.r.) The proton magnetic resonance spectra of the acetylated unknown at 60 and 100 mc.p.s. were considerably different to those of the unacetylated unknown. Certain of the protons, those which were situated on carbons whose free hydroxyl group had been acetylated, were shifted to lower f i e l d . The integral indicated eight hydrogen atoms in addition to two methoxyls and two acetates. This would be consistent with a loss of one mole of water from 2,3-di-O-methyl-D-glucitol to form an anhydro ring. There are several - 21 -po s s i b i l i t i e s as to the structure of such a dehydration product, an epoxide, a five membered ring or a six membered ring. Of these three p o s s i b i l i t i e s only the five membered ring is consistent with the p.m.r. spectra and the lack of chemical reaction (75). The 100 mc.p.s. proton magnetic resonance spectrum of the acetylated un-known in chloroform, disclosed a septet at T 4.76 consistent with a proton on C-5 geminal to an acetate and coupled with three hydrogens (H-6i, H-62 and H-M). The two C-6 hydrogens (H-6J and H-62) were also shifted downfield to T 5.42 and 5.87 indicating that the C-6 hydroxyl was also acetylated. The H-4 resonance at T 5.91 could be seen clearly^coupled to H-5. Irradiation of jH-S and H-6 separately confirmed the coupling between these four hydrogens. Since neither C-6 nor C-5 were involved in the formation of a ring, there • must be a furan type ring between C-l and C-4. Analysis of the ring structure can be carried no further since the environmental similarity of the remaining hydrogens introduces second order coupling and isotopic substitution would be necessary to further assign the remaining four hydrogens. The assignment of H-4, however, allows one to evaluate the configuration at C-4. The coupling constant " J " between H-4 and H-3 has a value of 3.7 c.p.s. This is consistent with an envelope conformation for a five membered ring in which C-4 is below the plane of the ring and the molecule has the gluco configuration. Figure 10. Envelope conformation of gluco configuration. - 22 -•r-H Figure 11. P.M.R. spectrum of 5,6-di-0-acetyl-l,4-anhydro-2,3-di-0-methyl-D-glucitol. -23 -(This s p l i t t i n g may be seen to occur in the 5,6 epoxide of isopropylidene-a-D-glucofuranose (76)). This is, in addition, chemically sound since formation of the 1,4-anhydride of sorbitol and mannitol proceeds with retention of configuration (77). One may also be certain that because of the relative s t a b i l i t y of the methoxyl blocking group, no migration of these functions w i l l have taken place. Hence i t is possible to assign the structure of the unknown compound as 1,4-anhydro-2,3-di-O-methyl-D-glucitol. Mass Spectra The mass spectrum of the acetylated unknown compound was determined and the fragmentation patterns are consistent with an assigned structure of 5,6-di-0-acetyl-l,4-anhydro-2,3-di-O-methyl-D-glucitol. Although'it is not possible to provide absolute identification of peaks since no labelling experiments were carried out, the volume of published spectra (74,78) allows assignments and interpretation, a l l of which are consistent with the proposed structure. In addition, i t is not possible to assign the mass spectrum to any other conceivable structure. The mass spectrum of l,4;3,6-dianhydro-2,5-di-0-methyl-D-glucitol was determined and the assignment of peaks as a result of previous work, allows one to indicate the correlation of known spectra with those of compounds not previously subjected to mass spectral analysis. The major peaks at m/e 58 and 69 can be seen to arise in other similar types of compounds in which the structure of these peaks has been assigned. The peak m/e 58 assigned as characteristic of methyl 3,6-anhydro-2,5-di-0-methyl-furanoside (78) sugars is seen to occur in the methylated dianhydrohexitol and presumably arises from the same functionality in the molecule. - 24 -TABLE 1 m/e 43 45 58 71 87 131 142 Molecular Ion Peaks f o r 5,6-di-0-acetyl-l,4-anhydro-2,5-di-O-methyl-D-glucitol Species CH3CO CH2=0-CH3 H CH3O C - CH 2 + CH30=CH-CH=.CH£ ,OCH3 Source acetylium ion. -base peak i n a i l published spectra f o r acetates. CV3, C-2 contribute to t h i s peak. C-2, C-l C-3-C-2-C-1 C-3 - C-2 AcO H -H CH-. CA loss of C-5 - C-6 C-6 —»• C-3 also M-CH3COOH, CK2=CO, MeOH Ref. (79) (80) (78) (78) (81) (82) 153 M-CH3COOH, CH30H, CH 30 184 203 M-CH3C00H, CH3OH 0 II M-CH2OCCH3 216 217 233 M-CH3COOH M-CH3COO* + M-CH3CO 244 M-CH3OH - 25 -CM ro CO-CM Si. CD" OJ co wl-o CO-CO tn- o CVI co-O O oo Figure 12. Mass spectrum of 5,6-di-0-acetyl-l,4-anhydro-2,3-di-0-methyl-• D-glucitol. - 26 -Figure 13. Source of m/e 58 peak. The peak m/e 69 assigned as c h a r a c t e r i s t i c of 1,4:3,6-dianhydro-2-0-methyl-3-Q-glucose (78) can be seen as well i n the 3,6-anhydrofu.ranoside and the 1,4:3,6-dianhydro-2,5-di-0-methyl-D-glucitol and t h i s arises from the f i v e membered anhydro rings. As mentioned, the absolute assignment of these peaks requires i s o t o p i c l a b e l l i n g - t h i s has not been c a r r i e d out. Figure 14. Source of m/e 69 peak. - 27 -TABLE 2 Molecular Ion Peaks f o r 1,4:3,6-dianhydro-2,5-di-0-methyl-D-glucitol m/e Species 58 CH3-C-C-CH2 + 59 0=CH2-CH=0 69-99 100 H 3 C O M-0CH2-CH0Me 101 142 M-CH3OH 143 M-CH30-174 M Source C 1 and C 2 C 5 and C 6 C 3 and C 4 C 1-C 4 and C 3 -C 6 C H 2 - 28 -O r - O Cvi o CM" 9 _ J O o r- CJ) Figure 15. Mass spectrum of 1,4:3,6-dianhydro-2,5-di-0-methyl-D-. g l u c i t b l . - 29 -D-2. A solution to hydrolysis problems. The extreme d i f f i c u l t y encountered in the hydrolysis of the periodate oxidized methyl 6-0-methyl-D-galactofuranosides and methyl 4-0-methyl-a-D-mannopyranoside indicates that this procedure at present cannot be used as a valuable tool in the identification of unknown par t i a l l y methylated sugar moieties. The degradation during acid hydrolysis is extreme and i t is questionable i f one could evaluate the structure of an unknown component or mixture of components using this method. It i s , however, well known that facile hydrolysis can be accomplished after reduction of the aldehydic functions formed by periodate oxidation. A problem s t i l l exists however, since one must evaluate the amount of a compound that may be present in an unknown mixture as well as its structure. The techniques needed for the" evaluation of structure and quantity are presently available and need only be applied to a series of compounds to show their general applicability. As noted, reduction of three different isomers leads to formation of the same t e t r i t o l . However, i f reduction were carried out employing sodium borodeuteride, the reduced end of the molecule would be . substituted with one deuterium. Mass spectral analysis of this compound, as i t s acetate or s i l y l ether, would clearly indicate the nature of the substitution of the deuterium in relation to the methyl ether function. Using the previous example one could find the existence of characteristic fragments. The remaining problem is the evaluation of the quantity of an unknown or known compound. The proposed procedure is determination of the molar response factor (M.R.F.) for a compound when i t is passed through the gas chromatograph. This allows one to give a quantitative estimate •of the amount of a compound in a mixture. - 30 -C H D O A c H ~ O C H 3 H — O A c CHDOAc r EXISTENCE OF THIS FRAGMENT C H D O A c H-H-- O C H 3 -OAc C H D O A c - EXISTENCE OF BOTH THESE FRAGMENTS C H D O A c H ~ O A c H—OCHo CHDOAc ISTENCE OF THIS FRAGMENT Figure 16. Characteristic fragments from labelled isomeric t e t r i t o l s . The standard procedure for determining the M.R.F. for a compound is to weigh a sample and add an internal standard to which a l l samples w i l l be related. A problem arises however, when new or unknown compounds are to be used. If the amount of material available is very small, is a syrup or a mixture, then large errors can occur as a result of inaccurate weights. For example, with a syrup i t is very d i f f i c u l t to remove a l l the solvent in which the compound was dissolved. To get around the previous problem, i t was decided to measure M.R.F. by weighing the sample eluted. The component was collected in a capillary tube after injection and resolution on the gas chromatograph. Hence the sample weight would not only give a molar response factor with regard to some internal standard, but would also give an absolute'response factor. - 31 -There are two possible sources of.error in this procedure. It is possible that degradation occurs on the column so that a specific amount is lost on each injection. This does not appear to be a significant factor with the amounts that could be measured. A twenty fold change in sample size did not show a significant fluctuation attributable to thermal degradation. The second possible source of error was the poss i b i l i t y of incomplete collection of the sample. Only in the case of the d i s i l y l ether of ethylene glycol did this factor appear to influence the amount of the sample collected. As previously mentioned,sample size variation did not affect the response factor found and this is also an indication of total collection. The response factor is calculated using the molecular weight of the sample collected and,in the case of an unknown, this would be obtained from the mass spectrum. A large percentage of the weight of sample is due to the TABLE 3 Molar Response Factors for some Sample Compounds Compound Av.R.F. Mol.Wt. . M.R.F.(abs.) as S i l y l ether (at ATT 4) gm/mole area/mole xlO 6 peak area/wt in ug Ethylene glycol 1.109 ± . 115 204 5.33 x 10~3 Butane 1,4-diol .395 ± .003 234 1.69 x 10"3 Erythritol .367 ± .015 410 0.895x lO" 3 2-0-Methyl erythritol .331 + .003 352 0.940x 10~3 4-0-Methyl threitol .303 011 352 0.861x l O - 3 * may be some loss of s i l y l ether during collection. s i l y l ethers, hence only small amounts of unknown compounds are required in order to determine the molar response factor. For example, 2-0-methyl-eryth-T.ito'1 has a'molecular weight of 136,while i t s s i l y l ether derivative has a - 32 -molecular weight of 352. Assuming that three, approximately 1 mg samples are required for the accurate determination of the M.R.F., then only 1.2 mg. of 2-0-methyl erythritol would be required. In addition, the mass spectrum could easily be obtained from one of the samples collected for the M.R.F. The presence of small or large amounts of impurity would not interfere with the determination of the M.R.F. since their weight would not be considered. The obvious extension of this technique to fields other than carbohydrates should prove invaluable to workers dealing with small quantities of compounds, where accuracy is hampered by d i f f i c u l t i e s in handling techniques, lack of pure standards or complicated reaction mixtures. D-3. Review and Discussion of Periodate Oxidation Fleury and Lange (83) pointed out that the reduction of periodic acid studied by Malaprande (84) could be considered selective for hydroxyl groups attached to adjacent carbon atoms. The carbon chain is broken and two aldehyde groups are produced. H - C - O H H - C - O H 1 H O i o ; •> + + I 0 3 + H 2 0 HO Figure 17. Periodate oxidation of adjacent hydroxyl groups. The scope of the reaction was extended to the oxidation of 1,2-di-ketones and a-hydroxy ketones by the work of Clutterbuck and Reuter (85). Nicolet and Shinn (86) extended the reaction to the oxidation of a series of a-amino acids and found that those containing an a-hydroxy-amine structure were oxidized rapidly. Compounds containing three or more hydroxyl groups on adjacent carbon atoms are oxidized by n-1 moles of periodate, where n is the number of adjacent hydroxyls, and are cleaved to yield two aldehyde groups and n-moles of formic acid. The formic acid may be estimated by ti t r a t i o n and can provide information about the arrangement of hydroxyl groups. H O •> H C 0 2 H + 2 I 0 3 + H 2 0 C H O Figure 18. Formation of formic acid. - 34 -If a primary alcohol group is involved in the oxidation, then form-aldehyde is produced. The amount may be estimated and used to determine the number of periodate oxidizable primary hydroxyls. C H 2 O H H-C-OH C H 2 0 + i o 4 + I 0 3 + H 2 0 C H O Figure 19. Periodate oxidation of a primary hydroxy1 Although the exact course of periodate oxidation is unknown, the most widely accepted interpretation is that suggested by Criegee (87,88). It is assumed that a cyclic ester is formed and the formation of a cyclic inter-mediate -has been confirmed by kinetic (89) and polarimetric measurements (90, 91). + 10J "1 > - 3 H-J>< :=o H-C-O Figure 20. Course of periodate oxidation. In agreement with the concept of the above oxidation mechanism, is the fact that the vicinal cis-hydroxyl groups in cyclohexanediol are oxidized more rapidly than the trans hydroxy 1 groups (92). Similarly,methyl aldohexo-pyranosides with cis-hydroxyl groups such as methyl cx-D-galactopyranoside and methyl a-D-mannopyranoside are oxidized faster than glycosides with trans-- 35 -hydroxyl groups, as occurs in methyl a-D-glucopyranoside (93). In cases where the trans ct-glycol groups are held rigidly apart by a fixed stereo-chemistry, as in l,6-anhydro-3-D-glucofure>nose (80) or methyl 4,6-0-benzy-lidene-a-D-altroside (89), no oxidation takes place because of the d i f f i c u l t y in forming the cyclic ester. In this work, the periodate oxidation of methyl 6-0-methyl-D-galactofuranosides proceeded at a much slower rate than normal for a periodate oxidation probably because of the steric requirements of the trans vicinal hydroxyls oxidized. Steric and perhaps electronic factors influence the rate of attack of periodate on vicinal hydroxyls. Smith and co-workers (95) have shown -that the position of cleavage of phenyl g-D-glucoside and methyl 6-0-trityl-a-D-glucoside when oxidized with one mole of periodate is subject to steric effects causing preferential cleavage between C-3, C-4 and C-2, C-3 respectively. However, interference by bulky substituents does not appear to fu l l y account for the retarded oxidative rate of the 1,6-di-O-trityl-D-fructofuranosides, as the corresponding 1,6-ditosyl derivative reacts readily and quantitatively (96). Schwarz (97),in his examination of the steric effects in the oxidation of hexitols with periodate, has shown that there is preferential attack at threo-glycol groups. This was accomplished by investigation of the intermediate com-pounds formed when galactitol (I), mannitol (II) and glucitol (III) were oxidized with 0.1 moles of periodate. For example, chromatographic examination of the oxidation products resulting from galactitol indicated formation of a tetrose (DL-threose) (oxidation at "b") while oxidation of mannitol gave mainly glyceraldehyde (oxidation at "c") and only a trace of tetrose. In addition, a trace of pentose was found, indicating some oxidation at "a". A similar oxidation of glucitol gave results suggesting comparable quantities of glycer-aldehyde and erythrose, as well as traces of xylose and arabinose. These results were confirmed by studies preformed by Courtois and Guernet (98). - 36 -CHoOH H-"HO-HO-H : -OH -OH CH-OH ••> a •> b ••> b > a HO-HO-H-<pH2OH -H -OH -OH C H 2 O H I ••> c H-H-C H 2 O H -OH -OH -OH CHgOH I •> b ••> c Figure 21. Positions of periodate oxidation i n various h e x i t o l s . Courtois and Guernet (99) extended the study made by Schwarz to include an expanded range of concentrations and compounds. The concentrations of periodate were varied from 5 to 40 % of the quantity reducible with an excess of oxidant. The compounds studied were g l y c e r o l , e r y t h r i t o l , mannitol, g l u c i t o l , g a l a c t i t o l , mannose, glucose and galactose. They found that attack on. the hexoses was more regular than attack on the p o l y o l s . Attack started p r e f e r e n t i a l l y at the reducing end and gave i n i t i a l l y the corresponding pentose. Tne degradation scheme was s i m i l a r to that of P e r l i n and Brice (100) f o r the lead tetraacetate oxidation of hexoses, i . e . hexose — > pentose — > tetrose. The small amount of formaldehyde formed i n d i c a t e d that there was l i t t l e attack between C-5 and C-6 at low concentrations of periodate. Periodate oxidation of e r y t h r i t o l lead to preferential., attack at one extremity. The glyceraldehyde producedwas then oxidized from the reducing end i n preference to the remaining e r y t h r i t o l . This sequence of oxidation was also observed for g l y c e r o l . . Oxidation y i e l d e d glyc'olic aldehyde which was subse-quently attacked at a f a s t e r rate than the i n i t i a l attack on g l y c e r o l i t s e l f . . the attack of periodate on the h e x i t o l s occurred most rapidly at the carbon-carbon bond j o i n i n g hydroxyls i n a threo configuration, as outlined . - 37 -previously. The extended range of periodate concentrations,however,allowed Courtois and Guernet to show that the composite aldehydes formed from the i n i t i a l attack were then attacked more rapidly than the polyol from which they were derived. In the present study, periodate oxidations were carried out, using one mole of periodate to oxidize 2,3-di-O-methyl-D-glucitol and 2,3-di-O-methyl-D-mannitol. The hydroxyls on C-4 and C-5 are in the erythro configuration and hence, might be expected to oxidize more slowly than the terminal C-5, C-6 position. This represents an analogous situation to the oxidation of erythritol performed by Courtois and Guernet (99). The further oxidation of the pentose •thus formed is complicated by two factors: the presence of a methyl group on C-3 and the cyclization of the aldehyde and the free hydroxyl at C-5 of the newly formed pentose to form a pyranose ring form. One might expect the oxidation of the pentose formed to occur more slowly than oxidation of the hexitol (101). The methyl ether on C-3 of the pentose might also be expected to interfere with oxidation between C-l and C-2 of the pentose in a manner similar to the steric hinderance of borohydride reduction (55). In an analogous argument, one might expect the C-3 methyl of the polyol to interfere with oxidation between C-4 and C-5. The formation of a considerable amount of pentopyranose structure prior to oxidation has been shown in this work by the formation of the corresponding formyl esters, 4-0-formyl-2,3-di-O-methyl-L-threose and 4-0-formyl-2,3-di-0-methyl-D-erythrose from the complete oxidation of the respective hexitols. Oxidation with one mole of peridate of 2,3-di-0-methyl-D-glucitol and 2,3-di-O-methyl-D-mannitol gave relatively high yields of the pentose products: 3,4-di-O-methyi-L-xylose and 3,4-di-O-methyl-D-arabinose respectively. - 38 -C H 2 O H 4-OChL H3CO-F- © CH 2 OH + O C H , H 3COf-H-OH -hOH ® CHoOH >C2 CHO + C H O t H 2 O H IO; HC0 2 H fast C H 2 0 -> KCO-f-CH 2 OH -T-OCH3 IO4 -j-OH CHO + C H 2 0 ~5> H( H,OH CH. I 0 4 H3CCH-C H O -T-OCH3 H 3C CH 20<JH O fH20 H H OCH 3 "b CHO + O C H , H3CO-T-CH 2 OH + HC0 2 H Figure 22. Periodate oxidation of 2,3-di -O-methyl -D-glucitol. - 39 -CH 2 OH H3CO-HJZO-- © -4-OH -hOH CH 2 OH >f2 C H 2 O H H 3 C O - f H 3 C O -C H O + C H O 6 H 2 O H 10; HC0 2 H fast -> -f C H 2 0 ® IO, CHoOH HoCO— H 3 C O -+ O H £HO -f C H 2 0 CHO H . C O — H X O - -CH20(Jf H O H 3 C H 3C H( H,OH CH. IQ '4 H 2 0 HH CHo X) CHO H X O - -K C O - -CH 2 OH + HC0 2 H Figure 23. Periodate oxidation of 2,3-di-O-methyl-D-mannitol, - 4 0 -... The periodate oxidation of carbohydrates can be complicated by incomplete (102) or over oxidation (103). Incomplete oxidation u s u a l l y r e s u l t s from formation of formyl esters or blockage of one of two v i c i n a l hydroxyls by the c y c l i c acetal (104) of the sugar formed. H,OH O C H 3 O C H 3 Figure 24. I n h i b i t i o n of periodate oxidations. Over oxidation r e s u l t s from the oxidation of the C-H bond of an active methylene such as occurs i n a malondialdehyde (105). H/:HO | 0 - HO/CHO 2IO4 ROH + c o 2 R - O-C > R-O-C X : H O X : HO +2HC0 2 H Figure 25. Periodate oxidation of a malondialdehyde. Over oxidation may be further complicated by the formation of elemental iodine (106,107). + 3 IO ; > HCOfeCH3 + C0 2 +3HC0 2 H + l 2 C H 3 Figure 26. Formation of elemental iodine on periodate o x i d a t i on. - 41 -The oxidation of ketoses is complicated by two different courses of oxidation (108, 109). © H-H-H-C H 2 O H <> OH OH OH t H 2 O H •>© •> (2 H-H-C H 2 0 C 0 2 H -OH OH H-I-OH C H 2 O H OH © H-H-C H 2 0 i O o H C H O OH OH C H 2 O H ^ 2 H C 0 2 H + C H 2 0 + C H O - C 0 2 H •> 3HCOoH +CHoO Figure 27. Course of periodate oxidation of ketoses. As discussed previously, periodate oxidation of pa r t i a l l y methylated sugars and sugar alcohols can lead to the formation of fragments which, i f identified, aid in the structural elucidation of the parent compound. However, one may look at periodate oxidation also as a preparative tool. While partially methylated derivatives have been prepared in this way, the preparation of D-erythrose from 4,6-0-benzylidene-D-glucitol (110), and L-xylose from 2,4-benzylidene-D-glucitol has been reported (111). Periodate oxidation has also been applied to sugar phosphates (112,113), benzyl ethers of sugars (114) and O-isopropylidene sugars (115,116). Formyl Ester - ' Measurement of the acidity produced in the periodate oxidation of carbohydrates is confined mainly to the estimation of formic acid. One of the - 42 -more d i f f i c u l t problems encountered i n obtaining a v a l i d formic a c i d assay i s the formation of formate esters. The formate i s formed from the c y c l i c , hemi-acetal structure which i s the equilibrium form of most sugars i n s o l u t i o n . The oxidation of D-glucose proceeds with consumption of three moles of periodate to an intermediate product, 2-0-formyl-D-glyceraldehyde. The rate of further oxidation i s dependent on the rate of hydrolysis of the formyl ester (117). : H 2 O H 3104 -> HoOH H O H-h O C H rate determining step in further oxidation CHoOH HPO 2 H C 0 2 H + C H 2 Q <-2 IO4 C H O H--OH C H 2 O H Figure 28. Periodate oxidation showing formate ester formation. The i s o l a t i o n of the 4-0-formyl esters of 2,3-di-O-methyl-D-erythrose and 2,3-di-O-methyl-L-threose provides further proof that oxidation occurs to a considerable extent between C-6 and C-5 of the respective h e x i t o l s . The pentoses thus formed are then oxidized, r e s u l t i n g i n the 4-0-formyl derivatives, The nature of the p o s i t i o n of the formyl proton produces a very low Tau (T) value nuclear magnetic resonance s i g n a l which allows one to confirm the presence of t h i s grouping. In addition, the s i g n a l f o r the hydrogen on the aldehyde may be observed at an even lower T value. 43 F O R M I C A C I D O. C-H CHCI3 SPINNING SIDE B A N D I l U t 0.0 1.0 2.0 Figure 29. P.M.R. spectrum of formyl and aldehyde hydrogens, Thin layer chromatography for monitoring carbohydrate reactions. One of the most useful techniques that has been developed in this laboratory is the monitoring of carbohydrate reactions with thin layer chromato-graphy (TLC) (118). Microscope slides developed in large weighing bottles allow one to rapidly follow the course of a reaction. The reaction mixture may be applied directly to the TLC plate and excess reagent may then be inactivated. For example, hydrolyses may be applied and the excess acid neutralized with a small amount of pyridine, or,when following borohydride reductions, the excess sodium borohydride is destroyed on the plate with acetic acid. One may follow virtu a l l y any reaction provided the concentration is such that a small number - 44 -o f a p p l i c a t i o n s o f t h e s o l u t i o n a r e a l l t h a t i s r e q u i r e d t o pr o d u c e a d e t e c t a b l e s p o t . The use o f a d e s t r u c t i v e s p r a y , such as 5 % n i t r i c a c i d i n s u l f u r i c a c i d , a l l o w s one t o d e t e c t a l l o r g a n i c compounds w h i c h a r e r e l a t i v e l y i n v o l a t i l e . In t h i s l a b o r a t o r y , a l l t h i n l a y e r chromatograms were r u n on s i l i c a g e l p l a t e s w h i c h were c o n d i t i o n e d a t 120°C f o r one h o u r and s t o r e d i n t h e open room. Table 4: Paper Chromatographic C h a r a c t e r i s t i c s of Known and New Tetroses, Pentoses and T e t r i t o l s . R F or R G / s o l . 2-0-Methyl-D-threose R E 0. 58/a [-28]2 3 (C, 2.0 i n H 20) 2-0-Methyl-D-erythritol R F 0. 165/a [+12°] (C, 0.8 i n H 20) R F 0.46/c 3-0-Methyl-D-threose (not characterized) 4-O-Methyl-D-threose RG 0. 80/b [+3°]1 8° (C, 2 i n H 20) 4-0-Methyl-D-threitol R F 0. 23/a [+4.7°] (C, 1.2 i n H 20) R F 0. ,51/c 2,3-Di-O-methyl-L-threose (not characterized 2,3-Di-O-methyl-L-threose R F 0. ,83/a [+12°] (C, 1.6-in MeOH) R F 0, .80/c 2,3-Di-O-methyl-D-erythrose ; R F 0 .69/a [-55.3] (C, 1.13 i n MeOH) 2,3-Di-O-methyl-L-threitol R F * unknown [+7.6°] (C, 1.4 i n MeOH) 2,3-Di-O-methylerythritol R F unknown* -2,4-Di-O-methyl-D-erythrose R F 0 . 70/a 21° [+60.1] (C, 1.4 in MeOH) R F 0 . 64/a 2,4-Di-O-methyl-L-erythrose R F 0 .70/a [-61.4°]2 1 (C, 4.9 i n MeOH) R F 0 .64/a 2,4-Di-O-methyl-D-threose R F 0 .66/a [+14.8°] (C, 1.17 i n MeOH R F 0 .57/a . 2,4-Di-O-methyl-L-threose • R F 0 .66/a [-14.3°]2 1 (C, 5.7 i n MeOH) R F 0 .57/a 3,4-Di-O-methyl-D-arabinose R F 0 . 195/a [-117°] D (C, 2.4 i n H 20) R F 0. .49/c 3,4-Di-O-methyl-L-xylose R F .55/a [-29°] D (C, 2.1 i n H 20) R F .65/c Ref. 31 R G values are r e l a t i v e to 2,3,4,6-tetra-O-methyl-D-glucose. Solvent (a) refer s to butanone-water azeotrope (b) r e f e r s to butan-l-ol-pyridine-benzene-water (4:2:1:1) (c) refers to butari-l-ol-ethanol-water (4:1:5) t Rp unknown due to the lack of a proper detection reagent. - 46 -EXPERIMENTAL Gas Liq u i d Chromatography A l l a n a l y t i c a l runs for the determination of M.R.F. were c a r r i e d out in an i d e n t i c a l manner. The gas chromatograph used was an F.and M.720 temperature programmed, dual column gas chromatograph equipped with a dis c integrator. The i n j e c t o r port was 270°C, the detector block was 290°C. The bridge power was set at 150 milliamps. The helium flow rate was 6.8 sec/10 mis. and the copper column was 8 feet (') x 1/4, packed with 20% SF-96 s i l i c o n e f l u i d on 60-80 mesh "Diataport-S". The column was run isothermally at 130°C fo r s i x minutes then programmed at 3° per minute. The programme stopped at 220°C and held at t h i s point. The gas chromatograph was employed r o u t i n e l y to analyse reaction mixtures, prepare samples f o r the mass spectrometer and determine p u r i t y of prepared samples. Samples were examined as acetates, s i l y l ethers and methyl ethers, generally using SF-96 as the l i q u i d phase. Preparative G.L.C. was performed using an 8' by 1/2 inch column packed with .20% SF-96 on 80-100 mesh "Diataport S". S i l y l a t i o n of Samples for Gas Chromatography Samples s i l y l a t e d f o r separation on the gas chromatograph were pre-pared by the method of Sweeley et a l . (119) with some increase i n the concen-t r a t i o n of sugar depending on the number of free hydroxy1 groups. Removal of the s i l y l ethers from the separated products was accomplished by r e f l u x i n g the sample i n methanol-water followed by evaporation under reduced pressure to obtain the s t a r t i n g material. Periodate Oxidations The course of periodate oxidations was followed by p i p e t t i n g samples into an excess of pH 6.98 buff e r (120) and s u f f i c i e n t 10% potassium iodide to - 47 -reduce a l l periodate to iodate. The liberated iodine was titrated with standardized sodium thiosulfate' using starch as the indicator. Evaporations were carried out under reduced pressure at a bath temperature of 35-45°. Optical rotations were equilibrium values measured on either a Bendix ETL-NPL.Automatic Polarimeter (Type 143 A) or a Rudolph Polarimeter(Model 219) at 21 + 2°. Melting points are uncorrected. Mass spectra were obtained at 70 eV on an Atlas MS-9 Mass Spectrometer. Synthesis of 2,3-di-O-methyl-L-threose Methyl 2,3-di-O-methyl-a-D-glucopyranoside was synthesized by the method of Irvine and Scott (49). The preparation of the benzylidene compound has been modified to avoid losses in the work-up of the reaction mixture. Methyl 4,6-benzylidene-a-D-glucoside Methyl a-D-glucopyranoside (144 g.) was added to a mixture of finely powdered, fused zinc chloride (116 g.) and freshly d i s t i l l e d benz-aldehyde (420 g.) and the mixture was shaken for ninety (90) hours at room temperature. The reaction was monitored by running thin layer chromatograms. The solution was poured in a fine stream into rapidly stirred ice and water (2 1.). The benzylidene compound and the benzaldehyde separated as a slurry. The ice water was decanted and sufficient sodium carbonate was added to neutralize any remaining zinc chloride and render the solution slightly basic. The resulting mixture was transferred to a round bottom flask and the benz-aldehyde was removed by steam d i s t i l l a t i o n . Insoluble zinc carbonate was f i l t e r e d from the hot solution and the methyl 4,6-0-benzylidene-a-D-glucoside crystallized from the cooled f i l t r a t e . The.product was recrystallized successively from water and 65-110° petroleum, (178.5 g.) yield 85% m.p. 163-164° [ a ] D +115.3 (c, 1.2 in CHC13). Lit.(121), m.p. 161-162°, [a]^+117-.5-(C, 1 in CHCI3). - 48 -Methyl 2,3-di-O-methyl-a-D-glucopyranoside Methyl 2,3-di-O-methyl-a-D-glucopyranoside was r e c r y s t a l l i z e d from ethyl acetate-petroleum ether (65-110°) m.p. 84-85°. [ a ] D +142.9 (C_, 2.5 i n EtOH). L i t . (122) m.p. 83-85°. [ a ] Q +143.1 (C, 5 i n EtOH). 2,3-Di-O-methyl-D-glucitol The hydrolysis of methyl 2,3-di-O-methyl-a-D-glucopyranoside r e s u l t e d i n some decomposition as discussed i n "Methods of Synthesis". As a r e s u l t i t was not possible to c r y s t a l l i z e 2,3-di-O-methyl-D-glucose. Recently however, a sample of 2,3-di-O-methyl-D-glucose st a r t e d to c r y s t a l l i z e spontan-eously and i t has been possible to i s o l a t e the 3 anomer of 2,3-di-O-methyl-D-glucose. R e c r y s t a l l i z a t i o n from anhydrous acetone gave 2,3-di-0-methyl-8-D-glucose m.p. 112° [ a ] Q +6.4'—> 59.6° (C, 2.1 i n H 20). L i t . (49) m.p. 110° [a] +10.6 +64.4° (C, 5.0 i n H 20) . Non-crystalline 2,3-di-O-methyl-D-glucose (3.0 g.) was dissolved i n water (50 ml.) and sodium borohydride (0.6 g.) was added. The so l u t i o n was ne u t r a l i z e d with a c e t i c acid a f t e r 36 hours. The s o l u t i o n was passed through a column of Amberlite IR-120 (H +) r e s i n to remove sodium ions. The eluant was evaporated to dryness under reduced pressure and the s o l i d (boric acid) and syrup were dissolved i n methanol and evaporated to dryness three times. The syrupy non-reducing product (3.1 g.) could" not be induced to c r y s t a l l i z e , [ a ] D + 1 5 (C, 3.0 i n H 20) . L i t . (123) [ a ] D +13 (C, 2.54 i n H 20). 2,3-Di-O-methyl-1,4,5,6-tetra-0-p_-nitrobenzoyl-Dr g l u c i t o l 2,3-Di-O-methyl-D-glucitol (100 mg.) was dissolved i n anhydrous pyridine and f r e s h l y d i s t i l l e d p-nitrobenzoyl chloride (400 mg.) was added. The reaction mixture was heated on a steam bath f o r one hour a f t e r which a few drops of water were added. The mixture was allowed to cool f o r f i f t e e n minutes. The cool s o l u t i o n was poured into a s t i r r e d saturated s o l u t i o n of - 49 -sodium bicarbonate. The reaction product, 2,3-di-0-methyl-l,4,5,6-tetra-0-p_-nitrobenzoyl-D-glucitol separated as a yellow gum (453 mg.) which was crystallized from acetone-methanol, m.p. 88-91°. Found: C, 52.3: H, 3.8. C 3 5H 3 0O 1 8N t f requires C, 52.0; H, 3.6 %. 2,3-Di-O-methyl-L-threose To non-crystalline 2,3-di-O-methyl-D-glucitol (2.02 g.) in water (1000 ml.) was added three molar equivalents of sodium metaperiodate (6.12 g.). The oxidation was allowed to proceed for 4 hours. The excess periodate and the iodate produced were removed by the addition of an excess of 10% barium acetate. The solution was fi l t e r e d and then de-ionized with Amberlite IR-120 (H+) and Duolite A-4 (OH ). The neutral solution was evaporated to give a mobile syrup (1.35 g.) containing two components R„ 0.67 and 0.59 r solvent A, T.L.C. Attempts to separate the formyl ester component on a cellulose-hydrocellulose column using solvent A were unsuccessful due to ester hydrolysis. The syrup was dissolved in water and one drop of pyridine was added. The product was evaporated to yield pure 2,3-di-O-methyl-L-threose, [a] D +12° (c, 1.6 in MeOH). Found: C, 48.4; H, 8.3. C 6H 1 20 4 requires C, 48.6; H, 8.2 %. • 2,3-Di-O-methyl-L-threitol 2,3-Di-O-methyl-L-threose (100 mg.) was treated with excess sodium borohydride (200 mg.) After 16 hours, the excess borohydride was.decomposed with a slight excess of acetic acid. The solution was deionized by passing through a column of Amberlite IR-120 (H +). Evaporation of the resulting solution followed by evaporation of methanol (10 ml.) three times removed boric acid, yielding a colourless syrup (65 mg.) of 2,3-di-O-methyl-L-threitol. [a] +76° (c, 1.4 in MeOH) . Found: C, 48.2; H, .9.5. CaH^O^ requires C, 48.0; H, 9.3 %. - 50 -2.3- Di-0-methyl-l,4-di-0-p_-nitrobenzoyl-L-threitol A portion of the 2,3-di-O-methyl-L-threitol (30 mg.) was dissolved in pyridine (1 ml.) and treated with p_-nitrobenzoyl chloride (100 mg.) in the usual manner, to give 2,3-di-0-methyl-l,4-di-0-p_-nitrobenzoyl-L-threitol. m.p.. 143-144°. Found: C, 53.3; H, 4.6. C 2 0H 2 0O 1 0N 2 requires C,'53.6; H, 4.5 1.4- Anhydro-2,3-Di-O-methyl-D-glucitol A mixture of methyl 2,3-di-O-methyl-a-g-glucopyranoside arid 2,3-di O-methyl-D-glucitol (3 g.) was treated with 2N. sulfuric acid (100 ml.) at 100° for 24.hours. The reaction was neutralized with barium carbonate and the neutral product was f i l t e r e d and subsequently deionized by passing through successive columns of Amberlite IR-120 (H+) and Duolite A-4 (OH ). The neutral solution was reduced with an excess of sodium borohydride for 24 hours. The excess borohydride was destroyed with acetic acid. Sodium ions were removed by passing the solution down a column of Amberlite IR-120 (H+) and the boric acid was removed by evaporation to dryness of the water solution followed by three evaporations of methanol solutions. The 1,4-anhydro-2,3-di-O-methyl-D-glucitol was removed from the 2,3-di-O-methyl-D-glucitol by continuous chloroform extraction of an aqueous solution of the mixed polyols. The chloroform solution was evaporated under reduced pressure to give a highly enriched fraction of l,4-anhydro-2,3-di-O-methyl-D-glucitol (0.8 g.). The enriched fraction was acetylated using acetic anhydride (2 ml.) in pyridine (10 ml.). The reaction after 16 hours was concentrated under reduced pressure to a volume of approximately 3 mis. This solution was subjected to separation on the preparative G.L.C. column to give a pure fraction of 5,6-di-0-acetyl-l,4-anhydro-2,3-di-O-methyl-D-glucitol (0.5 g.) - 51 -A portion of the 5,6-di-0-acetyl-l,4-anhydro-2,3-di-0-methyl-I> glucitol (0.2 g.) was deacetylated using a trace of sodium methoxide in methanol. The sodium ions were removed by treatment of the methanol solution with Amberlite IR-120 (H+) and water. The solution was evaporated under reduced pressure to give syrupy l,4-anhydro-2,3-di-O-methyl-D-glucitol (0.13). Found: C, 49.9; H, 8.4. C 8H 1 60 5 requires C, 50.0; H, 8.4%. l,4-Anhydro-2,3-di-0-methy 1-5,6-di-0-p_-nitrobenzoyl-D-glucitol A portion of the pure l,4-anhydro-2,3-di-O-methyl-D-glucitol (0.05 g.) was dissolved in pyridine (1.0 ml.) and treated with p_-nitrobenzoyl chloride (150 mg.) in the usual manner, to give l,4-anhydro-2,3-di-0-methyl-5,6-di-0-p_-nitrobenzoyl-D-glucitol.. m.p. 114°. Found: C, 53.5; H, 4.7; N, 5.5. c 2 i + H 2 2 N 2 ° l l requires C, 53.85; H, 4.5; N, 5.7%. Synthesis of 2,3-di-O-methyl-D-erythrose Methyl a-D-mannopyranoside D-Mannose'(180 g.) was added to 3 % methanolic hydrogen chloride (150 ml.) and ethylene dichloride (300 ml.). The mixture was refluxed on a steam bath for six hours. During the course of the reaction, a two phase liquid system formed, the lower layer of which formed a crystalline mass. The cooled mixture was fi l t e r e d and washed with a l i t t l e ice-cold methanol, followed by ether. The mother liquors and methanol washings were evaporated to dryness under reduced pressure and fresh 3 % methanolic hydrogen chloride (75 ml.) and ethylene dichloride (150 ml.) were added. The mixture was refluxed for a further three hours and a second crop of crystals was isolated. The methyl a-D-mannopyranoside (154 g.) was recrystallized from ethanol water, m.p. 190-191°. [a] +77.3 ( c , 1.2 in H20) L i t . (56) m.p. 191-192° [a]'n + 79.0 ( c , 1.0 in H20) . - 52 -Methyl 4,6-0-benzylidene-a-D-mannopyranoside Finely powdered methyl a-D-mannopyranoside (50 g.) was dissolv e d as r a p i d l y as possible i n 98-100 % formic acid (250 ml.) and f r e s h l y d i s t i l l e d benzaldehyde (250 ml.) was immediately added to the sol.ution. A f t e r being allowed to stand f i v e minutes, the s o l u t i o n was poured with s t i r r i n g into a mixture of water (11.) and anhydrous potassium carbonate (685 g.). The excess benzalde-hyde was removed by steam d i s t i l l a t i o n of the r e s u l t i n g s o l u t i o n . The aqueous phase was extracted with chloroform in. a continuous extractor. The methyl 4,6-O-benzylidene-a-D-mannoside (23.0 g.) was r e c r y s t a l l i z e d from benzene, m.p. 143-145°. L i t . (124), m.p. 147-148°. Methyl 4,6-0-benzylidene-2,3-di-O-methyl-a-D-mannopyranoside Methyl 4,6-benzylidene-a-D-mannopyranoside (5 g.) was dissolved i n dimethyl formamide (50 ml.) and methylated by Kuhn's method (69) using methyl iodide (20 ml.) and s i l v e r oxide (20 g.) added i n portions three times during the reaction, at the s t a r t , at 8 hours and at 32 hours. Methyl 4,6-benzy-lidene-2,3-di-O-methyl-a-D-mannopyranoside (4.3 g.) was i s o l a t e d as a syrup which could not be induced to c r y s t a l l i z e . [ a ] ^ +60.7 ( c , 2.4 i n CHC13) . L i t . (124), [ a ] D +62.7 ( c , 1.178 i n CKC1 3). 2,3-Di-O-methy1-D-mannitol Acid hydrolysis of methyl 4,6-benzylidene-2,3-di-0-methyl-a-D-mannopyranoside (3.0 g.) followed by reduction with sodium borohydride y i e l d e d c r y s t a l l i n e 2,3-di-O-methyl-D-mannitol ).1.8 g.). The poly o l was r e c r y s t a l l i z e d from ethyl acetate, m.p. 101-102°. L i t . (125),m.p. 101-103°. 2,3-Di-O-methyl-D-erythrose To 2,3-di-O-methyl-D-mannitol (1.00 g.) in water (500 ml.) was - 53 -added three molar equivalents of sodium metaperiodate (3.06 g.). The oxidation was allowed to proceed for four hours. The reaction was worked up in the usual manner with barium acetate. The neutral solution was evaporated to give a mobile syrup (885 mg.) containing two components, solvent A. (TLC S i l i c a gel). The faster running 4-0-formyl-2,3-di-O-methyl-D-erythrose could be converted to the slower component, 2,3-di-O-methyl-D-erythrose by treatment with mild base. The pure 2,3-di-O-methyl-D-erythrose was separated as a mobile syrup from trace impurities by column chromato-graphy on a cellulose-hydrocellulose column using solvent A. [a] D -55.3° ( c , in MeOH). Found: C, 48.4; H, 8.3. CgH^O^ requires C, 48.6; H, 8.2%. 2,3-Di-O-methylerythritol A sample of 2,3-di-O-methyl-D-erythrose (50 mg.) was reduced with sodium borohydride (100 mg.). The reaction was worked up in the usual manner after 16 hours. Evaporation of the neutral solution gave 2,3-di-0-methyl-erythritol (35 mg.). Found: C, 48.3; H, 9.25. CgH^O^ requires C, 48.0; II, 9.4 %. 2,3-Di-O-methyl-1,4-di-0-p_-nitrobenzoylerythritol Treatment of a portion of the syrupy 2,3-di-0-methylerythritol (25 mg.) with p_-nitrobenzoyl chloride (100 mg.) in the usual manner gave 2,3-di-0-methyl-l,4-di-0-p-nitrobenzoylerythritol. m.p. 178°. Found: 53.5; H, 4.4. C 2 0H 2 0O 1 0N 2 requires C. 53.55;.H, 4.5 %. Synthesis of 2-0-Methyl-D-erythritol Methyl 2,3-isopropylidene-a-D-mannoside. Methyl a-D-mannopyranoside (25 g.) was dissolved in 98-100 % formic acid (100 ml.) and acetone (750 ml.) was added. The reaction was allowed to proceed for fourteen days. "Drierite" (30 g.) drying agent was added on the - 54 -fourth day to aid i n maintaining anhydrous conditions. Undissolved methyl a-D-mannopyranoside and " D r i e r i t e " were removed by f i l t r a t i o n . The s o l u t i o n was n e u t r a l i z e d by pouring into a s l i g h t excess of 15 % sodium hydroxide. The aqueous mixture formed two layers and the excess acetone was removed by evaporation under reduced pressure. The s o l u t i o n was extracted with chloro-form (3 x 5,00 ml.) and ethyl ether (3 x 500 ml.). The extracts were combined and evaporated to y i e l d a syrup which was dissolved i n water. Extraction of the water with 30-60° petroleum (3 x 300 ml.) removed a l l the methyl 2,3;4,6-di-O-isopropylidene-a-D-mannopyranoside. The aqueous phase was evaporated under reduced pressure to y i e l d a syrup. The syrup was dissolved i n hot et h y l acetate and on cooling methyl 2,3-0-isopropylidene-a-D-mannopyranoside (10.92 g.) c r y s t a l l i z e d out. m.p. 107-108°. L i t . (68), m.p. 105°. Methyl 4-0-methyl- -D-mannopyranose • . . Methyl 4-0-methyl-a-D-mannopyranoside was prepared by the method of Smith (66). There was however, d e t r i t y l a t i o n during'the methylation and the methanolysis product was p u r i f i e d by column chromatography on a c e l l u l o s e -hydrocellulose column using solvent A. Methyl 4-0-methyl-a-D-mannopyranoside c r y s t a l l i z e d from butanone on prolonged standing m.p. 101-102°. L i t . (126), m.p. 101-103°. 2-0-Methyl-D-erythritol Methyl 4-0-methyl-a-D-mannopyranoside (0.8 g.) i n water (200 ml.) was oxidized with 1.5 mole equivalents of sodium metaperiodate (1.2 g.) f o r twenty-four hours. The reaction was worked up with barium acetate i n the usual manner. The neutral s o l u t i o n was'reduced with an excess of sodium borohydride f o r 24 hours and the excess borohydride was destroyed with a c e t i c a c i d . Sodium ions were removed by passing the s o l u t i o n down a column of Amberlite IR-120 (H +), the b o r i c a c i d was removed by repeated d i s t i l l a t i o n s ' - 55 -of methanol. Mild acid hydrolysis of the hemiacetal gave a sample of 2-0-methyl-D-erythritol (0.39 g.) [ a ] D +12° (c, 0.8 in H 20). Found: C, 44.2; H, 8.8. C5H120^ requires C, 44.1; H, 8.9 %. 2-0-Methyl-l, 3,4-tri-0-p_-nitrobenzoyl-D-erythritol Treatment of a portion of 2-0-methyl-D-erythritol (50 mg.) with p_-nitrobenzoyl chloride (200 mg.) in pyridine (2 ml.) in the usual manner gave 2-0-methyl-l,3,4-tri-0-£-nitrobenzoyl-D-erythritol m.p. 218-220° mixed m.p. with authentic sample 219-220°. Synthesis of 4-0-methyl-D-threose 6-0-Methyl-D-galactose . . - • 6-0-Methyl-D-galactose was synthesized by the methylation of diacetone galactose followed by mild acid hydrolysis. After recrystallization from ethanol i t had the following constants, m.p. 128° [a]^ +115° ( c , 2.1 in H 20). L i t . (62), m.p. 128° [ a ] D +114° ( c , 2.1 in H20) . Methyl 6-0-methyl-a-D-galactofuranoside Methyl 6-0-methyl-g-D-galactofuranoside Methyl 6-0-methyl-a-D-galactopyranoside A two.percent methanolic solution of 6-0-methyl-g-galactose (5 g.) containing 0.012 to 0.013 per cent hydrogen chloride was refluxed 18 hours until the free sugar content was very low, as indicated by tests on a sample with Fehling's solution. The hydrogen chloride was removed by the addition of silve r oxide. The f i l t e r e d solution was evaporated to dryness and chromato-graphed on a cellulose-hydrocellulose column with solvent A to yield three fractions. Fraction one, (2.475 g.) methyl 6-0-methyl-B-D-galactofuranoside [ a ] D = 117° ( c , 4.7 in MeOH). Fraction two, (0.735 g.) methyl 6-0-methyl-a-D-galactofuranoside. [ a]' n' + 9 5 ° (c-» 1 - 9 ^ i n MeOH). Fraction three (0.151 g.) methyl 6-0-methyl-a-D-galactopyranoside, m.p. 132-134° mixed m.p. with - 56 -authentic sample m.p. 132-134°. Methyl 6-0-methyl-2,3,5-tri-0-p_-nitrobenzoyl- a-D'-galactofuranoside Treatment of methyl 6-0-methyl-a-D-galactofuranoside (50 mg.) with p_-nitrobenzoyl chloride in the usual manner gave methyl 6-0-methyl-2,3,5-tri-0-p_-nitrobenzoyl-a-D-galactofuranoside. m.p. 107-110°. Found: C, 53.1; H, 3.8. C 2 9 H 2 5 O 1 5 N 3 requires C , 53.2; H, 3.85 %. . Methyl 6-0-methyl-2,3,5-tri-0-p-nitrobenzoyl- g-D-galactofuranoside Treatment of methyl 6-0-methyl-B-D-galactofuranoside (50 mg.) with p_-nitrobenzoyl chloride i n the usual manner gave methyl 6-0-methyl-2,3 35-tri-0-p_-nitrobenzoyl-6-D-galactofuranoside. m.p. 82-85°. Found C, 53.0; H, 3.75 %. 4-O-Methyl-D-threose-dimethylacetal Methyl 6-0-methyl-ag-D-galactofuranoside (3.0 g.) was oxidized with 1.5 mole equivalents of sodium metaperiodate (75 mls.^0.3M) i n water (1.2 1.). The reaction was allowed to proceed f o r three days. Excess sodium metaperiodate and iodate were removed in the usual manner with barium acetate and Amberlite IR-120 (H +). Evaporation of the deionized s o l u t i o n gave 2.873 g. of the periodate oxidized product. This mixture was subjected to a methanolysis with 3 % methanolic hydrogen chl o r i d e f o r 24 hours and no slow moving component on TLC (solvent A ) remained. Chromatography of t h i s mixture (1.67 g.) on the c e l l u l o s e - h y d r o c e l l u l o s e column with solvent A gave a f r a c t i o n (0.63 g.) with properties consistant with the dimethyl acetal of 4-0-methyl-D-threose, having three methoxyl and two free hydroxyls i n i t s N.M.R. spectra. [a] +5 ( c , 0.8 in MeOH). - 57 -4-0-Methyl-D-threose Hydrolysis of t h i s f r a c t i o n with d i l u t e sulphuric acid (.5N) gave 4-0-methyl-Q-threose. [ a ] n +5°(c, 1,3 i n H20). Lit.(47), [ o ] n +3° (c.,2 i n H 20). 4-0-Methyl-D-threitol Reduction of a portion of periodate oxidized furanosides (1.0 g.) with sodium borohydride, followed by mild acid hydrolysis gave a sample of 4-0-methyl-D-threitol. [a] +4.7° ( c , 1.2 i n H 20), Found: C, 44.1; H, 8.7. C s H ^ O i j requires C, 44.1; H, 8.9%. 4-0-Methyl-l,2, 3-tri-0-p_-nitrobenzoyl-D-threitol Treatment of 4-0-methyl-D-threitol(50 mg.) i n pyridine (1 ml.) with p_-nitrobenzoyl chloride (150 mg.) i n the usual manner gave 4-0-methyl-l,2,3-tri-O-p-nitrobenzoyl-D-threitol m.p. 129°. Found: C, 53.3 H, 3.6. C 2 6H 2 10 1 3N 3 requires C, 53.5; H, 3.6. Controlled periodate oxidations Controlled periodate oxidations were performed on 2,3-di-O-methyl-D-g l u c i t o l and on 2,3-di-O-methyl-D-mannitol. One mole of sodium metaperiodate was allowed to react completely with each h e x i t o l . 3,4-Di-O-methyl-L-xylose 2,3-Di-O-methyl-D-glucitol (1.11 g.) was added to a solution (175 ml.) containing one mole equivalent of sodium metaperiodate (1.12 g.) and allowed to react for twenty-four hours. Removal of the ions i n the usual manner yielded a neutral solution which on T.L.C. in solvent A showed one major and three lesser spots. The major spot was i s o l a t e d by column chromatography, Solvent A,to give a f r a c t i o n , 3,4-di-O-methyl-L-xylose (0.74 g.) which could not be induced to crystallize. [ a ] D -14.6° ( c , 2.1 in MeOH). L i t . (128) 0 isomer +13° (c., 1.6 in MeOH). Methyl 3,4-di-O-methyl-a-L-xyloside A portion of the 3,4-di-O-methyl-L-xylose (2.0 g.) was refluxed with 3% methanolic hydrogen chloride for twenty-four hours. The solution was neutralized with silve r oxide and evaporated to give a syrup of the two possible glyosides. The glycosides were then purified by preparative G.L.C. to give a crystalline sample of methyl 3,4-di-O-methyl-a-L-xylose, m.p. 87-89°. L i t . (127), m.p. 89-90°. Found: OMe, 48.15. C 8H 1 50 5 requires OMe, 48.35 %. 3,4-Di-O-methyl-D-arabinose 2.3- Di-O-methyl-D-mannitol (1.15 g.) was added to a solution containing one mole equivalent of sodium metaperiodate and allowed to react for twenty-four hours. The neutral solution obtained by the usual work-up showed on T.L.C. chromatography in solvent A one major and three lesser spots. The major spot was isolated by column chromatography using solvent A to give a fraction, 3,4-di-O-methyl-D-arabinose (0.83 g.) which could not be induced to crystallize. [ a ] D -118° ( c , 1.3 in H20) . L i t . (128) [ a ] D + 125° ( c , 2.4 in H20) (L-isomer) . 3,4-Di-O-methyl-D-arabonamide 3.4- Di-O-methyl-D-arabinose (50 mg.) was oxidized four days with bromine water. The lactone was recovered in the usual manner. 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