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UBC Theses and Dissertations

I. Reactions of omega-linked disaccharides. II. Synthesis of the 2,4-di-O-methyl Slessor, Keith Norman 1964

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I. REACTIONS OF o£-LINKED DISACCHARIDES II. SYNTHESIS OF THE 2,4-DI-O-METHYL TETROSES by KEITH NORMAN SLESSOR B.Sc., The University of B r i t i s h Columbia, 1960 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of CHEMISTRY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH August, 1964 COLUMBIA In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Bri t i sh Columbia, I-agree that the Library shall make i t freely available for reference and study* I further agree that per-mission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publi-cation of this thesis for financial gain shall not be allowed without my written permissions. V Department of The University of Bri t i sh Columbia, Vancouver 8, Canada The Uni v e r s i t y of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR. THE DEGREE OF DOCTOR OF PHILOSOPHY B.Sc, The University of B r i t i s h Columbia, 1960 TUESDAY3 SEPTEMBER 15th, 1964, AT 10i30 A.M. IN ROOM 261, CHEMISTRY BUILDING COMMITTEE IN CHARGE Chairman: I. McT. Cowan External Examiner: Professor J.K.N. Jones F.R.S. of KEITH NORMAN SLESSOR G.G.S, Dutton L.D. H a l l J.P. Kutney C,A. McDowell T„ Money A. Rosenthal G.M„ Tener J. Trotter Queens U n i v e r s i t y , Kingston I. Reactions of <X-Linked Disaccharides I I . Synthesis of the 2,4-Di-O-Methyl. Tetroses ABSTRACT L Reactions ofc<-Linked Disaccharides Through reaction of s p e c i f i c a l l y substituted maltoses, c<-glucosidic disaccharide derivatives have been pre-pared. C a t a l y t i c oxidation of benzyl^-maltoside yielded maltobiouronic a c i d . T r i t y l a t i o n of 1,6-anhydro-"maltose made possible the preparation of the 6'.-0= tos y l ester. Replacement of the tosylate with azide ion followed by reduction and hydrolysis y i e l d e d a small amount of 6 ' --amino^ .6 ' deoxy^maltose. Replacement of the t o s y l a t e with t h i o l a c e t a t e allowed the prepara-t i o n of 6'-deoxy-6 1-mercapto-maltose. Iodide replace-ment: of the sulphonyl ester followed by reduction gave the 6 1-deoxy-1,6-anhydro d e r i v a t i v e which was converted to 6 1-deoxy-maltose by a c e t o l y s i s and deacetylation. A route for the preparation of 4^0- (oC-D-glucopy-ranosyluronic acid)-D^xylose by s e l e c t i v e decarboxyla-t i o n of maltosyldiuronic acid was .attempted and found unfeasible. Attempts to prepare 6-substituted maltoses by reaction of benzyl. 4', 6 '-0~benzylidene- jS-maltoside with various reagents were unsuccessful. I I . Synthesis of the 2,4-Di-O-Methyl Tetroses The four isomeric 2,4-di-O-mefchyl tetroses were pre-pared by periodate oxidation o f known methylated sugars. 2,4-.Di-0-methyl-D"and L~erythroses were prepared from 4,6-di-O-methyl-D-glucose and 3j5-di-0-methyl-L-arabinose r e s p e c t i v e l y . 2,4-Di-0-methyl-D»and L-threoses were prepared from 3,5-di-0~methyl~D-xylose and 1,4,6-tri-O-methyl-L-sorbose. The tetroses were characterized as t h e i r c r y s t a l l i n e 2,4-di.nitrophenylhydrazones. The Rf and RQ values of the free sugars were recorded i n a v a r i e t y - o f solvents including a s i l i c a gel thin-layer chromatography system. GRADUATE STUDIES F i e l d of Study; Organic Chemistry Topics i n Organic Chemistry Carbohydrate Chemistry Physical Organic Chemistry Reaction Mechanisms Natural Products Newer Synthetic Methods Polysaccharide Chemistry J.P. Kutney D,E= McGreer R,E, Pincock G,G,S, Dutton L,D. Hayward A. Rosenthal R. Stewart R.E. Pincock A.I. Scott G.CS, Dutton A. Rosenthal G , G o S . Dutton Related Studies; Topics i n Physical Chemistry Topics i n Inorganic Chemistry Crystal Structure A, Bree J.R. Coope R0 Snider H. Bartlett: H.C. Clark W.R. Cullen N„ B a r t l e t t S„ Melzak J . T r o t t e r PUBLICATIONS S.A, Black, G.G.S. Dutton and K.N. Slessor, "Synthetic Disaccharides", Advances i n Carbohydrate Chemistry, accepted for p u b l i c a t i o n . G.G.S. Dutton and K.N. Slessor, "Synthesis of the 2,4,Di.-0-Methyl Tetroses", Canadian Journal of Chemistry, XLII (1964), pp. 614-619. G.G.S. Dutton and K.N. Slessor, "Synthesis of Maltobiouroni.c Acid (4-0(6<cD-Glucopyranosyl-uronic Acid)~D-gl ucose)", Canadian Journal of Chemistry, XLII (1,964), pp.11.10-1112. - i i -ABSTRACT Chairman: Professor G. G. S. Dutton I. REACTIONS OF ©<-LINKED DISACCHARIDES Through reaction of s p e c i f i c a l l y substituted maltoses, <X-glucosidic disaccharide derivatives have been prepared. Catalytic oxidation of benzyl ^-maltoside yielded maltobiouronic acid. T r i t y l a t i o n of 1,6-anhydro- f§ -maltose made possible the preparation of the 6*-0-tosyl ester. Replacement of the tosylate with azide ion followed by reduction and hydrolysis yielded a small amount of 6'-amino-61-deoxy-maltose. Replacement of the tosylate with thiolacetate allowed the preparation of 6 1-deoxy-6 1-mercapto-maltose. Iodide replacement of the sulphonyl ester followed by reduction gave the 6'-deoxy-l,6-anhydro derivative which was converted to 6 1-deoxy-maltose by acetolysis and deacetylation. A route f o r the preparation of 4-0-(0< -D-glucopyranosyl-uronic acid)-D-xylose by selective decarboxylation of maltosyldiuronic acid was attempted and found i n f e a s i b l e . Attempts to prepare 6-substituted maltoses by reaction of benzyl 4 1,6 1-O-benzylidene- ^ -maltoside with various reagents were unsuccessful. - i i i -I I . SYNTHESIS OF THE 2,4-DI-O-METHYL TETROSES The four isomeric 2,4-di-0-methyl tetroses were prepared by periodate oxidation of known methylated sugars. 2,4-Di-0-methyl-D-and L-erythroses were prepared from 4,6-di -0 -methyl-D-glucose and 5,5-di-0-methyl-L-arabinose respectively. 2,4-Bi-O-methyl-D- and L-threoses were prepared from 3,5-di-O-me thyl-D-xylose and 1,4,6-tri-O-methyl-L-sorbose. The tetroses were characterized as their c r y s t a l l i n e 2,4-dinitrophenylhydrazones. The Bj. and R Q values of the free sugars were recorded in a variety of solvents including a s i l i c a gel thin-layer chromatography system. - i v-TABLE OP CONTENTS I. REACTIONS OF -LINKED DISACCHARIDES Page No. INTRODUCTION 1 Purpose 1 Chemical Synthesis of c>_-Glucosidic Disaccharides 3 Koenigs-Knorr Synthesis 3 Inversion at Carbon 2 5 Condensation via 1,2-Anhydro Rings 5 Anomerization 7 Modification Reactions 8 Protection of the Reducing Function 9 Use of Anhydro Rings 12 Use of Esters 13 Use of Ethers 17 Use of Acetals and Ketals 20 Epimerizations and Inversions 21 METHODS OF SYNTHESIS 23 Maltobiouronic Acid (4-O-(0C-D-Glucopyranosyluronic Acid)-D-glucose 23 4-0-(oC-D-Glucopyranosyluronic Acid)-D-xylose 25 4-0-(6-Amino-6-deoxy- o£-D-glucopyranosyl)-D-glucose 28 4-0-(6-Deoxy-6-mercapto-6C-D-glucopyranosyl)-D-glucose 32 4-0-(6-Deoxy-o£-D=glucopyranosyl)-D-glucose 34 4-0-( 3-Amino-3-deoxy-<X.-D-glycopyranosyl)-D-glucose 36 Attempts to Synthesize 4-0-(o<. -B-Glucopyranosyl)-6-deoxy-6-substituted-D-glucose Derivatives 45 - V-Page No. DISCUSSION 46 Maltobiouronic Acid 46 Acid Hydrolysis of 1,6-Anhydrides 47 Acetolysis of 1,6-Anhydrides 48 Mercapto Sugars 51 Comparative Reactivity of Primary Hydroxyl Groups 53 EXPERIMENTAL 54 Benzyl Hepta-O-ace tyl-|?>-maltoside 54 Benzyl |?>-Maltoside 55 Platinum Oxidation Catalyst 55 Catalytic Oxidation of Benzyl |£-Maltoside 56 Methyl(Benzyl Hepta-O-acetyl- ^-maltosid)uronate 56 Barium(Benzyl |3 -Maltosid)uronate 57 Maltobiouronic Acid 58 Methyl(Hepta-0-acetyl- -maltosid)uronate 59 Constitution of Maltobiouronic Acid 59 Reaction of Benzyl Hepta-O-acetyl- &-maltoside with L i A l H 4 r 60 1,6-Anhydro- ^-maltose 61 Direct Tosylation of 1,6-Anhydro-^-maltose 61 l,6-Anhydro-6'-O-trityl-^ -maltose 62 Penta-0-acetyl-l,6-anhydro-6'-O-trityl- ^-maltose 62 2,2' ,3,3' ,4'-Penta-0-acetyl-l,6-anhydro- |5 -maltose 63 Penta-0-acetyl-l,6-anhydro-6 ' -0-tosyl- -maltose 63 - v i -Page No. Penta-0-acetyl-l,6-anhydro-6 1 -azido-6 1 -deoxy- (I -maltose 64 6 1-Amino-6 1-deoxy-maltose 6 5 Potassium Thiolacetate 66 Penta«=0-acetyl-6 1 -S-acetyl-l,6-anhydro-6 1 -deoxy-P-maltose 67 Hepta-O-acetyl-6'-S-acetyl-6'-deoxy- oC-maltose 67 6'-Deoxy-6'-mercapto-maltose 68 Penta-O-acetyl-l,6-anhydro-6 • -deoxy-6 ' -iodo-(5 -maltose 69 Penta-O-acetyl-l,6-anhydro-6'-deoxy- ^-maltose 69 6'-Deoxy-maltose 70 Hepta-O-acetyl-6'-deoxy- ^-maltose 71 Catalyzed Lead Tetraacetate Oxidations 71 Methyl ©i-D-Glucopyranoside 71 1,6-Anhydro-y3-D-glucose 72 Benzyl 4 1 ,6 '-0-Benzylidene-6-0-tosyl- |5 -maltoside 72 Benzyl 4 1,6'-0-Benzylidene-6-0-trityl-^ -maltoside 75 Benzyl 4 1 ,6 '-0-Benzylidene-6-0-mesyl-/3 -maltoside 73 BIBLIOGRAPHY 75 - v i i -Page No. II . SYNTHESIS OF THE 2,4-DI-O-METHYL TETROSES INTRODUCTION 82 Purpose 82 Background 83 METHODS OF SYNTHESIS 87 2,4-Di-O-methyl-D-erythrose 87 2,4-Di-0-methyl-L-erythrose 89 2,4-Di-O-methyl-D-threose 90 2,4-Di-0-methyl-L-threose 92 DISCUSSION 98 Incomplete Periodate Oxidation of Reducing Sugars 98 Chromatography 100 Anomalous Behaviour of Optical Isomers 102 Derivatives 105 EXPERIMENTAL 106 Preparation of the S i l i c a Gel Column 106 Synthesis of 2,4-Di-0-methyl-D-erythrose 107 4,6-Di-O-methyl-D-glucose 107 4,6-Di-O-methyl-B-glucitol 107 4,6-Di-0-methyl-D-glucitol phenylurethan 108 2,4-Di-O-methyl-D-erythrose 108 2.4- Dinitrophenylhydrazone of 2,4-di-0-methyl-B erythrose 109 Synthesis of 2,4-Di-0-methyl-L-erythrose 109 3.5- Di-0-methyl-L-arabinose 109 3,5-Di-O-methyl-L-arabinonolactone 110 - v i i i -Page No. 3,5-Di-O-me thyl-L-arabinonamide 110 3,5-Bi-O-methyl-L-arabinitol 110 2,4-Di-0-methyl-L-erythrose 111 2.4- Dinitrophenylhydrazone of 2,4-di-0-methyl-L-erythrose 111 Synthesis of 2,4-Di-O-methyl-D-threose 112 1.2- Isopropylidene-D-xylofuranose 112 3.5- Di-0-methyl-l,2-isopropylidene-D-xylose 112 3,5-Di-0-methyl-B-xylose 112 p-Bromophenylosazone of 3,5-di-0-methyl-D-xylose 113 3,5-Di-0-methyl-D-xylitol 113 2,4-Di-0-methyl-B-threose 113 2,4-Dinitrophenylhydrazone of 2,4-di-0-methyl-D-threose 114 Synthesis of 2,4-Di-0-methyl-L-threose 114 2.3- Isopropylidene-l,4,6-tri-0-methyl-L-sorbose 114 l,4,6-Tri-0-methyl-L-sorbose 115 l,4,6-Tri-0-methyl-hexitol 115 2.4- Di-0-methyl-L-threose 116 2,4-Dinitrophenylhydrazone of 2,4-di-0-methyl-L-threose 116 BIBLIOGRAPHY 117 - i x -LIST OF FIGURES AND TABLES I. REACTIONS OF o(-LINKED DISACCHARIDES Page No, Figure 1. Synthesis of Maltobiouronic Acid 24 Figure 2. Synthesis of 4-0-(o^-D-Glucopyranosyl uronic Acid)-D-xylose 26 Figure 3. Synthesis of 6•-Amino-6'-deoxy-maltose 29 Figure 4. Alternate Synthesis of 6 1-Amino-6 1-deoxy-maltose 31 Figure 5. Synthesis of 6'-Deoxy-6'-Mercapto-maltose 33 Figure 6. Synthesis of 6'-Deoxy-maltose 35 Figure 7. Synthesis of 3-Amino-3-deoxy-sugars 37 Figure 8. Synthesis of 4-0-(3-Amino-3-deoxy-c^-D-glycopyranosyl)-D-glucose 40 Figure 9. Conformational Models of l,6-Anhydro-/3 -maltose 42 Figure 10. Catalyzed Lead Tetraacetate Oxidation N 43 Figure 11. Thin Layer Chromatography of Acetolysis 50 Figure 12. Proposed Acetolysis Mechanism 52 Table 1. Catalyzed Lead Tetraacetate Oxidation 72 - X -II. SYNTHESIS OF THE 2,4-DI-O-METHYL TETROSES Page No. Figure 1. Synthesis of 2,4-Di-O-methyl-D-erythrose 88 Figure 2. Synthesis of 2,4-Di-O-methyl-L-erythrose 91 Figure 3. Synthesis of 2,4-Di-O-methyl-D-threose 93 Figure 4. Synthesis of 2,4-Di-0-methyl-L-threose 95 Figure 5. Reduction of 1,4,6-Tri-O-methyl-L-sorbose 97 Figure 6. 5-Aldo-l,2-0-isopropylidene-D-xylopentofuranose 104 Figure 7. Possible Structure of C r y s t a l l i n e 2,4-Di-O-me thyl-D-threose 104 Table 1. Physical Properties of 2,4-Di-0-methyl Tetroses 101 - x i -• ACKNOWLEDGEMENTS I would l i k e to g r a t e f u l l y acknowledge the e f f o r t s of two people on ray behalf. The f i r s t , Professor G. G. S. Dutton, has presided over ray studies with a lenient but steady hand. His leadership has been a deep source of ideas and encouragement. I t has been a pleasure to work under the influence of such a genial person. The second, my wife, Marie, has p a t i e n t l y endured the duration of ray studies. Her constant encouragement has been a welcome help during this time. I would l i k e to thank Dr. P h i l Reid for reading the i n i t i a l manuscript and off e r i n g many he l p f u l suggestions. I would also l i k e to thank the National Research Council of Canada f o r the award of studentships for the duration of my studies. I. REACTIONS OF at-LINKED DISACCHARIDES INTRODUCTION * Purpose In spite of the importance of starch and similar food polysaccharides, no s a t i s f a c t o r y method has been developed for synthesizing the oi -glucosidic linkage. Many approaches have been t r i e d , but i n nearly a l l cases the synthesis of oC-glucosides has been hindered by very low y i e l d s of the desired product. An obvious route remains for the synthesis of disaccharides containing o£ -linkages. This i s the modification of n a t u r a l l y occurring disaccharides having the oC -glucosidic configuration. The reactions commonly used in monosaccharide chemistry have seldom been e f f e c t i v e l y applied to disaccharides. Due to the ease of hydrolysis of the glycosidic bond, no reaction in a synthetic sequence may employ strongly acidic conditions. As disaccharides contain twice as many hydroxyl groups, reactions normally exhibiting s e l e c t i v i t y in simpler systems often f a i l when applied to disaccharides. The r e a c t i v i t y normally associated with a s p e c i f i c hydroxyl group may be d r a s t i c a l l y altered by s t e r i c factors originating from the proximity of the two sugar units. It i s obvious that # The following conventional abbreviations have been used throughout the textr t o s y l - Ts (p-toluenesulphonyl), mesyl = Ms (me thane sulphonyl), ana! t r i t y l = Tr ( t r i p h e n y l -methyl). A l l ring-hydrogen atoms are omitted in the Haworth structures. -2-indiscriminate use of general procedures employed i n monosaccharide chemistry w i l l not be successful. Reactions employed in modifying disaccharides may r e l y on p r e f e r e n t i a l e t h e r i f i c a t i o n of primary hydroxyl groups, or blocking of s p e c i f i c a l l y oriented hydroxyls by acetals or ketals. A rather unique functional group, the 1,6-anhydro-linkage, enables simultaneous blocking of the primary hydroxyl of the reducing sugar as well as the reducing function i t s e l f . The use of such reactions may permit the modification of disaccharides at s p e c i f i c positions. In an e f f o r t to elucidate some of the reactions that are applicable to disaccharide chemistry, attempts to prepare s p e c i f i c a l l y substituted maltose derivatives were undertaken. Maltose, 4-0-(oC-D-glucopyranosyl)-D-glucose, was chosen for three reasons. Maltose i s the most common oC-linked disaccharide containing glucose. Secondly, the action of various hydrolytic enzymes upon modified maltoses should provide some information on the s p e c i f i c i t y of these enzymes. This i n turn may provide information on the fine structure of starch. F i n a l l y , the introduction of reactive substituents into amylitol (1, 2) has provided derivatives with r a d i c a l l y d i f f e r e n t properties. Synthetically altered maltoses would provide not only model compounds for such studies, but also reference compounds for comparison of products obtained by p a r t i a l hydrolysis. Chemical Synthesis of Q< -Glucosidic Disaccharides Koenigs-Knorr Synthesis Although several methods exist for the preparation of disaccharides, the method generally used i s the elimination of hydrogen halide between a glycosyl halide and a hydroxyl group. The reaction i s c a r r i e d out in the presence of an acid acceptor which may take part d i r e c t l y i n a concerted mechanism. The reaction was introduced by Koenigs and Knorr ( 3 ) , who condensed acetobromglucose with methanol i n the presence of various acid acceptors. If the methanol i s replaced with a monosaccharide possessing one free hydroxyl group, a disaccharide r e s u l t s . This reaction and the other less common methods of synthesis have been the subject of three reviews (4, 5, 6 ) . Recently a tabular l i s t of synthetic disaccharides has been prepared ( 7 ) . The replacement of the bromide in the Koenigs-Knorr reaction generally proceeds with an inversion of carbon atom one. The product of the reaction possesses therefore, the opposite configuration to the reactant glycosyl halide. The configuration of the halide in acetobromglucose has been found to be cK. • The reasons for t h i s have been summarized by Lemieux ( 8 ) , "in the absence of large a x i a l substituents at the 3- or 5-positions, the thermodynamically stable anoiaer for glycopyranosyl halides i s the anomer which -4 -has the halogen in a x i a l orientation". Thus, the Koenigs-Knorr reaction proceeds with acetobromo glucose to give mostly the |8 - glucoside. A -glucosyl halide might be expected to give the desired d. -anomer upon reaction, but studies have shown that the 1,2-trans halide-acetoxy system reacts by way of an orthoacetate intermediate, preserving the i n i t i a l configuration (9). The use of a n i t r o ester as a non-p a r t i c i p a t i n g group at position two gave isomaltose (6-0-(c< -D-glucopyranosyl)-D-glucose) in high y i e l d (10). This y i e l d was l a t e r attributed to a very active s i l v e r perchlorate c a t a l y s t , the synthesis of which could not be repeated (11). The t r i c h l o r o a c e t y l ester has been used as a non-participating group, since the electron withdrawing power of the three chlorine atoms of the t r i c h l o r o a c e t y l function should deactivate the carbonyl to such an extent that i t would be unable to a s s i s t in the displacement of the halide (11, 12). The e f f e c t of c a t a l y s t s , condensing agents, and r a t i o of reactants has been extensively studied in the case of the non-participating ^ - g l u c o s y l chlorides, in an attempt to obtain reproducible high y i e l d s . Condensations to the primary 6 position were reasonably successful. However, condensations at secondary positions, for example kojibiose (2-0-(©C -D-glucopyranosyl)-D-glucose), gave very low y i e l d s (11). -5-Future investigations of more r e l i a b l e catalysts may provide higher y i e l d i n g condensations. At present, the preparation of oC-linked glucoside disaccharides by the Koenigs-Knorr condensation must be considered impractical. Inversion at Carbon Atom 2 An i n t e r e s t i n g approach to oC-glue opyrano sides was reported early t h i s year (13). An attempt was made to replace iodide in a l k y l 2-deoxy-2-iodo-o£ -D-mannopyranoside tri a c e t a t e s with acetate. The iodo compound i s reported to be r e a d i l y prepared. Thus a Sjj.g> replacement of iodo by acetate would r e s u l t in an oC-glucopyranosyl configuration. Unfortunately, the iodide was found highly r e s i s t a n t to nucleophilic attack on carbon. Condensation Via 1,2-Anhydro Rings Alcoholysis of tri-O-acetyl-l,2-anhydro-pC-D-glucopyranose ( B r i g l ' s anhydride) at room temperature has been shown to produce /3 -glucosides i n good y i e l d (14, 15). However, the s t e r i c outcome of the reaction i s changed i f the reaction i s c a r r i e d out at elevated temperatures. The reaction of phenol with B r i g l ' s anhydride at 100° yielded only phenyl 3,4,6-tri-0-acetyl-o£-D-glucopyranoside (14), Lemieux (16) v i s u a l i z e s the reaction as proceeding through a 1,6-^3-D- c y c l i c ion which i s capable of reacting at carbon atom one to form - 6 the <?C -glucoside, Haworth and Hickinbottom (17) were the f i r s t to u t i l i z e B r i g l ' s anhydride i n the synthesis of disaccharides. Condensation of t h i s anhydride with 2,3,4,6-tetra-0-acetyl-ytf-D-glucose i n benzene at 90-100° gave an eight percent y i e l d of oC , j3-trehalose. In a similar manner, Lemieux (18, 19) has succeeded i n synthesizing maltose octaacetate and sucrose octaacetate by condensation of B r i g l ' s anhydride with l,2,3,6-tetra-0-acetyl-/3-D-glucopyranose and 1,3,4,6-te tra-0-acetyl-|S-D-f ructose respectively. The synthesis of sucrose, although erroneously reported by P i c t e t and Vogel (20, 21), had not been previously accomplished. Kojibiose was i s o l a t e d from a mixture obtained by heating B r i g l ' s anhydride at 116° for several days (22). Two other disaccharide products were present, one of which was chromatographically i d e n t i c a l to o£ , c<-trehalose. Condensations with B r i g l ' s anhydride at elevated temperatures give products in which the oC-isomer predominates. Unfortunately, side reactions such as self-condensation of the anhydride are known to occur under these conditions. As a r e s u l t , in a l l cases studied, the yields of disaccharide were low. -7-Anomerization The anomerization of a l k y l glycosides was f i r s t reported by Pacsu (23) i n 1928. Refluxing solutions of stannic chloride or titanium tetrachloride converted methyl t e t r a -O-acetyl- j3 -D-glucopyranoside into the corresponding oC-anoraer. Although varied opinions ex i s t as to the mechanism of th i s reaction, Lindberg (24) and Lemieux (25) have shown that the g l y c o s i d i c linkage i s not completely broken during the anomerization. Lemieux (16) has discussed the mechanism postulated by Lindberg (24), and has suggested an alternate mechanism which he f e e l s meets fewer objections. A r e f l u x i n g solution of titanium tetrachloride in chloroform anomerized gentiobiose octaacetate to the oC-anomer, isomaltose (26). A similar reaction using antimony pentachloride as the c a t a l y s t gave yie l d s of one half that of the titanium tetrachloride c a t a l y s i s (27). A unique combination of reactions, including anomerization, led to the synthesis of 6-0-(oC-D-glucopyranosyl)-D-galactose (28). A Koenigs-Knorr condensation of tetra-O-acetyl-OC-D-glucopyranosyl bromide and 1,2:3,4-diisopropylidene galactose gave only the j3 -1,6-linked disaccharide. Anomerization of the disaccharide octaacetate with titanium tetrachloride yielded an equilibrium mixture of the oL - and & - glycosidic - 8 -forms. De-O-acetylation and digestion of the mixture with ^-glucosidase l e f t only the c^-l,6-disaccharide. Anomerization has not been widely used i n the synthesis of ai.-disaccharides for two reasons. The f i r s t of these i s that recent extension of anomerization to disaccharides has not allowed time for the f u l l u t i l i z a t i o n of t h i s procedure. Secondly, the reaction seems generally applicable only to disaccharides i n which the glycosidic bond i s to a primary hydroxyl. It i s possible that new catalysts for the anomerization of disaccharides linked i n secondary positions w i l l be developed. Such an advancement would enable preparation of a l l types of oC -disaccharides. Modification Reactions Due to d i f f i c u l t i e s involved i n the synthesis of cC-disaccharides, the p o s s i b i l i t y of modifying n a t u r a l l y occurring o^-glucosyl-disaccharides was investigated. Modifications which allow the a l t e r a t i o n of a position on the sugar unit require the reaction to be s p e c i f i c . The means of achieving t h i s s p e c i f i c i t y usually requires the use of blocking groups on a l l positions where reaction i s undesired. The judicious choice of blocking groups may allow t h e i r p r e f e r e n t i a l removal, thus permitting a series of consecutive reactions to be c a r r i e d out on d i f f e r e n t positions. The following discussion outlines the d i f f e r e n t types of blocking groups and describes their synthetic applications i n disaccharide chemistry. Protection of the Reducing Function The reducing function of a disaccharide i s an aldehyde usually e x i s t i n g in a hemiacetal form. The extreme ease of oxidation and reduction of t h i s group necessitates i t s protection during most reactions. Chemically, blocking groups for the reducing function can be described as either aldehyde or hemiacetal derivatives. The l a t t e r retains the ri n g structure of the sugar, whereas the former opens the r i n g to give an a c y c l i c derivative. Emphasis w i l l not be placed on t h i s d i s t i n c t i o n however, since the chemical properties of the members within these two groups vary so widely. The benzyl group has been most frequently used i n synthetic a l t e r a t i o n s of disaccharides because of i t s s t a b i l i t y and ease of removal. Benzyl glycosides are base stable and r e s i s t a n t to mildly acidic conditions. Synthesis of the glycoside i s generally achieved by Koenigs-Knorr condensation and removal i s e a s i l y accomplished by hydrogenation under very mild conditions. In his studies on the p a r t i a l l y methylated derivatives of maltose and cellobiose (29), Hess used the benzyl group to block the reducing function. Jayme and Demmig (30) obtained cellobiouronic acid, 4-0-(£ -B-glucopyranosyl uronic -10-acid)-D-glucose, by c a t a l y t i c oxidation of benzyl ( 5 -cell o b i o s i d e and subsequent cleavage of the glycoside. Kremer and Gundlach (31) used benzyl ^-maltoside as starting material i n the synthesis of a branched tetrasaccharide. Although a great variety of other glycosides of disaccharides have been prepared (see for example 32), few have been used as synthetic intermediates. Workers have generally regarded glycosides such as methyl biosides as poor intermediates, due to the s i m i l a r i t y i n ease of hydrolysis of the glycosidic and disaccharide linkages. Compton (33) has shown, however, that a c e t o l y s i s of the glycoside i s considerably faster than the cleavage of the disaccharide. The synthesis of c e l l o -biomethylose (4-0-(6-deoxy--D-glucopyranosyl)-6-deoxy-glucose) was achieved i n good y i e l d from the acetolysis of the methyl ^3-glycoside (33). Other than this synthesis and that of sophorose (2-0-(^ -D-glucopyranosyl)-D-glucose) (34), acetolysis has been disregarded in synthetic procedures. Caution should be employed when the reaction i s to be applied to disaccharides linked in the primary p o s i t i o n , i . e . 1—6, as Matsuda and co-workers (35) have shown that disaccharides with 1—2, 1—3, and 1—4 linkages are considerably more stable to acetolysis than are 1—6 linkages. Alkaline degradation of a r y l biosides has been shown to y i e l d 1,6-anhydro rings (36), which may be then hydrolyzed -11-under weakly acidic conditions to give the parent sugars. At present, no synthetic route has u t i l i z e d the a r y l glycoside as a blocking group with removal via the anhydro sugar. Two important syntheses have proceeded from a disaccharide blocked with a 1,6-anhydro r i n g . 1,6-Anhydro-^3-cellobiose has been the starting material for the synthesis of both cellobiouronic acid (37) and pseudo-cellobiouronic acid, 4-G-( -D-glucopyranosyl)-D-glucuronic acid (38). The 1,6-anhydride of maltose was f i r s t reported by P i c t e t and Marfort (39) as a product of the thermal decomposition of maltose. Karrer and Kamienski (40) l a t e r reported i t s synthesis from the base elimination of the methiodide of N,N-dimethyl-amino hepta-0-acetyl-^3-maltoside. Asp and Lindberg (41) synthesized the hexaacetate by a base elimination of phenyl hepta-0-acetyl-^3 -maltoside. Acetolysis of l,6-anhydro-|S-maltose was f i r s t reported by Freudenberg and Soff (48). These authors showed that the anhydro linkage was cleaved much faster than the disaccharide linkage. It should be noted that although a n i l i d e s (43) and di t h i o a c e t a l s (44, 45) of disaccharides have been prepared, these derivatives have not been used as synthetic intermediates. There exists an ample selection of glycosidic blocking groups that can be removed under a variety of experimental -12 conditions. Judicious choice of these blocking agents permits the reaction of other portions of the molecule without destruction of the reducing group. Use of Anhydro Rings Anhydro derivatives may be useful as blocking groups as well as reactive centers. The opening of acetylated 1,6-anhydrides with titanium tetrachloride chlorinates on the C-l p o s i t i o n , giving an <^-chloride with a free C-6 hydroxyl (46). Opening of ethylene oxide type anhydro rings can be accomplished with a variety of reagents. The chemistry of the anhydro sugars was reviewed in 1946 by Peat (47) and more recently by Newth (48). The synthesis of 1,6-anhydro- ^ -maltose (41) was described e a r l i e r i n the discussion on blocking groups for the reducing function. Formation of 1,2,2»,3,3',41,6•-hepta-0-acetyl-^3 -maltose through opening of the anhydride with titanium t e t r a c h l o r i d e , and subsequent reaction of the chloride with mercuric acetate has l e d to the synthesis of branched t r i -saccharides (41, 49). Methyl 5,613 1,6 ' -di-anhydro-/3-maltoside and / 3-cellobioside have been prepared by the action of base on the i r di-mesylates (50). In spite of the in t e r e s t i n g reactions undergone by 3,6-anhydrides, their use in the synthesis of modified -13-disaccharides i s severely r e s t r i c t e d . The opening of the anhydride requires conditions so severe that cleavage of the disaccharide would surely r e s u l t (51, 52). The synthesis of ethylene oxide type rings in the disaccharide series may provide a route for configurational change i n the constituent monosaccharides, e s p e c i a l l y those on the non-reducing portion of the molecule. Such syntheses must await the freeing of a s p e c i f i c hydroxyl within the molecule, as the anhydride i s usually prepared by treatment of a sulphonyl ester with a l k a l i . Use of Esters There e x i s t many examples of the use of acetates as blocking groups in disaccharide investigations. Three such examples have already been described. Two of these involved the opening of f u l l y acetylated 1,6-anhydro-disaccharides with titanium t e t r a c h l o r i d e . Condensation of the halide at C-l with mercuric acetate leaves only one free hydroxyl in the molecule. This hydroxyl at C-6 has been oxidized i n cellobiose (38). In maltose, i t has been condensed with glucose to give the anomeric trisaccharides (41, 49). The i s o l a t i o n of primary hydroxyls in disaccharides has been achieved through t r i t y l a t i o n , a cetylation, and d e t r i t y l a t i o n . This synthetic route was used in two syntheses already -14-mentioned: the synthesis of cellobiouronic acid (37), and a tetrasaccharide formed from maltose (31). The synthesis of cyclohexyl 4-0-( o^-D-glucopyranosyluronic acid)-/-? -D-glucopyranosiduronic acid via this d i t r i t y l ether was reported (53). To prepare the 6,6 1-di-0-tosyl ester of oC ,oC-trehalose, Bredereck (54) f i r s t prepared the d i t r i t y l ether. In 1923, B r i g l and Mistele (55) reported that the action of phosphorus pentachloride on octaacetyl-yS -maltose produced hexa-0-acetyl-2-trichloroacetyl - y 8 -maltosyl chloride. No further work seems to have been reported on t h i s compound which would be expected to undergo reactions similar to i t s glucose analog. From these reactions, the synthesis of the 1,2-anhydro derivative as well as 1,2',3,31,4•,6,6'-hepta-0-acetyl-o(-maltose.could be expected. By the reaction of aqueous sodium acetate with acetobrom sugars, acetates of the cx!-series with only the 2 hydroxyl unsubstituted have been prepared (56, 57). In p a r t i a l l y acetylated sugars, the migration of acetate groups occurs r e a d i l y i n d i l u t e base (58). Soft glass i s reported to be s u f f i c i e n t l y alkaline to catalyze t h i s migration (59). This rearrangement could probably be u t i l i z e d to provide disaccharides with only the 4' position free as i s reported for monosaccharide derivatives (60). -15 Examples i n the discussion above have i l l u s t r a t e d the use of acetates as a blocking group only. No attempt has been made to include a review of acetate or benzoate derivatives. A r t i c l e s on the properties and reactions of esters have been published (61, 62). General methods for the preparation and removal of carboxylic acid esters have recently been described (63). The chemistry of t r i f l u o r o a c e t a t e esters as applied to carbohydrate chemistry has been recently reviewed (64). Sulphonyl esters are unique because of their manner of reaction with nucleophiles. Whereas carboxylic acid esters undergo acyl-oxygen f i s s i o n upon reaction, sulphonyl esters undergo alkyl-oxygen cleavage. Nucleophilic attack occurs at the a l k y l carbon causing inversion of configuration at that center. Reactions of sulphonate esters of secondary alcohols proceed much slower than those of primary. Under c l a s s i c a l reaction conditions (refluxing acetone) secondary esters did not generally undergo replacement, but with the use of high d i e l e c t r i c aprotic solvents such reactions have been used more frequently. A short discussion of SJJ^ sulphonyl replacements has been published recently (65). Replacement of sulphonates with a variety of reagents has led to the synthesis of substituted deoxy sugars. For example, replacement with azide ion followed by reduction -16-yields an amine. Replacement with iodide and subsequent reduction gives the unsubstituted deoxy sugar. Coupled with their a b i l i t y to invert configurations, replacement reactions undergone by sulphonates hold unlimited p o s s i b i l i t i e s for modifications i n disaccharide molecules. Although sulphonates show some s p e c i f i c i t y in reacting p r e f e r e n t i a l l y with primary hydroxyls, the presence of so many hydroxyl groups in disaccharides does not allow t h i s s e l e c t i v i t y to be operative. In the d i r e c t e s t e r i f i c a t i o n of a disaccharide, a complex mixture of sulphonates i s obtained. For t h i s reason many workers prefer to i s o l a t e the free primary hydroxyl by formation of the t r i t y l ether. The 6,6'-di-O-tosyl ester of hexa-O-acetyl-</,oC 1-trehalose was prepared i n t h i s manner (54). Replacement of the tosylates with iodide gave the 6,6'-diiodo compound which upon treatment with s i l v e r f l u o r i d e in pyridine yielded the d i - 5 , 6 - ^ - D -glucopyranoseen. Benzyl hexa-0-acetyl-6 1-O-tosyl-^-maltoside was reacted with sodium iodide in acetone to give the 6-iodo compound (66). Reduction with Raney n i c k e l reduced and deacetylated the iodo acetate to y i e l d benzyl 6 1-deoxy- ^ -maltoside. The l a t t e r was not r e a d i l y cleaved by a brewers yeast preparation which r a p i d l y s p l i t benzyl -maltoside, in d i c a t i n g the importance of the 6'-OH i n the enzyme hydrolysis. -17-By treatment of the 6,6'-di-O-mesylates with a l k a l i , the 3,6:3',6'-di-anhydro derivatives of methyl /3 - c e l l o b i o s i d e and f3 -maltoside were prepared (50). Although mesylation gave a product which could be r e c r y s t a l l i z e d , the t o s y l a t i o n product was amorphous. An excellent review on sulphonyl esters was prepared by Tipson i n 1953 (67). Due to the many advances in the f i e l d , the review i s unfortunately out of date. In spite of the extensive use of sulphonates in recent monosaccharide chemistry, there are few examples of their use i n the disaccharide f i e l d . The development of s p e c i f i c a l l y blocked disaccharides w i l l undoubtedly awaken in t e r e s t in such esters and their reactions. Use of Ethers The general use of ethers as blocking groups i s l i m i t e d by the severe conditions generally necessary f o r their removal. Two types of ethers, benzyl and t r i t y l , are useful because of c h a r a c t e r i s t i c chemical properties which allow their removal under mild conditions. Benzyl ethers are removed by hydrogenation i n the presence of a palladium c a t a l y s t . T r i t y l ethers are removed under weakly a c i d i c conditions. These two ethers may be used advantageously i n modifications of disaccharides. The chemistry of the benzyl ethers has been reviewed by McCloskey (68). This a r t i c l e describes the general methods -18-of synthesis and the s t a b i l i t y of the ethers to certain reagents. The synthesis of 3-0-benzyl-D-glucose has been reported by benzylation of 1,2:5,6-di-0-isopropylidene-D-glucose with benzyl bromide and s i l v e r oxide. More recently (69), benzylation has been achieved with benzyl chloride and sodium hydride at 130° (70). This l a t t e r paper describes a thin layer chromatographic system for these derivatives, as well as a spectrophotometric method of determining the number of benzyl residues per molecule. One unfortunate property of carbohydrate benzyl ethers seems to be t h e i r reluctance to c r y s t a l l i z e . McCloskey has stated that benzylation appears to be somewhat selective when benzyl chloride and potassium hydroxide are employed. Benzylation of 1,6-anhydro-^S-D-glucopyranose under these conditions gave varying y i e l d s of the 2,4-di-0-benzyl ether. This ' s e l e c t i v i t y ' i s more l i k e l y due to s t e r i c hindrance of the attacking species by the 1,6-anhydro bridge. Jeanloz (71) has shown that sulphonation of 1,6-anhydro-yS -D-glucopyranose also y i e l d s the 2,4-di-0 substituted ester. S e l e c t i v i t y has been shown in the preparation of 2-0-benzyl-4,5-0-isopropylidene-D-fucose dimethyl acetal (72). The synthesis was achieved through the reaction of one mole of sodium and subsequent treatment with benzyl chloride to give a 42$ y i e l d . This reaction must be considered a p r e f e r e n t i a l alkoxide formation 19-rather than selective benzylation, since the sodium reacts p r e f e r e n t i a l l y with the more aci d i c hydroxyl group at carbon two. Benzyl ethers have not been used to block positions other than the reducing function i n disaccharides. Their use in t h i s capacity was outlined e a r l i e r . Helferich has reviewed the chemistry and applications of t r i t y l ethers (60). Although t h i s review was published in 1948, the basic applications of t r i t y l ethers have remained unchanged. Two new developments, however, are worthy of mention. Synthesis of disaccharides by Koenigs-Knorr condensations has been achieved by In s i t u replacement of primary t r i t y l ethers using s i l v e r perchlorate as a c a t a l y s t . Thus the reaction of tetra-0-acetyl-o£-D-gluco-pyranosyl bromide with 1,2,3,4-tetra-0-acetyl-6-0-trityl-^-D-glucose i n nitromethane containing s i l v e r perchlorate gave acetylated gentiobiose in 55-60$ y i e l d (75). An attempt to p r e f e r e n t i a l l y remove a primary t r i t y l ether in the presence of a secondary tetrahydropyranyl ether i n a substituted ribofuranoside was unsuccessful (74). To circumvent t h i s problem, Khorana and co-workers employed para-methoxy substituted t r i t y l ethers. They report that for each methoxyl group substituted on the t r i t y l residue the rate of hydrolysis increases by a factor of about 10. Thus -20 the t r i s u b s t i t u t e d derivative, tri-p-anisylmethyl ether, hydrolases about 1000 times as fas t as the parent t r i p h e n y l -methyl derivative. Introduction of p-nitro groups into the t r i t y l residue should increase i t s resistance to hydrolysis in comparison with the parent compound. With such substitution, a t r i t y l ether of any acid s t a b i l i t y should be available. Examples of the few uses of t r i t y l ethers i n reactions of disaccharides have been considered previously under the discussion of esters. T r i t y l ethers' major contribution i s thei r a b i l i t y to react s e l e c t i v e l y with primary hydroxyls even i n the presence of a large number of secondary hydroxyls. The reaction of maltose with excess t r i t y l chloride gave 6,6' - d i - O - t r i t y l maltose (75). Recent workers have shown, however, that i n s u f f i c i e n t t r i t y l chloride y i e l d s the 6 ' -0 - t r i t y l derivative (76). T r i t y l a t i o n may then lead to d i f f e r e n t i a t i o n between not only primary and secondary hydroxyls,but also primary hydroxyls of d i f f e r e n t environments. Use of Acetals and Ketals Acetals and ketals d i f f e r from other blocking groups i n that they block two hydroxyl groups simultaneously, and the formation of the c y c l i c structure i s highly dependent on the stereochemistry of the hydroxyl groups involved. The r i n g may contain 5 or 6 members depending on the configuration of the hydroxyls and the reagent employed. Ketones w i l l not -21 e a s i l y form six membered rings due to the 1,3-diaxial i n t e r -action between the a l k y l of the ketone and the two a x i a l hydrogens of the sugar. Benzaldehyde prefers a six membered ring where the large aromatic portion can remain equatorial. In such a compound, the conformation of the fused rings can be considered f i x e d since f l i p p i n g would necessitate the bulky aromatic r i n g to assume an a x i a l orientation. Few examples exist of the use of acetals and ketals i n disaccharide chemistry. Sutra condensed acetaldehyde with maltose to form a sirupy compound which he regarded as a d i -O-acetal (77). Under the conditions reported for t h i s condensation, thin layer chromatography indicated the formation of four major products (78). A c r y s t a l l i n e mono-O-benzylidene compound has been reported to form by condensation of benzaldehyde with lactose dibenzyl d i t h i o a c e t a l (44). No structure was assigned to t h i s compound. The removal of most acetals and ketals can be achieved with weakly a c i d i c conditions. The replacement of 1,2-0-isopropylidene groups by acetolysis has been reported (79, 80, 81). Such mild conditions should enable the removal of these blocking groups without damage to the disaccharide structure. Epimerizations and Inversions In their review on the synthesis of oligosaccharides, Evans, Reynolds, and Talley (4) give a description of the various -22-methods of changing configurations within the monosaccharide units. A l l of these methods involve change of configuration of the C=2 or C-5 hydroxyl of the reducing sugar. In a recent paper describing the inversion of carbohydrates upon acetolysis of isopropylidene derivatives (82), the author indicates the s i m i l a r i t y of the acid induced inversions to the aluminum chloride and hydrogen f l u o r i d e epimerizations. Although acid catalyzed rearrangements of t h i s type are now more f u l l y understood (85), no major developments applicable to disaccharides have been reported since the review was written. The judicious application of the reactions described above w i l l undoubtedly lead to the i s o l a t i o n of many important disaccharides. Because of the d i f f i c u l t i e s involved i n the synthesis of glucose disaccharides of ^ - c o n f i g u r a t i o n , modification offers a p r a c t i c a l and chemically i n t e r e s t i n g way of obtaining these compounds. -23-METHODS OF SYNTHESIS Maltobiouronic Acid 4-0-(o^-D-Glucopyranosyluronic Acid)-D- glucose. Jayme and Demmig (30) synthesized cellobiouronic acid by c a t a l y t i c a e r i a l oxidation of benzyl y 3-cellobioside. The c a t a l y t i c oxidation of carbohydrates has recently been reviewed (84). The oxidation has been shown to be s t e r e o s p e c i f i c , oxidizing primary hydroxyls faster than secondary, and a x i a l hydroxyls more r a p i d l y than equatorial. A system of confor-mational analysis has been developed from the application of these p r i n c i p l e s (85). Since c a t a l y t i c oxidation i s highly dependent on stereochemistry, i t was i n t e r e s t i n g to compare the oxidation product of benzyl y_?-maltoside with that of benzyl ft - c e l l o b i o s i d e (Fig. l ) . Benzyl hepta-O-acetyl-yd?-maltoside was synthesized from acetobromomaltose (86) and benzyl alcohol by the condensation method described by Helferichand Berger (87). Deacetylation with methanolic ammonia gave the c r y s t a l l i n e benzyl /3-maltoside (88). Catalytic oxidation gave a mixture of a c i d i c sugars, which upon e s t e r i f i c a t i o n with diazomethane and acetylation gave c r y s t a l l i n e methyl (benzyl hexa-O-acetyl-P-maltosid)uronate. The methyl ester hexaacetate was obtained in 40$ y i e l d , based on the benzyl ^-maltoside consumed. Saponification was achieved by warming with aqueous barium hydroxide to y i e l d the barium (benzyl /S -maltosid)uronate. -24--25-Hydrogenolysis using palladium on barium sulphate (89), followed by a c i d i f i c a t i o n with Amberlite IR 120 (H +) yielded maltobiouronic acid. E s t e r i f i c a t i o n of maltobiouronic acid with diazomethane, followed by acetylation with acetic anhydride i n pyridine, yielded the c r y s t a l l i n e methyl ester heptaacetate• Borohydride reduction of a sample of the acid, followed by acid hydrolysis indicated only glucuronic acid and s o r b i t o l upon paper chromatography. The structure of the synthetic uronic acid i s therefore 4-0-(oC-B-glucopyranosyluronic a c i d ) -D-glucose. The i s o l a t i o n of maltobiouronic acid from the c a t a l y t i c oxidation of benzyl ^-maltoside indicates that the difference glucosidic derivative (cellobiose) i s not s u f f i c i e n t to change the site of oxidation. The 6'-position i s p r e f e r e n t i a l l y oxidized i n both compounds, yi e l d i n g the respective aldo-biouronic acids. Due to the importance of aldobiouronic acids, e s p e c i a l l y those containing D-glucuronic acid and D-xylose, in structural investigations of natural products, the following scheme for the synthesis of 4-0-(oC-D-glucopyranosyluronic acid)-D-xylose was proposed (Fig. 2). between the oC -glucosidic derivative 4-0(ol -D-Glucopyranosyluronic Acid)-D-xylose. F i g . 2 -27 Prolonged c a t a l y t i c oxidation of benzyl /s-maltoside or permanganate oxidation of benzyl 2,2',3,3' ,4-penta-O-acetyl-yS maltoside should give the di-uronic acid. Removal of the benzyl glycoside by c a t a l y t i c hydrogenation would r e s u l t i n 4-0-(oC-D-glucopyranosyluronic acld)-D-glucuronic acid. Treatment of t h i s d i - a c i d with ni c k e l acetate i n pyridine at 80° as outlined by Zweifel and Deuel (90) should cause decarboxylation of the reducing sugar residue, y i e l d i n g the desired compound. The c r u c i a l step of t h i s synthetic route i s the decarboxylation reaction which, although f i r s t reported i n 1956, has not been u t i l i z e d since. Although hot pyridine has been used to e f f e c t rearrangements i n sugars to t h e i r epimers and corresponding ketoses (91), these authors employed pyridine solutions of nickel s a l t s at 80° for decarboxylation. Substitution of the uronate at C-l as a glycoside prevents the decarboxylation, but e s t e r i f i c a t i o n of the acid i s reported not to hinder the reaction. The mechanism of the reaction i s suggested to be a complexing of the C-l oxygen lone pair by the metal ion employed as a c a t a l y s t (90). It i s then postulated that the hydrogen of the C-l hydroxyl can participate in an electron transfer in which the carboxyl function i s eliminated. 28 Although Zweifel and Deuel were able to i s o l a t e c r y s t a l l i n e L-arabinose from the decarboxylation of D-galacturonic a c i d , no arabinose was detectable by paper chromatography upon r e p e t i t i o n of th i s experiment under the reported condi t ions . Several more attempts to repeat t h i s work were unsuccessful , and for this reason the proposed synthesis of 4-Q-(oi -D-glucopyranosyluronic ac id)-D-xylose was discont inued. 4-0-(6-Amino-6-deoxy-oC-D-glucopyranosyl)-D-glucose. A poss ible synthetic route to 6 *-amino-6•-deoxy-maltose (4-0-(6-amino-6-deoxy-^-D-glucopyranosyl)-D-glucose) i s out-l i n e d below ( F i g . 3) . The methyl ester hexaacetate benzyl g lycos ide , an intermediate i n the synthesis of maltobiouronic a c i d , upon treatment with methanolic ammonia would deacetylate and form the amide. Reduction of the amide with l i th ium 0 F i g . 3 -50 aluminum hydride would give the primary amine (92), from which the amino sugar could be i s o l a t e d by c a t a l y t i c debenzylation. Although benzyl ethers are stable to lithium aluminum hydride under moderate conditions (95), the reaction of benzyl glycosides under similar conditions has not been investigated. For t h i s reason, the action of lithium aluminum hydride in refluxing ether on benzyl hepta-O-acetyl-|8-maltoside was investigated. Acetylation of the reaction product aft e r one hour yielded material which indicated two major components on s i l i c a gel thin layer chromatography (94). The faster spot corresponded to the s t a r t i n g material, benzyl hepta-O-acetyl- |3-maltoside, and the slower spot was presumably nona-O-acetyl-maltitol. The presence of compounds other than benzyl hepta-O-acetyl- |3 -maltoside indicated that the benzyl glycoside was unstable to conditions necessary for the reduction of the amide. For t h i s reason, t h i s synthetic route was not considered p r a c t i c a l and was abandoned. An alternate route f o r the synthesis of 6'-amino-6f-deoxy-maltose i s the azide replacement of a 6'-O-sulphonate ester. Reduction of a primary azide yields a primary amine. 1,6-Anhydro- |S-maltose was selected as the st a r t i n g material. It was hoped that tosylation of t h i s material would give p r e f e r e n t i a l l y the 6'-O-tosylate. Unfortunately t o s y l chloride F i g , 4 -32-was not s u f f i c i e n t l y s p e c i f i c and thin layer chromatography indicated two monotosylates and several ditosylates i n addition to s t a r t i n g material. T r i t y l a t i o n followed by acetylation and d e t r i t y l a t i o n with 80$ acetic acid gave 2,2*,3,3',4'-penta-0-acetyl-l,6-anhydro-p -maltose. Tosylation of t h i s substrate was then carried out quickly and i n good y i e l d by the use of excess t o s y l chloride. Replacement of the t o s y l ester was accomplished by heating the ester with sodium azide in N,N-dimethylformamide. Deacetylation followed by c a t a l y t i c reduction yielded chromatographically pure 6'-araino-l,6-anhydro-6'-deoxy-p -maltose. The a b i l i t y of the 6-amino group to hinder acid hydrolysis (95) would be expected to s t a b i l i z e the glycosidic linkage of a 6-amino glucoside. This has, i n f a c t , been reported by Cramer and co-workers (96). Thus mild acid hydrolysis should open the 1,6-anhydro r i n g without cleavage of the disaccharide, y i e l d i n g 6'-amino-6*-deoxy-maltose. 4-0-(6-Deoxy-6-mereapto-ol-D-glucopyranosyl)-D-glucose The replacement of sulphonyl esters by potassium thiolacetate was investigated by Chapman and Owen (97) and found to be a convenient method of introducing a sulfhydryl group into a molecule. Reaction of 2,2» j S ^ 1 , . ' -penta-0-acetyl-l,6-anhydro-6'-0-tosyl-p -maltose with potassium thiolacetate i n N,N--33-CH2SAc CH_OAc Pig. 5 KSAc DMFA H 2 S 0 4 A c 20, Ac OH ISSaOMe &-MeOH -34-dimethylformamide gave 2,2',3,3',4'-penta-0-acetyl-6'-S-acetyl-l,6-anhydro-/#-maltose in good y i e l d . Acetolysis was used to open the 1,6-anhydro rin g without cleavage of the disaccharide linkage. Deacetylation of the octaacetate yielded the free thio sugar, which oxidized very r a p i d l y with atmospheric oxygen to give the disulphide. Addition of excess 2-mercaptoethanol regenerated the 6'-deoxy-61-mercapto maltose. 4-0-(6-De6xy-g<!-D-glucopyranosyl)-D-glucose Although benzyl 6 1-deoxy -^3-maltoside had been synthesiz (66) for enzyme studies, the removal of the benzyl glycoside was not attempted. An attempt to prepare 6-deoxy-, 6'-deoxy-, and 6,6'-dideoxy-maltose v i a tosylation of methyl ^-maltoside f a i l e d to y i e l d characterizable products (98). The synthesis of 6 * -deoxy-maltose (4-0-(6-deoxy-<X^-D-glucopyranosyl)-D-glucose) was attempted by the replacement of the 6 1-0-tosylate by iodide. The c a t a l y t i c reduction of the iodide yielded 2,2*,3,3',4*-penta-O-acetyl-1,6-anhydro-6 ' -deoxy- {$>-maltose. Acetolysis and deacetylation yielded 6'-deoxy-maltose. -35-Fig. 6 -36-4-0-(5-AmiDO-3-deoxy-o<-D-glycopyranosyl)-D-glucose A general synthesis of 3-amino-3-deoxy-sugars was f i r s t introduced to carbohydrate chemistry by Baer and Fischer (99) and has been investigated i n a very thorough manner by Baer i n subsequent publications (100). The subject has been recently reviewed by Lichtenthaler (101). The synthesis i s a modification of the Sowden-Fischer synthesis i n which two aldehyde groups within the same molecule react with n i t r o -methane i n a t y p i c a l a l d o l condensation. The dialdehyde i s produced from gly c o l oxidation with periodate or lead tetraacetate (Fig. 7). Condensation of the dialdehyde with nitromethane under the influence of base leads to a C-nitro alcohol. Upon a c i d i f i c a t i o n , the n i t r o group in v a r i a b l y takes up an equatorial orientation. The configurations of carbons 2 and 4 can give r i s e to four possible isomers but in practice, manipulation of the condensation conditions allows the p r e f e r e n t i a l formation of certain of the configurations. Subsequent reduction of the C-nitro-alcohol yields the 3-amino-3-deoxy-glycoside. Recent nuclear magnetic resonance work (102) has shown that 2!,3 f4-tri-0-acetyl-l,6-anhydro-^-D-glucose exists primarily in a IC rather than a 3B conformation. -37 -38-0(\c OF\c AcO IC 3B Replacement of the r e l a t i v e l y bulky acetyl groups with hydrogen, would s t a b i l i z e the IC conformation as there would be less crowding of the a x i a l C3 substituent against the 1,6-anhydro bridge and less C2, C4 1,3-diaxial i n t e r a c t i o n . 1,6-Anhydro-^-D-glucose would be expected then, to possess the IC conformation. Reeves (103) has deduced from the o p t i c a l rotation of cuprammonium solutions of l,6rra"nhydro-y5 -D-glucose that the sugar does indeed exist in a IC conformation. There i s l i t t l e difference in the o p t i c a l rotation of the complex formed from either l,6-anhydro-3-0-methyl- /^-D-glucose or i t s parent 1,6-anhydro-^-D-glucose. This indicates that hydroxyl groups 2 and 4 are involved i n the complex formation which they could only do i f they are in a d i a x i a l ( i . e . IC) conformation. 1,2-Biols are oxidatively cleaved by lead tetraacetate at a rate which i s dependent upon the geometry of the d i o l system (104). The closer the two hydroxyl groups approach each other the faster the rate of oxidation. For example, cis-cyclohexandiol i s oxidized 23 times more quickly than trans-cyclohexandiol (105) and 1,6-anhydro- /3 -D-glucofuranose i s not attacked at a l l (106). In the l a t t e r the 1,2-diol system i s locked and the projected bond angle between the hydroxyl groups i s very near 120°. From the information discussed above, a synthesis of 4-0-(3-amino=3-deoxy-o(-D-glycopyranosyl)-D-glucose was proposed (Fig. 8). Lead tetraacetate oxidation at 5° of 1,6-anhydro^ f& -maltose prepared by deacetylation of the known hexaacetate (41), should give the dialdehyde, as the hydroxyl groups on the non-reducing residue being trans diequatorial should oxidize more quickly than the trans d i a x i a l orientation of hydroxyls on the anhydro glucose portion of the molecule (Fig. 9). Condensation of the dialdehyde with nitromethane would then give a C-3' nitro-disaccharide which after reduction could be hydrogenolyzed to the free amino sugar. Before attempting the tetraacetate oxidation of 1,6-anhydro- ^-maltose, the oxidation of model compounds was attempted. The two compounds selected as models of both -41-portions of the 1,6-anhydro- p-maltose molecule were methyl O^-D-glucoside and l,6-anhydro-^_? -D-glucose (Fig. 9 ) . It was rather surprising, therefore, to f i n d that 1,6-anhydro- fZ -D-glucose oxidized at a faster rate than methyl o_-D-glucoside (Fig. 10). From consideration of the ex i s t i n g data outlined above, 1,6-anhydro-y_?-D-glucose would be expected to oxidize at ^ / ^ Q or less the rate of methyl oL-D-glucoside due to the orientations of the respective hydroxyl groups. Such i s not the case, i n f a c t , the two molecules oxidize at e s s e n t i a l l y the same rate. Three p o s s i b i l i t i e s allow explanation of the oxidation r e s u l t s ; a) Lead tetraacetate oxidation i s not s p e c i f i c and oxidizes g l y c o l groups without regard to t h e i r geometry. While t h i s p o s s i b i l i t y must be kept i n mind i t i s extremely u n l i k e l y . The basis for the use of lead tetraacetate has depended on the s p e c i f i c i t y of i t s oxidative cleavage and i t i s d i f f i c u l t to see why l,6-anhydro-y3-D-glucose should pose an exception. b) The 1,6-anhydro-linkage i s hydrolyzed very quickly in g l a c i a l acetic acid, which i s the solvent used for lead tetraacetate oxidations, giving D-glucose. D-Glucose would then be attacked very quickly by the oxidant with a t o t a l Catalyzed Lead Tetraacetate Oxidation -44-rate for the two reactions s l i g h t l y greater than the rate for the glycoside. This a t t r a c t i v e p o s s i b i l i t y was eliminated by observing the o p t i c a l rotation of a solution of 1,6-anhydro-|S-C-glucose in g l a c i a l acetic acid. If any hydrolysis occurred the rotation would change from a strongly negative value (ca. -70°) to a positive value (ca. +53°). In 24 hours no change in rotation of the levorotatory solution was observed. c) l,6-Anhydro-/5-D-glucose i n g l a c i a l acetic acid a c t u a l l y exists i n a 3B conformation. Such a conformation would allow tetraacetate oxidation to proceed at a rate equal to that of methyl o(-D-glucopyranoside. The s l i g h t l y slower rate of oxidation of the glucoside may possibly be explained by the formation of a 6-0-formyl ester which slows the rate of oxidation of formic acid to carbon dioxide. This e s t e r i f i c a t i o n cannot occur i n 1,6-anhydro-^-D-glucose because the anhydro linkage blocks the 6-position. Change i n conformation with solvent i s not a common occurrence but has been observed in methyl 2-deoxy- c/-D-ribopyranoside (107). However, in these compounds, the r i n g remains i n a chair with only the di s p o s i t i o n of the substituents varying. The proposed synthesis of the amino disaccharide was discontinued at t h i s point due to the lack of information -45-on the oxidation step. Further work i s to be carried out in an e f f o r t to determine the cause of the anomalous oxidative behaviour of l,6-anhydro-/3-D-glucose. Attempts to Synthesize 4-0-(oC-D-Glucopyranosyl)-6-deoxy-6- substituted-D-glucose Derivatives In an attempt to synthesize 6-substituted derivatives of maltose, benzyl 4 *,6 1-O-benzylidene-j5 -maltoside was employed. In t h i s molecule only one primary hydroxyl remains free and attempts to s e l e c t i v e l y tosylate t h i s hydroxyl group were unsuccessful. The reaction of a more s p e c i f i c reagent, t r i t y l chloride, was investigated. Although removal of t r i t y l i n the presence of benzylidene would not have been possible, the use of a trimethoxy t r i t y l derivative (74) would have permitted such a manipulation. Had t r i t y l a t i o n proceeded in a normal fashion, the corresponding methoxy derivative would have been prepared. Sp e c i f i c t r i t y l a t i o n of the 6-position was not achieved. The lack of reaction of the t r i t y l chloride may have been due to s t e r i c hindrance and for this reason mesylation was attempted. Mesylation was the most e f f e c t i v e of the three methods t r i e d . However, the reaction was abandoned as a preparative method, because sulphonation occurred i n only about 50$ y i e l d as indicated by thin layer chromatography. -46-BISCUSSION Maltobiouronic Acid The synthesis of maltobiouronic acid by the c a t a l y t i c oxidation of benzyl |3 -maltoside was accomplished s l i g h t l y i n advance of t h i s work by Hirasaka (108). Knowledge of his r e s u l t s was obtained shortly after publication of our work early t h i s year (109). The physical constants of a l l compounds common to both publications were i n agreement. Hirasaka (110) reported, at the same time, the synthesis of maltobiouronic acid by the permanganate oxidation of 1,2, 2' ,3,3 1,4* ,6-hepta-0-acetyl- [3 -maltose. This acetate was also treated i n acetic acid with chromium tri o x i d e followed by permanganate. Acid chrornate oxidation of the alcohol i s presumably faster than permanganate, whereas the reverse i s true of the oxidation of the aldehyde. By using both reagents a faster o v e r a l l oxidation was accomplished. Maltobiouronic acid would be expected to be one of the products obtained from the p a r t i a l acid hydrolysis of oxidized amylose. P a r t i a l l y acetylated amylose was prepared from amylose by t r . i t y l a t i o n , acetylation and d e t r i t y l a t i o n . Chromate-permanganate oxidation of t h i s substrate yielded material oxidized at 50% of the primary alcoholic positions (111). Although p a r t i a l hydrolysis was not reported, the authors -47-stated that the oxidized amylose had physical constants quite d i f f e r e n t from n i t r i c acid oxidized amylose. The non-reducing residue i n maltose has been assigned both skew (112) and chair (113) conformations by various authors. Had c a t a l y t i c oxidation of benzyl ^-maltoside given a major product other than maltobiouronic acid, a conformation might have been assigned to the starting material. With the i s o l a t i o n of maltobiouronic acid as the major product nothing can be said regarding the conformation of benzyl ^S-maltoside. Acid Hydrolysis of 1,6-Anhydrides Acid hydrolysis of 1,6-anhydro rings has been reported i n a number of communications (114, 115, 116). 0.2N Hydrochloric acid for 4 hours on a steam bath (115) indicates the severity of conditions necessary to e f f e c t t h i s hydrolysis. Under such conditions the rates of hydrolysis of the 1,4-/2 linkage and the anhydro r i n g of l,6-anhydro-/3 -cellobiose were found to be nearly equal (116). In studies of 6-amino-6-deoxy-D-glucose (96), methyl 6-amino-6-deoxy-o^-D-glucoside was not hydrolyzed by 0.2N hydrochloric acid at 100° for 8 hours. Extending these conditions to e'-amino-l,6-anhydro-6'-deoxy-^S-maltose, the anhydro rin g should hydrolyze while the glycosidic linkage should not, because of the s t a b i l i z a t i o n by the 6-amino function. -48-Hydrolysis of 61-amino-l,6-anhydro-6•-deoxy-/3 -maltose with 0.15N hydrochloric acid for 13 hours at 100° yielded, as indicated by paper chromatography, glucose, the expected amino disaccharide, some degradation products, and a small amount of sta r t i n g material. The presence of glucose was somewhat mysterious as there did not appear to be an equal amount of 6-amino-6-deoxy-glucose present i n the hydrolysate. The reluctance of the anhydro r i n g to open may be a further example of the d i f f i c u l t y with which amino sugars are hydrolyzed (95). This i s probably due to the necessity of double protonation for hydrolysis, as the amine r e a d i l y accepts the i n i t i a l proton and further protonation i s hindered by the molecule's positive charge. The i s o l a t i o n of a f r a c t i o n recognizable as the amino-disaccharide indicates a d e f i n i t e s t a b i l i z a t i o n of the glycosidic linkage by the 6-amino function. Acetolysis of 1,6-Anhydrides Acetolysis of 1,6-anhydro rings was f i r s t reported by Freudenberg and Soff (42). These authors showed that the reaction gave an equilibrium mixture of the and £ forms with the oL predominating (88$ for D-glucose). Haskins, Hann and Hudson showed that the 1,6-anhydro linkage was acetolyzed much faster than the 1,4-^6 disaccharide linkage. These procedures were incorporated i n the chemical syntheses of cellobiose (117) and lactose (118). -49-Thin layer chromatography of carbohydrate acetates has been described by a number of authors (94, 119). In an attempt to follow the acetolysis of 1,6-anhydro derivatives by thin layer chromatography, samples were removed during the course of the reaction and spotted d i r e c t l y onto thin layer plates. The addition of two drops of pyridine to the plate was s u f f i c i e n t to neutralize the sulphuric acid present. The spot was then dried at room temperature. The plates were developed with ethyl ether-toluene (2:1, v/v., solvent D), a solvent described by Gee (120) for thin layer chromatography of f u l l y methylated sugars. The acetates were detected by spraying with sulphuric acid and heating. A t y p i c a l pattern observed i n the acetolysis of penta-0-acetyl-6'-S-acetyl-l,6-anhydro-6'-deoxy- jS-maltose i s shown in Figure 11. Obviously, two consecutive reactions are taking place. The f i r s t i s the acetolysis of the anhydro r i n g to an approximately equal mixture of the c£ and /3 acetates. The second reaction i s the anomerization of the two acetates to give an equilibrium mixture i n which the oC predominates. The i d e n t i t y of the second reaction was confirmed by the formation of the equilibrium mixture upon dissolving octa-O-acetyl-yS-maltose in the acetolysis solution. I t i s quite l i k e l y the opening of the anhydro rin g i s i n i t i a t e d by the formation of a - 5 0 -\ Reaction Time (min.) F i g . II -51-1,6"p -D-eyclic ion which rearranges to a more stable carboniura ion. This ion reacts with a molecule of acetic acid to form an equal mixture of the and /3 anomers. Anomerization, catalyzed by the sulphuric acid, then s h i f t s the r a t i o of isomers to the equilibrium value (Fig. 12). Mercapto Sugars Although the replacement of sulphonates by thiolacetate i s a convenient method of introducing sulphur into a sugar, complications arise upon deacetylation. Thiols are known to undergo atmospheric oxidation and t h i s reaction i s accelerated i n the presence of a l k a l i . Acid catalyzed deacetylation has been suggested (121) but these conditions do not seem advisable for disaccharide derivatives. Regeneration of the t h i o l from the disulphide can be accomplished by addition of excess 2-mercapto-ethanol (122). Several alternate methods e x i s t for the introduction of sulphur into sugar molecules and these have been reviewed by Hutson and Horton (65). Possibly the most promising method i s the replacement of sulphonates by thiosulphates forming the 'Bunte s a l t 1 . As t h i s molecule now possesses a negative charge, ion exchange chromatography can be conveniently ca r r i e d out. Generation of the free mercapto sugar i s accomplished by addition of excess low molecular weight t h i o l s such as 2-mercapto-ethanol. The use of thiosulphate CH2OAc ™ ° 0 AcO'X.OAc PAc-H 2 S 0 4 AcOH AcO' XiOAc OAc H 20Ac-H,OAc 'OAc c I F i g . 12 -53-derivatives was f i r s t introduced by Swan (122) for the study of sulphur containing proteins. Comparative Reactivity of Primary Hydroxyl Groups As investigations on disaccharides continue, one f a c t i s becoming apparent. The primary hydroxyl group on the reducing residue of 1,4 linked glucose disaccharides i s less reactive than the other primary hydroxyl. Lindberg (38) has remarked on the d i f f i c u l t y of oxidizing the 6-position with respect to the 6'-position. The p a r t i a l t r i t y l a t i o n of maltose gives only the 6'-derivative, and no 6-substituted derivative (110). Catalytic oxidation of maltose or cellobiose derivatives lead to 6'-oxidized products (30, 108, 109). The unsuccessful attempts to prepare 6-substituted derivatives as described i n t h i s work serve as additional examples of what may be a general e f f e c t i n 1,4 linked disaccharides. -54-EXPERIMENTAL Evaporations were carr i e d out under reduced pressure at a bath temperature of 40-45°. Optical rotations are equilibrium values measured on a Bendix-Ericson ETL-NPL Automatic Polarimeter (Type 143A) at 21- - 2°. Melting points quoted are uncorrected. Chromatograms were run by the descending technique in the following solvents: (A) ethyl acetate -acetic acid - formic acid - water (18:3:1:4); (B) 1-butanol -ethanol - water (4:l:5)j (C) ethyl acetate - acetic acid -water (18:7:8). S i l i c a gel thin layer plates were run by the ascending technique in the following solvents: (D) ethyl ether - toluene ( 2 : l ) j (E) benzene - methanol (19:l)j (F) butanone - water azeotrope; (G) 1-butanol - acetic acid -ethyl ether - water (9:6:3:1). Sugars were detected on paper chromatograms by the p-anisidine-trichloroacetate spray (123) f o r reducing sugars and periodate-permanganate spray (124) for non-reducing sugars. Detection of sugars on thin layer plates was accomplished by spraying with concentrated sulphuric acid and heating the plate at 150°. Benzyl Hepta-O-acetyl-p -maltoside To acetobromomaltose (16.6 g) prepared from maltose (10 g) by the method of Barczai-Martos and Korosy (86), was added f r e s h l y d i s t i l l e d benzyl alcohol (32 ml) and mercuric cyanide (6 g). The mixture was heated to 85° on a water bath and -55-s t i r r e d vigorously for 45 minutes. The r e s u l t i n g solution was poured with s t i r r i n g into ethanol (90 ml) and cooled to 5° for 3 hours. The crude product (10.5 g, 61% based on the acetobrom sugar) was col l e c t e d by suction f i l t r a t i o n . R e c r y s t a l l i z a t i o n from ethanol yielded material melting at 124-125! L i t . (88), m.p. 125°. Benzyl j3 -Maltoside Deacetylation of benzyl hepta-O-acetyl-^-maltoside was accomplished with methanol saturated with ammonia as described by Fischer and Kogl (88). R e c r y s t a l l i z a t i o n from methanol -ethyl acetate gave the c r y s t a l l i n e glycoside, m.p. 148-149°. L i t . (87), m.p. 147-148.° Platinum Oxidation Catalyst Modification of a method of Brown (125) f a c i l i t a t e d the preparation of an active platinum c a t a l y s t . To carbon (10 g, Darco G-60), digested overnight in 6N hydrochloric acid, washed free of chloride with d i s t i l l e d water and dried at 120° for four hours, a solution of c h l o r o p l a t i n i c acid (5 g) in 5 0 % aqueous ethanol (50 ml) was added. To t h i s mixture, a solution of sodium borohydride (2.5 g) in ethanol (100 ml) was added slowly with s t i r r i n g . After 5 minutes 6N hydrochloric acid (10 ml) was added and the suspension suction f i l t e r e d . The precipitate was washed free of chloride with d i s t i l l e d -56-water and dried i n vacuum over phosphorus pentoxide. The product (12 g) was pyrophoric and in l a t e r experiments drying was not carried out and the product was kept as a moist paste. Catalytic Oxidation of Benzyl |4-Maltoside To a solution of benzyl ^-maltoside (5 g) and sodium bicarbonate (1 g) i n d i s t i l l e d water (100 ml) was added platinum catalyst (5 g). The mixture was maintained at 65° and magnetically s t i r r e d for 6 hours during which time a stream of oxygen was passed into the solution through a gas dispersion tube. After cooling the mixture was f i l t e r e d through C e l i t e , a c i d i f i e d with Amberlite IR 120 (H ) and passed slowly through a column of Duolite A-4 (0H~). The Duolite r e s i n was washed with water (500 ml) and the combined eluates evaporated to recover unreacted benzyl ^-maltoside (3-4 g). The Duolite r e s i n was eluted with N NaOH (15 ml), washed with water (100 ml) and the combined eluate a c i d i f i e d by passage through Amberlite IR-120 (H V). The acid mixture obtained was evaporated to a brown sirup ( l g ) . Methyl(Benzyl Hepta-O-acetyl- ^°maltosid)uronate I To the acid mixture (1.5 g) dissolved i n methanol (15 ml) excess diazomethane in ether was added. After ten minutes the excess diazomethane was decomposed with g l a c i a l acetic acid, and the solution evaporated to a sirup. This material was acetylated in pyridine (20 ml) with acetic anhydride (50 ml) overnight. Recovery in the normal way yielded a sirup which c r y s t a l l i z e d . R e c r y s t a l l i z a t i o n from ethanol or methanol yielded the methyl ester hexaacetate (0.8 g) m.p. 164-165°, C, 55.95j H, 5.62#. Found: C, 53.80; H, 5.71#. Barium (Benzyl ^-Maltosid)uronate To a solution of the methyl ester hexaacetate (415 mg) in methanol (20 ml) a saturated aqueous barium hydroxide solution (40 ml) was added and the solution refluxed on a steam bath for 90 minutes. The solution was neutralized with carbon dioxide, f i l t e r e d , and the precipitate washed with water (15 ml). The combined f i l t r a t e s were a c i d i f i e d by passage through Amberlite IR-120 (H +) and evaporated to a glassy s o l i d . The s o l i d was dissolved in water (2 ml) and neutralized with saturated aqueous barium hydroxide. The prec i p i t a t e obtained upon the addition of ethanol (30 ml) was centrifuged, washed with ethanol (10 ml) and was dried by solvent exchange with ether to give a white powder (300 mg) 2.04 i n CHC1 3). Calculated for c3„ H4o°l8 ! 1.53 i n H g0). -58-Maltobiouronic Acid A small scale experiment (8.25 mg of barium s a l t ) was conducted i n a Warburg type microhydrogenator. Hydrogenolysis was complete after two hours with the hydrogen uptake constant at 1.04 moles. To a solution of the barium s a l t (202 mg) i n water (10 ml) was added palladium oxide on barium sulphate catalyst (270 mg). The mixture was hydrogenolyzed at room temperature i n a Paar apparatus at a pressure of one atmosphere. After two hours the mixture was f i l t e r e d , the precipitate washed with water (10 ml) and the combined f i l t r a t e s a c i d i f i e d by passage through Amberlite IR-120 (H +) and evaporated to a sirup (138 mg). Freeze-drying yielded a product which analyzed as a monohydrate. After drying overnight i_n vacuo (0.02 mm) at 80° the product had l o s t a weight equivalent to 1.2 moles of water. Upon drying at 100° for 36 hours a further 0.5 moles of water was l o s t . Drying was discontinued at this stage as the acid began to darken in d i c a t i n g decomposition. Neutralization equivalent: calculated for cig H2g°_3 ( m o n o n y d r a t e ) , 374; found 367, L ^ J D 1 1 6 ° 2 , 5 2 i n H2 0)« Calculated for C12 H22°13 ( m o n o h y d r a t e ) J c» 38.50; H, 5.88$. Found: C, 38.26; H, 5.76$. Rf 0.051, R - 0.33, R 0.90, solvent ' —± 9 —glucose ' —maltose system A. -59-Methyl (Hepta-O-acetyl- j2 -maltosidjuronate To maltobiouronic acid (55 mg) in methanol (5 ml) was added excess diazomethane in ether, and after 10 minutes the solution was evaporated to dryness. Acetic anhydride (10 ml) and anhydrous sodium acetate (0.5 g) were added and the mixture heated on a steam bath for two hours. The mixture was poured into ice water and extracted into chloroform in the usual manner. Crystals (50 mg) were obtained which upon r e c r y s t a l l i z a t i o n from methanol melted at 197-198°. [o£\ D 77° (c, 0.54 i n CHC1 3). Calculated for C g 7 H 3 6 0 1 9 J C, 48.80} H, 5.42$. Found: C, 4 9 . 2 9 j H, 5.53$. Hydrogenolysis of methyl (benzyl-hexa-O-acetyl-p-maltosid)-uronate (145 mg) i n g l a c i a l acetic acid (5 ml) with Pd/C catalyst (75 mg) for 10 hours at atmospheric pressure gave a material which upon acetylation as described above gave methyl (hepta-O-acetyl- p-maltosid)uronate (43 mg). Melting point and mixed melting point 197-198 . Constitution of Maltobiouronic Acid To maltobiouronic acid (6 mg) in water (5 ml) sodium borohydride (30 mg) was added. After seventeen hours at room temperature the solution was neutralized with acetic acid and evaporated to dryness. The sirup was evaporated with 3$ HC1 in methanol ( 3 x 5 ml) and the residue dissolved o -60-in water (5 ml), passed through Amberlite IR-120 (H +) and evaporated to dryness. The r e s u l t i n g material was dissolved in N H 2S0 4 (3 ml) and hydrolyzed at 100° in a sealed tube overnight. Neutralization of the hydrolyzate with washed barium carbonate gave a f i l t r a t e which upon a c i d i f i c a t i o n by passing through Amberlite IR-120 (H +) indicated only glucuronic acid and s o r b i t o l upon paper chromatography. Reaction of Benzyl Hepta-O-acetyl-^-maltoside with LiAlH^ To a suspension of L i A l H 4 (0.2 g) i n dry tetrahydrofuran (5 ml) was added a solution of benzyl hepta-O-acetyl-(?> -maltoside (54 mg) i n tetrahydrofuran (5 ml). The mixture was s t i r r e d under ref l u x for 1 hour, after which time excess L i A l H 4 was destroyed with ethyl acetate. The mixture was then neutralized with g l a c i a l acetic acid and evaporated to dryness. To the residue anhydrous sodium acetate (0.2 g) and acetic anhydride (5 ml) were added and the mixture l e f t at room temperature overnight. The acetylation mixture was poured into ice water (50 ml), extracted with chloroform (3 x 20 ml), extracts washed i n the usual way and the dried extract evaporated to a sirup. The sirup upon s i l i c a gel thin layer chromatography (solvent E) indicated two major components. The faster of the two corresponded to the sta r t i n g material, benzyl hepta-O-acetyl-^ -maltoside. The second spot was more intense and was presumably nona-O-acetyl m a l t i t o l . -61-1,6-Anhydro-^3 -maltose To a suspension of hexa-0-acetyl~l,6-anhydro-^-maltose (41) (7 g) i n methanol (95 ml) was added a solution of 0.1M sodium methoxide i n methanol (5 ml). After 4 hours the acetate had dissolved, and aft e r a further 14 hours the solution was neutralized with IR 120 (H f). The solution was f i l t e r e d and evaporated to give a sirup which was dissolved in r e f l u x i n g methanol (50 ml). The hot solution was thinned with ethyl acetate (100 ml) and allowed to cool. The c r y s t a l s (5.2 g) were f i l t e r e d and r e c r y s t a l l i z e d in the same manner. M.p. 156-158°. [_o£~\ _ 108° (c, 7.3 i n H 20). L i t . (40), m.p. 150°. D 7 9 ° ' C a l c u l a t e d f o r C12 H20°10 : G» 4 4 * 4 4 5 H, 6.17$. Found: C, 44.02; H, 5.93$. Direct Tosylation of 1,6-Anhydro-^-maltose To 1,6-anhydro-^-maltose (100 mg) dissolved i n pyridine (1 ml) was added t o s y l chloride (53 mg) i n pyridine ( l ml). The solution was l e f t 17 hours at 5°, aft e r which time thin layer chromatography (solvent F) indicated three monotosylates and two ditosylates i n addition to starting material. Varying the temperature of the reaction and concentration of the reagents did not seem to change the r e l a t i v e amounts of the products as indicated by thin layer chromatography. Direct acetylation of the tosylation mixture indicated only two tosylated products on thin layer chromatography in solvent D, -62-presumably due to the i n a b i l i t y of the chromatographic system to separate i n d i v i d u a l isomers. Tosyl esters were i d e n t i f i e d by spraying with diphenylamine in ethanol and viewing under u l t r a v i o l e t radiation (126). l,6-Anhydro-6'-O-trityl-^-maltose To 1,6-anhydro-^3-maltose (2.2 g) powdered and dried over PgOg i£ vacuo f r e s h l y d i s t i l l e d pyridine (25 ml) was added. The mixture was warmed to e f f e c t solution and cooled to room temperature. Recrystallized t r i t y l chloride (2.85 g., 1.5 mole equiv.) was added and the solution l e f t at room temperature for 5 hours. The pyridine solution was added dropwise to ice water (500 ml) with continuous s t i r r i n g . The mixture was then extracted with chloroform (2 x 100 ml), and the chloroform extracts were amalgamated and f i l t e r e d through C e l i t e . The clear solution was thinned with an equal volume of petroleum ether (30-60°) and the precipitate c o l l e c t e d (1.9 g). The t r i t y l ether was r e c r y s t a l l i z e d three times from ethanol-water (1*2, 75 ml/g) to y i e l d material melting at 141-142°. J D 40.8° (c, 2.53 i n MeOH). Calculated for monohydrate C3iH34Oio*HgO: C, 63.70j H, 6.16$. Found: C, 63.78j H, 6.26$. Penta-0-acetyl-l,6-anhydro-6'-O-trityl-^-maltose To l,6-anhydro-6 1-O-trityl-^3-maltose (1.5 g) dissolved -63-in pyridine (15 ml) acetic anhydride (15 ml) was added and the solution l e f t overnight at room temperature. Dropwise addition of the acetylation solution into ice water (1 L) gave the acetylated ether (2.2 g). R e c r y s t a l l i z a t i o n from ethanol - water (3:1, 30 ml/g) gave material melting at 101-102°. ^ c ^ J D S 5 « ° ° (c> 1-55 i n CHClg). Calculated for C4i H44°i5 : c> 63.40j H, 5.67$. Found: C, 62.98j H, 5.84$. 2,2',3,5',4'-Penta-0-acetyl-l,6-anhydro-^ff -maltose Penta-0-acetyl-l,6-anhydro-6 »-0-trityl-^_t-maltose ( l g) was dissolved in 80$ aqueous acetic acid (40 ml) and the solution was kept 3 hours at 50°. To the warm solution, water (90 ml) was added and the mixture kept at 5° for 18 hours. The mixture was f i l t e r e d , the pr e c i p i t a t e washed with 25$ aqueous acetic acid (10 ml), and the combined f i l t r a t e s evaporated to a sirup. Crystals (665 mg) were obtained by dissolving the sirup in hot 2-propanol (25 ml). R e c r y s t a l l i z a t i o n from the same solvent (40 ml/g) gave c r y s t a l s melting at 82-83°. j ^ o - J D 43.4° (c, 2.43 i n CHC1 3). Calculated for C22 H30°15 : C » 4 9 « 4 4 J H» 5.62$. Found: C, 49.87; H, 5.70$. Penta-0-acetyl-l ,6-anhydro-6 ' -0-tosyl-y£?-maltose To the pentaacetate (4 g) dissolved i n dry pyridine (18 ml) t o s y l chloride (7.5 g) was added. After 5 hours at room -64-temperature, 3 drops of water were added to hydrolyze excess tosyl chloride and the solution was poured into ice water (80 ml). This mixture was extracted with chloroform (3 x 100 ml), and the chloroform extracts were combined and washed with water (7 x 100 ml). The solution was dried with calcium chloride, f i l t e r e d and evaporated to give a sirup which, when dissolved in hot 2-propanol (155 ml), gave the c r y s t a l l i n e tosylate (4.25 g). R e c r y s t a l l i z a t i o n from the same solvent (36 ml/g) gave material melting at 170-171°. C, 50.58; H , 5.23j S , 4.65$. Found: C, 50.48; H , 5.11j S, 4.36$. Penta-0-acetyl-l,6-anhydro-6'-azido-6 1-deoxy-p-maltose Penta-O-acetyl-l,6-anhydro-6'-0-tosyl-p -maltose (1.5 g) dissolved in N, N-dimethylformamide (75 ml) was heated on a steam bath with sodium azide (1.5 g) for one hour and was s t i r r e d frequently. After cooling, the solution was poured into an equal volume of water and the mixture extracted with chloroform (4 x 100 ml). The combined chloroform extract was thoroughly washed with water (8 x 200 ml), dried over calcium chloride, f i l t e r e d , and evaporated to a sirup. The sirup was dissolved in hot 2-propanol and the solution cooled to give c r y s t a l s (1.06 g) of the 6'-azide. Recrystal-l i z a t i o n from the same solvent (50 ml/g) yielded c r y s t a l s 1.00 in CHC1 3). Calculated f o r cj?9 H 5 6°i7 S* -65-melting at 151-152°. \j>C^\ 47.9° (c, 2.55 i n CHClg). Calculated for C 2gHg 90 1 4N3: C, 47.23j H, 5.19; N, 7.51$. Found: C, 47.68; H, 5.49; N, 7.58$. 6'-Amino-6'-deoxy-maltose To a solution of penta-0-acetyl-l,6-anhydro-6•-azido-6 1-deoxy- ^ -maltose (560 mg) i n methanol (20 ml) was added 0.1 M sodium methoxide in methanol (4 ml). After 18 hours at room temperature, the solution was neutralized with IR 120 (H +) and evaporated to a sirup which p l a i n l y showed an in f r a r e d absorption band near 2100 ora"^ c h a r a c t e r i s t i c of the azide group (127). The sirup was dissolved i n ethanol (15 ml) and 10$ palladium on charcoal (300 mg) was added. The mixture was maintained at 65° by means of a water bath while a fine stream of hydrogen was bubbled through the solution. After 45 minutes the solution was made s l i g h t l y a c i d i c with 3$ hydrogen chloride in methanol and the hydrogenation continued f o r a further 2 hours. The c a t a l y s t was removed by f i l t r a t i o n and the solution evaporated to give a chromatographically pure sirup R^ 0.054, solvent B, detected with ninhydrin. This material was dissolved in 0.15N hydrochloric o acid and heated i n a sealed tube for 13 hours at 100 . The hydrolysate was neutralized with s i l v e r carbonate, f i l t e r e d and adjusted to pH 5 with 0.3N HC1. Chromatography i n solvent B indicated only two ninhydrin positive materials -66 Rf 0.041 and 0.054. However p-anisidine-trichloroacetate spray indicated glucose, the material showing R f 0.041 and slower running degradation products. The presence of glucose was indicated by comparison with standards i n solvent systems A, B, C, and also by thin layer chromatography (128) i n solvent G. Preparative paper chromatography on Whatman 3MM paper in solvent B gave a small amount (10 mg) of the R^  0.041 material. Hydrolysis of a sample of t h i s material indicated glucose and 6-amino-6-deoxy-glucose, i d e n t i c a l to material prepared by the method of Cramer and co-workers (96). This indicates that the R f 0.041 material i s 6'-araino-6•-deoxy-maltose. The very low y i e l d prevented any (c, 0.3 i n HgO). Calculated for C 1 2 H 2 3 0 _ 0 N » H C 1 : N, 3.70$. Found: N, 3.97$. Potassium Thiolacetate To a solution of potassium carbonate (36 g) in water (500 ml) was added r e d i s t i l l e d t h i o l a c e t i c acid (22 ml). The solution was evaporated to dryness and the residue was extracted with ethanol (2 x 250 ml). The combined extract was f i l t e r e d and evaporated to dryness. The residue was dissolved in hot absolute ethanol (150 ml), decolourizing carbon (1 g) was added, the solution f i l t e r e d and allowed to attempts to prepare suitable derivatives. D -6 7-off cool. After 4 hours at 5°, the s a l t was f i l t e r e d / a n d dried over calcium chloride. T i e l d 14.6 g (129). Penta-O-acetyl-6 1-S-acetyl-l,6-anhydro-6'-deoxy-^ -maltose To a solution of penta-0-acetyl-l,6-anhydro-6 1-O-tosyl-^-maltose (500 mg) i n N,N-dimethylformamide (5 ml) was added potassium thiolacetate (220 mg). The mixture was heated on a steam bath for half an hour. After cooling, water (50 ml) was added and the mixture extracted with chloroform (4 x 50 ml). The combined chloroform solution was thoroughly washed with water (7 x 100 ml), dried over calcium chloride, f i l t e r e d and evaporated to a sirup. The sirup was dissolved i n hot 2-propanol (45 ml) which upon cooling gave c r y s t a l s (300 mg) of the S-acetate. Recrystal-l i z a t i o n of t h i s material from 2-propanol (80 ml/g) yielded c r y s t a l s melting at 219-220°. \^oC~\ D 34.6° (c_, 0.72 i n CHGlg). Calculated for C^H^O^-Si C, 48.66j H, 5.41j S, 5.41$. Found; C, 48.74; H, 5.57; S, 5.24$. Hepta-O-acetyl-6'-S-acetyl-6 1-deoxy- Q^-maltose Penta-O-acetyl-6'-S-acetyl-l,6-anhydro-6 1-deoxy-^-maltose (150 mg) was dissolved i n the acetolysis mixture (3 ml) (HgS04:Ac20:AcOH = 1:70:30). After 3 hours at room temperature the solution was added dropwise to ice water (25 ml). The white powder so obtained was f i l t e r e d and a portion r e c r y s t a l l i z e d -6 8 from ethanol-water (1:5, 100 ml/g). Further r e c r y s t a l l i z a t i o n did not remove a small amount of the /& -anomer which was apparent on thin layer chromatography in solvent D. M.p. C 2 8 H 5 8 ° 1 8 S : C» 4 8 ' 4 2 ? H» 5.49$. Found: C, 48.67j H, 5.79$. 6'-Deoxy-6'-mercapto-maltose To hepta-O-acetyl-6'-S-acetyl-6'-deoxy-0(-maltose (120 mg) in methanol (4 ml) was added a solution of 0.1M sodium methoxide methanol (1 ml). After the solution had been kept at room temperature overnight, i t was neutralized with IR 120 (H*), f i l t e r e d and evaporated to a sirup. Chromatography in solvent A showed two products R^ 0.018 and 0.25 which were the disulphide and the free t h i o l r espectively. When excess 2-mercapto-ethanol and 1 drop of concentrated ammonia were added, only the more mobile compound was present. Chromatography of the acid hydrolysate of 6'-deoxy-6 1-mercapto-maltose indicated glucose and 6-deoxy-6-mercapto-glucose. The l a t t e r was prepared by a route similar to that employed by Akagi, Tejima and Haga (150). Borohydride reduction followed by hydrolysis indicated s o r b i t o l and 6-deoxy-6-mercapto-glucose. Attempts to c r y s t a l l i z e the chromatographically pure disaccharide were unsuccessful. I n s u f f i c i e n t material was recovered to obtain suitable analyses. D 1 5 7 » 6 ° (£> 2 * 0 2 i n Hp0^* D 115° (c, 0.79 in CHClj). Calculated for -69-Penta-0-acetyl-l,6-anhydro-6 1 -deoxy-6 ' -iodo-^p -maltose To a solution of the 6'-O-tosylate (1.2 g) in N,N-dimethylformamide (50 ml) was added f i n e l y powdered sodium iodide (4 g). The solution was heated on a steam bath for 1_- hours. After cooling, the solution was diluted with water (100 ml), and extracted with chloroform (4 x 50 ml). The combined chloroform extract was washed with water (6 x 100 ml), dried over calcium chloride, f i l t e r e d and evaporated. The sirup obtained was dissolved in hot 2-propanol (100 ml) from which c r y s t a l s (1.04 g) were obtained on cooling. R e c r y s t a l l i z a t i o n from the same solvent (100 ml/g) gave material melting at 194-195°. _J>^ 3 D 4 2 « 0 ° 1 « 0 5 i n CHC1 5). Calculated for C^H O u I i C, 40.99j H, 4.50$. Found; C, 41.09; H, 4.58$. Penta-0-acetyl-l,6-anhydro-6'-deoxy- j$ -maltose To a solution containing penta-O-acetyl-1,6-anhydro-6'-deoxy-6*-iodo-^S-maltose (0.5 g) and pyridine (0.1 ml) in ethanol (10 ml) was added 10$ palladium on charcoal (0.5 g). o The mixture was kept at 60 b y means of a water bath and a stream of hydrogen was introduced through a c a p i l l a r y . After four hours the catalyst was f i l t e r e d and the solution evaporated. The r e s u l t i n g sirup was dissolved in chloroform (30 ml) washed with 1$ sodium thiosulphate (15 ml) and f i n a l l y washed with water (4 x 30 ml). The chloroform solution was -70-dried over calcium chloride and evaporated to give a faintly-yellow sirup. The sirup was dissolved in hot 2-propanol (25 ml) which upon cooling gave the c r y s t a l l i n e deoxy sugar (252 mg). R e c r y s t a l l i z a t i o n from 2-propanol (40 ml/g) gave material melting at 141-142 . [ j ^ l D 44«5° (c, 0.88 i n CHC1 3). Calculated for 0 2 2 H 3 0 0 1 4 : C, 50.96J H, 5.77$. Found: C, 50.87; H, 5.89$. 6 1-Deoxy-maltose Penta-0-acetyl-l,6-anhydro-6'-deoxy- p-maltose (485 mg) was dissolved in the acetolysis mixture (10 ml) (H 2S0 4:AcgO: AcOH = 1:70:30). The o p t i c a l rotation varied from £ j ^ J D 89.2° (5 min) to 138.8° (2 h) after which time i t was constant. The f l a s k was cooled i n ice after 3 hours and ice (5 g) was added. The sulphuric acid was neutralized by the addition of M barium acetate and the precipitate removed by centrifugation. The solution was evaporated to give a sirup which was evaporated to dryness with ethanol (3 x 10 ml). The hepta-O-acetate could be obtained as an amorphous powder on p r e c i p i t a t i o n from alcohol-water. The sirupy acetate was dissolved in methanol (10 ml) and a solution of 0.1 M sodium methoxide i n methanol (2 ml) was added. After 17 hours at room temperature the solution was neutralized with IR 120 (H*), f i l t e r e d and evaporated to a sirup. The sirup, which was chromatographically pure, could not be induced to c r y s t a l l i z e even after chromatography -71-on Dowex 50 ¥ x 2 ( L i v s a l t ) (131). Hydrolysis of a sample of the sugar with 2 N sulphuric acid indicated glucose and material i d e n t i c a l to 6-deoxy-glucose prepared by the procedure of Fischer and Zach (132). A second sample, which had been reduced with sodium borohydride before hydrolysis, indicated A portion of the 6'-deoxy-maltose (23.3 mg) was dissolved in pyridine (1 ml) and acetic anhydride (4 ml) was added. The solution was l e f t 30 hours at room temperature, poured into ice water (40 ml), and extracted with chloroform (3 x 30 ml). The combined extract was washed thoroughly with water (6 x 50 ml), dried over calcium chloride, f i l t e r e d and evaporated. The r e s u l t i n g sirup was dissolved in hot ethanol (2 ml) and the solution, upon cooling, yielded c r y s t a l l i n e hepta-0-acetyl-6 '-deoxy- B -maltose. M.p. 183-185°. ("(^ "1 n 64 (£, 0.68 i n CHC1 3). Calculated for C26 H36°17 : C» 5 0 » 3 2 J H, 5.80$. Found: C, 49.57; H, 5.55$, Catalyzed Lead Tetraacetate Oxidations Methyl o^-D-Glucopyranoside To a solution of methyl ^-D-glucopyranoside (26.4 mg., a -72-1.36 x 10""* moles) in g l a c i a l acetic acid (5 ml) was added 50$ aqueous potassium acetate (0.2 ml) and 0.0622 M lead tetraacetate i n g l a c i a l acetic acid. Lead tetraacetate consumption was estimated by iodimetry as outlined by P e r l i n (104). 1,6-Anhydro- ji -D-glucose 1,6-Anhydro- j§-D-glucose m.p. 174°, l i t . (133), m.p. 172°, (19.0 mg., 1.17 x 10~ 4 moles) was oxidized using the same quantities as for methyl ^-D-glucopyranoside. Table 1 Time Methyl o(-D-glucopyranoside 1,6-Anhydro-^-D-glucose (min) moles of tetraacetate consumed per mole of sugar 30 0.07 0.03 120 0.34 0.33 300 0.66 0.75 420 0.91 1.04 1500 2.02 2.12 Benzyl 4',6 t-0-Benzylidene-6-0-tosyl-^-maltoside To a solution of benzyl 4 1,6 1-O-benzylidene- ^-maltoside (650 mg) (134) in dry pyridine (5 ml) was added r e c r y s t a l l i z e d t o s y l chloride (286 mg) in dry pyridine (5 ml). The reaction proceeded 40 hours, during which time samples were removed f o r 73-thin layer chromatography (solvent F). Chromatography indicated that three major products were formed. Two of the products t r a v e l l e d at a rate corresponding to mono-tosylates, the other corresponding to a ditosylate. It was obvious that the s e l e c t i v i t y of tosylation was not s u f f i c i e n t l y s p e c i f i c to obtain the 6-tosyl derivative. Benzyl 4',6'-O-Benzylidene-6 - 0 -trityl - y f f -maltoside To a solution of benzyl 4 ' ,6 '-O-benzylidene-^-maltoside (35 mg) i n dry pyridine (0.35 ml) was added t r i t y l chloride (25 mg). The reaction was followed by thin layer chromatography in solvent D, which indicated no reaction had occurred even after 3 days at room temperature. Benzyl 4',6 1 -0-Benzylidene-6 -0-mesyl-^ -maltoside To a solution of benzyl 4 1,6 '-O-benzylidene-^-maltoside (300 mg) in dry chloroform (25 ml) was added dry pyridine (5 ml) and the solution cooled to 5°. Mesyl chloride (0.06 ml) was then added. The solution remained at 5° for 5 hours, and a drop of water was then added to hydrolyze any excess reagent. The solution was extracted with water (6 x 30 ml), dried over calcium chloride, and evaporated. Crystals which melted at 86-89° were obtained from ethyl acetate - petroleum ether (30-60°). 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Ber. £6, 27 (1933). 117. W. T. Haskins, R. M. Hann and C. S. Hudson. J. Am. Chem. Soc. £4, 1289 (1942). 118. ¥ . T. Haskins, R. M. Hann, and C. S. Hudson. J. Am. Chem. Soc. £4, 1852 (1942). 119. M. L. Wolfrom, R. M. de Lederkremer and L. E. Anderson. Anal. Chem. £5, 1357 (1963). 120. M. Gee. Anal. Chem. 35, 350 (1963). 121. Y. U. Zhdanov, G. A. Korol'chenko and G. N. Dorofeenko. Dokl. Akad. Nauk. SSSR. 143, 852 (1962). C.A. 57, 4747 (1962). 81 122. J. M. Swan. Nature 180, 643 (1957). 123. L. Hough, J. K. N. Jones and ¥. L. Wadman. J. Chem. Soc. 1702 (1950). 124. R. U. Lemieux and H. F. Bauer. Anal. Chem. 2_3, 920 (1954). 125. H. G. Brown and C. A. Brown. J. Am. Chem. Soc. 84, 2827 (1962). 126. M. Jackson and L. D. Hayward. J. Chromatog. _5, 166 (1961). 127. R. D. Guthrie and D. Murphy. J. Chem. Soc. 5288 (1963). 128. G. W. Hay, B. A. Lewis and F. Smith. J. Chromatog. 11, 479 (1963). 129. B. Bannister and F. Kagan. J. Am. Chem. Soc. 82, 3363 (1960). — 130. M. Akagi, S. Tejima and M. Haga. Chem. Pharm. B u l l . (Tokyo) 10, 562 (1962). 131. J. K. N. Jones, R. A. Wall and A. 0. P i t t e t . Can. J. Chem. J58, 2285 (1960). 132. E. Fischer and K. Zach. Ber. _U5, 3761 (1912). 133. R. B. Ward. Methods in Carbohydrate Chem. 2, 394 (1963). 134. A. Klemer. Chem. Ber. 92, 218 (1959). SYNTHESIS OF THE 2,4-DI-0-METHYL TETROSES - 8 2 -INTRODUCTION Purpose The aim of preparing p a r t i a l l y methylated tetroses was to f a c i l i t a t e the i d e n t i f i c a t i o n of small quantities of p a r t i a l l y methylated sugars by characterization of their periodate oxidation products. Periodate oxidation may supply not only information on the number of free v i c i n a l hydroxyls present, but also, analysis of the products for formic acid and formaldehyde gives information as to their l o c a t i o n . Formaldehyde then, indicates the presence of a primary alcohol in a 1,2-glycol system, whereas formic acid indicates a secondary alcohol flanked by two other alcohol groups. I d e n t i f i c a t i o n of the fragments might allow f u l l characterization of the sugar. Two recent communications have emphasized the need for reference compounds in the tetrose series. Had i d e n t i f i c a t i o n of the tetroses produced by periodate oxidation been possible, the intensive investigation necessary to determine the parent sugar would not have been necessary. Stephen ( l ) , in his structural investigation of V i r g i n i a  oroboides gum, used periodate oxidation to establish the positions of substitution on p a r t i a l l y methylated sugars. -83-Several chromatographically f a s t running products were obtained which, from consideration of the assigned structure of the parent sugar, were believed to be of the tetrose series. Stephen assigned probable structures for these products, only after extensive investigation to establish the structure of the parent sugar. P a r t i a l l y methylated glucoses, obtained from hydrolysis of a methylated glucan (2), gave, upon periodate oxidation, methylated reducing fragments, believed to be tetroses. To provide reference compounds for structural determinations of t h i s type, the synthesis of the 2,4-di-0-methyl tetroses was undertaken. Background Tetrose i s the generic name describing four carbon sugars. The class of compounds belonging to th i s family, which contain a straight carbon chain terminated by an aldehydic functional group, possess two asymmetric carbon atoms and therefore consists of four stereoisomeric compounds. The synthesis of tetroses can be approached from two dir e c t i o n s . Starting with D or L-glyceraldehyde, an ascent of the series can be carried out through addition of a further carbon atom by various chemical means, increasing the length of the three carbon sugar by one. The other major approach -84-i s through degradation of higher carbon sugars by cleavage of a carbon-carbon bond. Ascent of the series can be accomplished by the c l a s s i c a l K i l i a n i synthesis (3), i n which the aldose i s treated with hydrogen cyanide forming a n i t r i l e , which, through hydrolysis and reduction, regenerates an aldehyde containing one more hydroxy-methylene group than i t s precursor. A newer method, developed by Sowden and Fischer (4, 5), involves the condensation of the aldose with nitromethane. Subsequent hydrolysis of the n i t r o compound gives the new aldehyde in good y i e l d . An inherent drawback in any method f o r ascent of the series i s the introduction of a further asymmetric carbon atom into the molecule, giving r i s e to the p o s s i b i l i t y of two products. Generally, both possible products are formed, one usually i n higher y i e l d . The separation of these epimers constitutes the major drawback to methods which involve ascent of the serie s . Several methods are available for the degradation of the carbon chain of a sugar. A c l a s s i c a l method such as the Wohl degradation (6) requires the elimination of hydrogen cyanide from the oC-hydroxyl n i t r i l e . A newer method (7) involves the oxidation of di e t h y l dithio acetals with per-acids. Subsequent treatment with base eliminates the disulfone, -8 5-y i e l d i n g an aldehyde of one carbon l e s s . Probably one of the most useful and highest y i e l d i n g methods i s degradation through glycol cleavage. Cleavage between suitably disposed 1.2- d i o l systems by periodate (8), or less frequently lead tetraacetate, r e s u l t s i n two aldehydes. The use of both of these reagents has been recently reviewed (9, 10). The yiel d s from periodate cleavage are quantitative, in f a c t Malaprade (8) o r i g i n a l l y introduced this reaction as a method of quant i t a t i v e l y estimating periodate. Limitations i n degradative methods are r e a l i z e d when i t i s seen that a suitably orientated and substituted pentose must be used in the synthesis of a tetrose by the C-l elimination method, and that contiguous hydroxyl groups should not be present in a product expected to be produced by glycol cleavage. The chemistry of the twenty-four p a r t i a l l y O-methylated tetroses has only been b r i e f l y investigated. Pri o r to t h i s work, only three reports of synthetic p a r t i a l l y methylated tetroses had been published. Periodate oxidation of 3-0-methyl-D-xylose gave 2-0-methyl-D-threose (11), and s i m i l a r l y 2.3- di-O-methyl-D-arabinitol gave 2,3-di-0-methyl-D-threose (12). Although the preparation of 3-0-methyl-L-threose has been described (13), no physical properties of the product were described at that time. Previous workers in th i s group have attempted synthesis of 2,3-di-O-methyl-L-threose (14), -86-2,3-di-O-methyl-D-erythrose (14), 2-0-inethyl-D-erythrose (15), and 2,4-di-O-methyl-L-erythrose (16). -87-METHODS OF SYNTHESIS 2,4-Di-O-methyl-D-erythrose The synthesis of 2,4-di-O-methyl-D-erythrose was achieved by periodate oxidation of 4,6-di-O-methyl-D-glucitol. The g l u c i t o l was prepared by borohydride reduction of 4,6-di-0-methyl-D-glucose. 4,6-Di-O-methyl-D-glucose was synthesized by the method of B e l l and Lorber (17) which depends on the formation of a 4,6-benzylidene group on methyl ^-D-glucoside allowing blockage of hydroxyls 2 and 3 as benzyl ethers. The l a b i l i t y of the benzaldehyde acetal to weak acid allows p r e f e r e n t i a l removal of t h i s group. Subsequent methylation gives the di-O-methyl derivative. Reductive cleavage of the benzyl ethers and acid hydrolysis of the glycoside y i e l d the free sugar (Fig. 1). It i s i n t e r e s t i n g to note that while B e l l and Lorber*s method begins with the r e a d i l y available methyl ^-D-glueoside, the intermediate compounds are either sirups or d i f f i c u l t l y c r y s t a l l i z a b l e compounds. Dennison and McGilvray (18) on the other hand, start with the less common methyl /S -D-glucoside using the same sequence of reactions to give the ^-glycoside. In t h i s anomeric series a l l the intermediates were found to be c r y s t a l l i n e . This further substantiates - 8 8 -CH 2 OH / 0 C H 2 0CH HO'X .OH OCH-Z n C t g 0CHO 0CH 2 C1 KOH C H 2 0 H 0 C ^ O C H 2 \ = = TOCH 2 0 Na/Hg EttOH ' 9 H 2 O C H 3 0 C H 3 O l \ | O H H,OH OH 10 4 "0, O C H 2 0 t y » O C H 3 OCH 2 0 A g 2 0 CH :H 2 OCH 3 ^ O ' X ^ O H . / 1 0 C H 3 OH N a B H 4 0 CH }-OCH3 f-OH C H 2 O C H 3 OCH 3 H* ; H 2 O C H 3 ™"o< 3« C H 3 0 ' \ 0 £ H | 5 / 1 0 C H 3 OCH 2 0 H + CH 2 OH L 0 H HOH 0 C H 3 J-OH C H 2 0 C H 3 Fig. I -89-Zemplen's statement (19) that compounds containing ^ - l i n k a g e s are more d i f f i c u l t to c r y s t a l l i z e than the corresponding compounds with the j!> -linkage. Periodate oxidation of the reduced 4,6-di-0-methyl-D-glucose resulted in a 2.03 molar uptake of periodate per mole of sugar. After p r e c i p i t a t i o n of the iodate and excess periodate as barium s a l t s , the solution was evaporated to give a sirup. Characterization was achieved by formation of the 2,4-dinitrophenylhydrazone and measurement of the o p t i c a l r o t a t i o n . 2,4-Di-O-methyl-L-erythro se 2.4- Bi-O-methyl-L-erythrose was synthesized by periodate oxidation of 3,5-di-O-methyl-L-arabinitol. This alcohol was obtained through the borohydride reduction of 3,5-di-0-methyl-L-arabinose. 3.5- Di-O-methyl-L-arabinose was synthesized according to the procedure of H i r s t , Jones and Williams (20). This route r e l i e s on the acid s t a b i l i t y of the 5-tosyl ester formed by treating an o(,/3 mixture of methyl arabinofuranosides with t o s y l chloride in pyridine. The acid s t a b i l i t y allows the replacement of the methyl glycoside with 1,2-isopropylidene without concurrent ring expansion. In this way 1,2-isopropy-- 9 0 -lidene-5-tosyl-arabinose i s i s o l a t e d . Removal of the 5-to s y l group by reductive cleavage leaves the 3 and 5 hydroxyls free to be methylated. Acid hydrolysis of the isopropylidene group of the methylated derivative y i e l d s 3,5-di-0-methyl-L-arabinose (Fig. 2). A possible source of 3,5-di-O-methyl-L-arabinose i s f u l l y methylated mesquite gum. Several workers (21, 22) have hydrolyzed t h i s methylated gum with weak acid to cleave the arabinofuranose linkages. The dimethyl sugar was is o l a t e d from the hydrolyzate mixture by f r a c t i o n a l d i s t i l l a t i o n of the methyl glycosides. Unfortunately, traces of 2,5-di-O-methyl-L-arabinose aire present i n such preparations (23), and cannot e a s i l y be removed. 2,4-Di-O-methyl-L-erythrose i s i d e n t i c a l to i t s o p t i c a l isomer 2,4-di-O-methyl-D-erythrose, in a l l i t s properties except o p t i c a l rotation. The s p e c i f i c rotation was equal in magnitude but opposite i n sign as would be expected. 2,4-Di-O-methyl-D-threose Periodate oxidation of 3,5-di-O-methyl-D-xylitol yielded 2.4- di-O-methyl-D-threose which, upon p u r i f i c a t i o n , yielded a c r y s t a l l i n e product. The p e n t i t o l was obtained from the known 3.5- di-0-methyl-D-xylose (24) by borohydride reduction. - 9 1 -0 CH 1 Q4~ r CH3Q-I H0-| ( C H 2 O C H 3 Fig. 2 -92-3,5-Di-O-methyl-B-xylose was prepared by the method of Levene and Raymond (24) (Fig. 3). D-Xylose was condensed with two molecules of acetone in the presence of concentrated sulphuric acid to give 1,2-: 3,5-diisopropylidene-D-xylose. P a r t i a l hydrolysis of the diisopropylidene compound with dilute aqueous acid yielded 1,2-isopropylidene-D-xylo-furanose which was r e c r y s t a l l i z e d for the f i r s t time to give the pure compound. Methylation of 1,2-isopropylidene-D-xylofuranose by the method of Kuhn e_t a l (25) yielded a sirup which was p u r i f i e d by vacuum d i s t i l l a t i o n . Hydrolysis of the isopropylidene group with 25$ aqueous acetic acid yielded chromatographically pure 3,5-di-0-methyl-B~xylose. Borohydride reduction followed by periodate oxidation yielded 2,4-di-0-methyl-D-threose which c r y s t a l l i z e d after p u r i f i c a t i o n by s i l i c a gel column chromatog-raphy. 2,4-Di-O-methyl-L-threose 2,4-Di-O-methyl-L-threose was obtained from the periodate oxidation of the borohydride reduction product of 1,4,6-tri-0-methyl-L-sorbose. This sugar was synthesized by the method of Schlubach and Olters (26). Condensation of L-sorbose with acetone yielded 2,3:4,6-diisopropylidene-L-sorbose. P a r t i a l acid hydrolysis yielded 2,3-isopropylidene-L-sorbofuranose which, upon methylation, yielded the 1,4,6-tri-0-methyl -94-derivative. The trimethyl ether was obtained as a c r y s t a l l i n e compound, but no attempt was made to r e c r y s t a l l i z e i t due to i t s low melting point (m. p. 15-17°). Acid hydrolysis of 2,3-isopropylidene-l,4,6-tri-0-methyl-L-sorbose l i b e r a t e d the trimethyl ether as the free sugar. Borohydride reduction of 1,4,6-tri-O-methyl-L-sorbose followed by periodate oxidation yielded 2,4-di-0-methyl-L-threose (Fig. 4). The sirup could not be induced to c r y s t a l l i z e even when seeded with i t s o p t i c a l isomer. It i s important to note that reduction of a ketose such as sorbose may y i e l d two h e x i t o l s . In the reduction of 1,4,6-tri-O-methyl-L-sorbose, 1,4,6-tri-0-methyl-L-gulitol (or 1,3,6-tri-O-methyl-D-glucitol) and l,4,6-tri-0-methyl-L-i d i t o l (or 1,3,6-tri-O-methyl-L-iditol) may be formed. The configuration of the derived tetrose has no bearing on the configuration of the carbon atom at position 2, as i t i s eliminated in the oxidative cleavage. S i l i c a gel thin layer chromatography of the reduction mixture indicated only one major component, the stereochemistry of which can possibly be infe r r e d . Bragg and Hough have shown that borohydride reduction of aldoses and ketoses proceeds through the a c y c l i c staggered zig-zag conformation (27). 1,4,6-Tri-O-methyl-L-sorbose, when represented in t h i s fashion -95--96-(Fi g . 5), has the bulky methoxyl above the plane of the paper. Approach of the borohydride w i l l come then, predominantly, from below the plane of the paper. Such attack w i l l leave the newly formed hydroxyl group above the plane of the paper. The derivative so formed i s 1,4,6-tri-O-methyl-L-iditol as represented in F i g . 5. Since evidence of the configuration of carbon atom 2 i s not e a s i l y obtained, the v a l i d i t y of t h i s prediction cannot be evaluated. - 9 7 -H 0 X 0 H , C H 2 0 C H 3 CH3OH2C 0CH3 CH2OCH3 Ho^ HO hOCH: CH2OCH3 CH,OCH~ H OCH, \r 3 9 ^ c / C \ C / C \ H OH H OH ' H OCH-2 H • OH CH 3OCH 2 „C C CH20CH3 H OH C H OH CH2OCH3 CH2OCH3 T-OH -HO' •OCH 3 CH 2 OCH 3 Fig. 5 -98-DISCUSSION Incomplete Periodate Oxidation of Reducing Sugars Incomplete periodate oxidation of reducing sugars has been reported (1) and was substantiated in t h i s work. The reason for t h i s resistance to periodate oxidation i s l i k e l y due to the formation of formyl esters and c y c l i c inner acetals. Oxidative cleavage of the C-l carbon of an aldopyranose or aldofuranose sugar yields a formyl ester s t i l l attached to the sugar. The removal of t h i s formyl ester must precede any cleavage that involves the e s t e r i f i e d hydroxyl. Inner c y c l i c acetals have been shown to form (28) whenever a free hemiacetal formed contains a six membered r i n g . In t h i s way a hydroxyl group which would be expected to undergo periodate oxidation can be inactivated by the formation of such an inner acetal. Stephen (1), circumvented the danger of formyl ester formation by reduction of the free sugar to the corresponding h e x i t o l . In a similar manner, to obtain t h e o r e t i c a l periodate uptake values i n the synthesis of the 2,4-di-0-methyl tetroses, i t was found necessary to reduce the free sugar to the alcohol p r i o r to the glycol cleavage. aldehyde that i s , the c y c l i c -99 One of the most convenient methods of reducing sugars i s treatment with aqueous sodium borohydride. As discussed e a r l i e r (see page 94 ) Bragg and Hough (27) have shown that borohydride reduction i s preceded by opening of the sugar into i t s a c y c l i c zig-zag conformation. In t h i s conformation function w i l l retard the rate of reduction. They attribute t h i s to the s t e r i c hindrance of approach of the borohydride ion to the 1,3-system. Although 3 - 0-substituted aldoses constitute the majority of examples of such rate reduction, 4 - 0 -substituted ketoses belong to t h i s stereochemical class and show the same e f f e c t . This was discussed e a r l i e r i n the reduction of l , 4 , 6 - t r i - 0 -methyl-L-sorbose. In the preparation of 3,5-di - 0-methylpentitols from the parent pentoses and l,3,6-tri - 0-methylhexitols from 1,4,6-tri-O-methyl-L-sorbose, borohydride reduction was not complete afte r 24 hours. If longer times were used, detectable amounts of degradation products were formed; these were, presumably, due to Lobry de Bruyn - van Ekenstein transformations. Reductions were carr i e d out overnight and subsequent separation of the product from the unreduced material was effected by chromatography on a s i l i c a gel column using butanone-water azeotrope as the solvent. any bulky substituent in the position -100-Chromatography The use of s i l i c a gel columns originated with B e l l (29), who estimated the chain length of methylated polysaccharides by use of this method. Elution of the f u l l y methylated sugars, formerly non-reducing end groups, allowed estimation of the r a t i o of non-reducing end group to backbone residues. With the advent of cellulo s e chromatography, the use of s i l i c a gel columns was neglected. In p u r i f i c a t i o n s , such as described in this work, s i l i c a gel column chromatography offers comparable resolution in approximately one-fourth of the time. It should be noted that thin layer chromatography i s a f a s t , e f f i c i e n t method of analyzing methylated sugar fractions (30) obtained from either cell u l o s e or s i l i c a gel columns. Because the importance of p a r t i a l l y methylated tetroses l i e s in t h e i r chromatographic recognition, and R Q values were recorded in a large number of solvent systems (Table 1). - J . CI-T A B L E I Physical properties of 2,4-di-O-methyI tetroses 2,4-Di-O-methyl 2,4-Di-O-methyl 2,4-Di-O-methyl 2,4-Di-O-methyl D-erythrose L-erythrose D-threose L-threose Melting point [<*]D of free sugar 2,4-Dinitrophenyl-hydrazone melting point Solvent system1 Butanone Water Azeotrope Ethyl acetate Pyridine Water (8:2:1) Butan-l-ol Ethanol Water (4:1:5) upper layer Ethyl acetate Acetic acid Formic acid Water (18:3:1:4) Sirup 60.1° (c, 1.4 in MeOH) 105-106° RF 0.70, 0.64 Ro 0.86, 0.78 RF 0.77 Ro 0.92 RF 0.82 Ro 0.96 RF 0.80, 0.75 Ro 0.95, 0.SS Sirup -61.4° (c, 4.< in MeOH) 107-108° Silica gel T. L. C. RF 0.62, 0.50 114-116° (dimer?) - 1 4 . 8 ° (e, 1.17 in MeOH) - 9 ° (3 min) -> + 0.9° (15 min) (c, 0.35 in 1 iVHjSOO 14S-1490 RF 0.66, 0.57 R0 0.89, 0.76 RF 0.81 Ro 0.96 RF 0.84 Ro 0.98 RF 0.86, 0.75 Ro 1.02, 0.S8 Sirup - 1 4 . 3 ° (c, 5.7 in MeOH) - 1 7 ° (5 min) -> - 3 ° (15 min) (c, 0.55 in 1 ATHjSOO 149-150° Silica gel T. L. C. RF 6.65, 0*.50 ^Descending paper chromatography was carried out using Whatman No. 1 paper and Ro values are relative to 2.3,4,0 tetra-O-methyl-D-glucose. Sugars were detected by use of p-anisidine trichloracetate spray reagent. Optical isomers showed identical chro-matographic patterns in all solvents. RF and Ro values quoted for D-threose refer to the sirup before crystallization. The crystalline modification gave only the slower running component. Ascending silica gel thin-layer chromatography was adopted for rapid examination of column fractions. Detection was achieved by use of 6% H N O i in HiSO*. Subsequent heating at 150 was sufficient for development. -102-Anomalous Behaviour of Optical Isomers Paper chromatography of the tetroses i n some solvents and chromatography on a s i l i c a gel thin-layer system indicated the existence of two modifications of the tetroses, which were inseparable by s i l i c a gel column chromatography. Upon standing f o r extended lengths of time (3 months), sirupy tetroses were observed to become more viscous. Infrared spectra of the viscous sirup showed reduction i n the carbonyl stretching band at 1750 cm""^  and enhancement of the hydroxyl band at 3400 cm~^ ", with respect to the more mobile sirup. The infrared spectrum of the c r y s t a l l i n e 2,4-di-0-methyl-D-threose showed no carbonyl stretching band but contained a hydroxyl band which was s p l i t into two equal-intensity absorptions at 3375 and 3480 cm"^. The anomalous o p t i c a l rotation of the threose isomers (D, -14.8°; L, -14.3°) in methanol i s attributed to the existence of two or more modifications present in the solution of the sirupy L-threose compound. Upon solution of the isomers in 1 N sulphuric acid, the rotations dropped to near-zero values with opposite signs. These res u l t s suggest that 2,4-di-0-methyltetroses e a s i l y undergo dimerization to a compound of structure similar to that from 5-aldo-l,2-0-isopropylidene-D-xylopentofuranose (31) (Fig. 6). The structure of the c r y s t a l l i n e modification 103-of Z,4-di-0-methyl-D-threose may therefore be of a c y c l i c acetal nature (Fig. 7). Thin layer chromatography of the tetroses i s o l a t e d as sirups contained appreciable quantities of modifications of this type. Because of the small amounts of product, molecular weight and nuclear magnetic resonance studies were not undertaken. - 1 0 4 -H0^ H C <\ OH CH30"i H-C - \ 1 J - Q / 0CH3 CH2OCH3 CH2OGH3 Fig J ' - 1 0 5 -Derivative s Attempts to prepare c r y s t a l l i n e derivatives of the g l y c i t o l s were unsuccessful. Although some acetates and p-nitrobenzoates were prepared and shown to be homogeneous by thin-layer chromatography (32), c r y s t a l l i z a t i o n could not be induced. Acceptable a n a l y t i c a l values were seldom obtained for the sirupy di-O-methyltetroses presumably because of occluded solvent. No attempt was made to d i s t i l them because of the small amounts obtained and t h e i r v o l a t i l i t y . Several d i f f e r e n t reagents were t r i e d for characterizing the tetroses but only 2,4-dinitrophenylhydrazine gave s a t i s f a c t o r y c r y s t a l l i n e derivatives in a l l four cases. This reagent was used in neutral solution to give the phenylhydrazone rather than the osazone. In retrospect, the d i f f i c u l t y of making c r y s t a l l i n e derivatives may be due to the equilibrium with a dimeric form. -106-EXPERIMENTAL Evaporations were carried out under reduced pressure at a bath temperature of 40-45°. Optical rotations are equilibrium values measured on either a Bendix ETL-NPL Automatic Polarimeter (Type 143 A) or a Rudolph Polarimeter (Model 219) at 21 i 2°. Melting points quoted are uncorrected. Periodate oxidation estimations were carried out by quenching oxidation aliquots in buffered arsenite and b a c k - t i t r a t i n g with iodine. Solvent system A: butanone - water azeotrope. Preparation of the S i l i c a Gel Column To s i l i c a gel (Fisher S-157, 200 g) that had been screened to pass through 60 mesh was added butanone - water azeotrope (500 ml) with s t i r r i n g . The s l u r r y was allowed to s i t for 1 hour with occasional s t i r r i n g . The mixture was then s l u r r i e d into a column (3 x 36 cm), f i t t e d with a f r i t t e d disk, and the s i l i c a was packed t i g h t l y by tapping the outside of the column u n t i l no further s e t t l i n g was apparent. A 1 cm layer of sand was c a r e f u l l y placed on top of the s i l i c a gel. The column was equilibrated by passing butanone - water azeotrope through the column for two days. At the end of this time the flow rate was constant at 1.8 ml per minute. The movement of the solvent can be approximated with Sudan IV dye which shows an R_> of about 0.95 on thin layer -107-plates. Using t h i s as a marker the front time f o r the column was about 1 hour. Fractions from the column were analyzed by running samples on s i l i c a gel thin layer plates using the same solvent system as that used on the column. Synthesis of 2,4-Di-O-methyl-D-erythrose 4,6-Di-O-methyl-D-glucose 4,6-Di-O-methyl-D-glucose was prepared by the method of B e l l and Lorber (17). After r e c r y s t a l l i z a t i o n from ethyl acetate the constants were: m.p. 154-156 °. C°0 n 116° * 4,6-Di-O-methyl-D-glucitol (1,3-Di-O-methyl-L-gulitol) 4,6-Di-O-methyl-D-glucose (2.7 g) was dissolved i n water (50 ml) and sodium borohydride (0.5 g) was added. The solution was neutralized with acetic acid a f t e r 18 hours, and evaporated to dryness. The r e s u l t i n g s o l i d was treated with Z% HG1 in methanol (3 x 15 ml) and evaporated to dryness. The residue was dissolved in water (25 ml) and the solution de-ionized with Amberlite IR-120 (H f) and Duolite A-4 (0H~). Evaporation of the r e s u l t i n g solution gave a non-reducing sirup (2.6 g), 70.5° (c, 1.23 i n HgO). L i t . 1 0 8 ° >- 65.7° (c, 4 in H g0). -108-4,6-Di-0-methyl-D-glucitol Phenylurethan To 4,6-di-O-methyl-D-glucitol (100 mg) in pyridine (1 ml) was added phenylisocyanate (0.3 ml) and the solution was heated on a steam bath for 3 hours. Anhydrous methanol (1 ml) was added and heating continued f o r a further 15 minutes. The cooled solution was poured dropwise into cold water (25 ml) and the pr e c i p i t a t e f i l t e r e d . R e c y r s t a l l i z a t i o n from ethanol-acetone gave a product melting at 195-199°. By r e c r y s t a l -l i z a t i o n from ethyl acetate - petroleum ether (30-60°) the melting point could be raised to 203-205°. The mixed melting point with diphenylurea (m.p. 238°) was depressed but nitrogen analyses were always high. Calculated for C3g H 3g 0 _ o N4 J N, 8.16$. Found: N, 9.57, 9.61$. 2,4,Di-0-methyl-D-erythrose 4,6-Bi-0-methyl-D-glucitol (524 mg, 2.5 mmoles) in water (25 ml) was added to 0.12 M sodium periodate (50 ml, 6 mmoles). The oxidation was complete in 25 minutes with an uptake of 2.03 moles of periodate per mole of h e x i t o l . The solution was neutralized with washed barium carbonate, and diluted with an equal volume of methanol, f i l t e r e d and evaporated to give a mobile sirup which was p u r i f i e d by chromatography on a s i l i c a gel column to give 2,4-di-0-methyl-D-erythrose (297 rag) 1.4 in MeOH). -109-2 ,4-Dinitrophenylhydrazone of 2 ,4-Di-0-methyl-D-erythrose 2.4- Di-O-methyl-D-erythrose (100 rag) was dissolved in 100$ ethanol (5 ml) and r e c r y s t a l l i z e d 2,4-dinitrophenylhydrazine (110 mg) was added. The mixture was refluxed on a steam bath for 5 hours, evaporated to dryness, dissolved in ethyl acetate (10 ml) and petroleum ether (30-60°) (10 ml) was added to pr e c i p i t a t e excess reagent. The mixture was f i l t e r e d after 10 minutes, the f i l t r a t e evaporated to a sirup, dissolved in chloroform (2 ml) and applied to an alumina column (3 x 20 cm). Colle c t i o n of the l i g h t yellow band eluted with chloroform afforded yellow-orange c r y s t a l s melting at 102-103°. Subsequent r e c r y s t a l l i z a t i o n from ethyl acetate - petroleum ether (30-60°) gave c r y s t a l s melting at 105-106°. Calculated for C 1 2H 1 60 7N 4: N, 17.07; OCHj, 18.90$. Found: N, 17.04; 0CH5, 19.23$. Synthesis of 2,4-Di-0-methyl-L-erythrose 3.5- Di-0-methyl-L-arabinose 3,5-Di-O-methyl-L-arabinose was obtained by the procedure of H i r s t , Jones and Williams (20). The sirup showed only one component (R^ 0.59) upon paper chromatography in solvent system 6.7 in MeOH). L i t . (20), D 39° (25$ aqueous acetic acid). -110-3,5-Di-O-methyl-L-arabinonolactone 3,5-Di-0-methyl-L-arabinose (75 mg) in water (4 ml) was treated with bromine (4 drops) and the mixture l e f t at room temperature overnight. Aeration and treatment with s i l v e r carbonate followed by f i l t r a t i o n gave a solution which was passed through IR-120 (H*), and evaporated to a sirup. Sublimation at 65-70° (0.05 mm) gave c r y s t a l s of 3,5-di-0-methyl-L-arabinonolactone, m.p. 72-73°. L i t . (20), m.p. 73°. 3,5-Di-O-methyl-L-arabinonamide 3,5-Di—O-methyl-L-arabinonolactone (6 mg) was dissolved in methanol saturated with ammonia (1 ml) and the solution was l e f t overnight at room temperature. The solution was evaporated to give c r y s t a l s , which after r e c r y s t a l l i z a t i o n from acetone, melted at 144°. L i t . (20), m.p. 144°. 3,5-Di-O-methyl-L-arabinitol (1,3-Di-O-methyl-L-lyxitol) 3,5-Di-O-methyl-L-arabinose (1.7 g) dissolved in water (30 ml) was treated with excess sodium borohydride (1 g) and l e f t at room temperature overnight. The solution was worked up in the same manner as described for 4,6-di-0-methyl-D-g l u c i t o l . The sirup containing 3,5-di-0-methyl-L-arabinitol and 3,5-di-O-methyl-L-arabinose ( c a . 9:1) was separated completely on the s i l i c a gel column. The arabinose (R_« 0.56 -111-T.L.C. same solvent system) was found in the eluate, 175-300 mis after the dye marking the front. The a r a b i n i t o l (R^ 0.40) was found in the 675-1000 ml f r a c t i o n . Evaporation of the f r a c t i o n containing a r a b i n i t o l yielded a non-reducing sirup (1.4 g) J D -7° (c, 5.8 in CHClj). 2.4- Di-O-methyl-L-erythrose 5.5- Di-O-methyl-L-arabinitol (527 mg, 2.93 mmoles) i n water (200 ml) was treated with 0.08 M sodium periodate (50 ml, 4.0 mmoles). The oxidation was complete in 10 minutes and was worked up as described for i t s o p t i c a l isomer. The f i n a l periodate uptake was 1.04 moles per mole of p e n t i t o l . P u r i f i c a t i o n on s i l i c a gel gave a colourless sirup (365 mg) D -61.4° (c, 4.85 in MeOH). 2,4-Dinitrophenylhydrazone of 2,4-Di-0-methyl-L-erythrose The hydrazone was prepared using the same quantities as that for i t s o p t i c a l isomer, giving c r y s t a l s , which upon r e c r y s t a l l i z a t i o n from ethyl acetate - petroleum ether (30-60°), melted at 107-108°. Calculated f o r C_2H_g0_N4: N, 17.07; 0CH3, 18.90$. Found: N, 16.97; 0CH3, 19.08$. -112-Synthesis of 2,4-Di-Q-methyl-D-threose l,2-Isopropylidene~D-xylofuranose 1,2-Isopropylidene-D-xylofuranose was prepared by the acid hydrolysis of l,2:3,5-diisopropylidene-D-xylose. The product was r e c r y s t a l l i z e d from ethyl acetate - petroleum H 20). Calculated f o r C gH 1 40 5: C, 50.52; H, 7.57$. Found? C, 50.68; H, 7.65$. 3,5-Di-0-methyl-l,2-isopropylidene-D-xylose 1,2-Isopropylidene-B-xylofuranose was methylated by the method of Kuhn ejb a l (25). The compound was i s o l a t e d as a sirup, which was d i s t i l l e d under vacuum, c o l l e c t i n g the f r a c t i o n b o i l i n g at 80-82° (0.05 mm). The mobile l i q u i d had a s p e c i f i c 3,5-Di-0-methyl-D-xylose 3,5-Di-0=methyl-l,2-isopropylidene-D-xylose (5 g) was dissolved in 25$ aqueous acetic acid and heated on a steam bath for 5 hours after which time the o p t i c a l rotation was. constant. After evaporation a yellow sirup (2.1 g) remained which showed only one component (Rf 0.57) upon paper ether (30-60°) (1:4) m.p. 67-69° in H 20). L i t . (33), m.p. 41-43° rotation 5.2 in CHC1 3). L i t . (34), -113-chromatography in solvent system A, Q ^ - ^ ] p 23.5° (£, 3.9 in H 20). L i t . (34), D 25° (c, 1.13 in HO). p-Bromophenylosazone of 5,5-Di-O-methyl-D-xylose The osazone was prepared by the method of Applegarth, Dutton and Tanaka (55). 5,5-Di-O-methyl-D-xylose (75 mg) and p-bromophenylhydrazine (280 mg) were dissolved in g l a c i a l acetic acid (4.5 ml). Water (2.2 ml) was added and the solution heated 6 minutes on a steam bath and allowed to cool. After 2 hours at 5° the cr y s t a l s were f i l t e r e d and r e c r y s t a l l i z e d from ethyl acetate - petroleum ether (30-60°) (1:7) m.p. 106.5-107.5°. L i t . (24), m.p. 107-108°. 5,5-Di-0-methyl-D-xylitol (l,3-Di-0-methyl-L-xylitol) 5,5-Di-0-methyl-D-xylose (590 mg) dissolved in water (15 ml) was treated with excess sodium borohydride (200 mg). The solution was l e f t at room temperature overnight, and worked up in the manner described f o r the a r a b i n i t o l . The sirup (550 mg) obtained showed no reducing properties ^oC\ -5.4° (c, 6.6 i n CHClg). 2.4- Di-0-methyl-D-threose 3.5- Di-0-methyl-D-xylitol (212 mg, 1.17 mmoles) in water (40 ml) was treated with 0.203 M periodic acid (10 ml, 2.03 mmoles). The oxidation was complete in 10 minutes and the -114-f i n a l periodate uptake amounted to 0.91 moles per mole of p e n t i t o l . Work up was i d e n t i c a l to that described e a r l i e r for the erythroses to give a mobile sirup (124 mg). P u r i f i c a t i o n of this material on a s i l i c a gel column yielded c r y s t a l l i n e (dimeric?) 2,4-di-O-methyl-D-threose. R e c r y s t a l l i z a t i o n from ethyl acetate - petroleum ether (30-60°) (1:5) gave c r y s t a l s 2,4-Dinitrophenylhydrazone of 2,4-Di-O-methyl-D-threose The hydrazone was prepared i n the same manner and using the same quantities as for the 2,4-di-0-methyl erythroses. The derivative a f t e r r e c r y s t a l l i z a t i o n from ethyl acetate -petroleum ether (30-60°) melted at 148-149°. Calculated for C 1 2 H 1 6 ° 7 N 4 J N> 17.07; 0CH3, 18.90$. Found: N, 17.26} 0CH5, 18.43$. Synthesis of 2,4-Di-0-methyl-L-threose 2,3-Isopropylidene-l,4,6-tri-O-methyl-L-sorbose 2,3-Isopropylidene-l,4,6-tri-0-methyl-_-sorbose was prepared by the method of Schlubach and Olters (26). Vacuum d i s t i l l a t i o n ---->• 0.9° (15 min) (c, 0.35 in 1 N HgS0 4). Calculated for C 6 H 1 2 ° 4 : C » 4 8 » 6 4 J H> 8.11; 0CH3, 41.89$. Found: C, 48.78; H, 8.22; 0CH3, 41.51$. -115-(0.05 mm) gave a f r a c t i o n b o i l i n g at 1 0 5 - 1 1 5 ° . The sirup c r y s t a l l i z e d upon being kept in a freezer ( - 1 0 ° ) for six months, m.p. 1 5 - 1 7 ° . No attempt was made to r e c r y s t a l l i z e th i s mater ia l . \^<^-J D 3 4 . 2 ° (c , 4.05 in C;HC13). L i t . (26), CKJ D 2 9 * 6 ° ( - ' 1 , 0 i n C H C 1 3 ) * 1 ,4 ,6 -Tri -O-methyl -L-sorbose 1 ,4 ,6-Tri -O-methyl -L-sorbose was prepared by mild ac id hydro lys i s of- 2 , 3 - i sopropy l idene- l ,4 ,6 - tr i -0 -me thyl -L-sorbose (26). The sirup showed only one component (R^ 0.72 upon paper chromatography in solvent system A.^ ^ - 1 . 8 5 ° — » ~ 3 . 1 6 ° (c , 7.2 in CHC1-). L i t . (26), D 3 . 8 ° (c, 1.5 in CHC1 5 ) . 1 , 4 , 6 - T r i - O - m e t h y l - h e x i t o l ( 1 , 3 , 6 - T r i - O - m e t h y l - D - g l u c i t o l or 1 , 3 , 6 - T r i - O - m e t h y l - L - i d i t o l ) 1 ,4 ,6-Tri -O-methyl -L-sorbose (520 mg) i n water (15 ml) was treated with excess sodium borohydride (1 g) and l e f t overnight at room temperature. The methylated hex i to l s were i s o l a t e d in the same manner as previous ly described. The r e s u l t i n g s irup showed only one major component (R .^ 0.45) on s i l i c a gel thin layer chromatography using solvent system A. This component was i s o l a t e d by chromatography on a s i l i c a gel column to give a s irup (280 mg) having a ro ta t ion ^ 5 . 6 ° (c , 1.5 in CHC1-). -116-2,4-Di-O-methyl-L-threose To the tri-O-methyl hex i t o l (650 mg, 2.9 mmoles) dissolved in water (5 ml), 0.2 M sodium periodate (20 ml, 4.0 mmoles) was added. The oxidation was complete in 5 minutes with an uptake of 0.98 moles of periodate per mole of h e x i t o l . Recovery of the tetrose i n the manner described above and subsequent p u r i f i c a t i o n on a s i l i c a gel column yielded a 2,4-Dinitrophenylhydrazone of 2,4-Di-0-methyl-L-threose The hydrazone was prepared using the same quantities as that of i t s o p t i c a l isomer, giving the derivative, which upon r e c r y s t a l l i z a t i o n from ethyl acetate - petroleum ether N, 17.07j 0CH 3, 18.90$. Found: N, 16.87; OCHj, 19.16$. colourless sirup (50-60°) melted at 149-150°. Calculated for c i g H 1 6 ° 7 N 4 s -117-BIBLIOGRAPHY 1. A. M. Stephen. J. Chem. Soc. 2030 (1962). 2. G. G. S. Dutton and A. M. Unrau. Can. J. Chem. 41, 2439 (1963). — 3. H. K i l i a n i . Ber. 18, 3066 (1885). 4. J. C. Sowden and H. 0. L. Fischer. J. Am. Chem. Soc. 69, 1963 (1947). — 5. J. C. Sowden. Advances in Carbohydrate Chem. 6, 291 (1951). 6. A. Wohl. Ber. _26, 730 (1893). 7. D. L. MacDonald and H. 0. L. Fischer. J. Am. Chem. Soc. 74, 2087 (1952). 8. L. Malaprade. B u l l . Soc. chim. (France) 43, 683 (1928). 9. J. M. Bobbitt. Advances in Carbohydrate Chem. 11, 1 (1956). 10. A. S. P e r l i n . Advances i n Carbohydrate Chem. 14, 9 (1959). 11. G. W. Huffman, B. A. Lewis, F. Smith and D. R. Spriesters-bach. J. Am. Chem. Soc. _77, 4346 (1955). 12. I. J. Goldstein, H. Sorger-Domenigg and F. Smith. J. Am. Chem. Soc. 81, 444 (1959). 13. K. Gatzi. Helv. Chim. Acta. 21, 195 (1938). 14. K. E. Pierre. M.Sc. Thesis. University of B r i t i s h Columbia. (1962). 15. S. C. Ho. B.Sc. Thesis. University of B r i t i s h Columbia. (1959). 16. A. E. Barlay. M.Sc. Thesis. University of B r i t i s h Columbia. (1961). 17. D. J. B e l l and J. Lorber. J. Chem. Soc. 453 (1940). 18. J. C. Dennison and D. I. McGilvray. J. Chem. Soc. 1616 (1951). 19. G. Zemplen and Z. Bruckner. Ber. 64, 1852 (1931) as quoted i n : W. L. Evans, D. D. Reynolds anc[ E. A. Talley. Advances in Carbohydrate Chem. 6, 27 (1951). -118-20. E. L. H i r s t , J. K. N. Jones and E. Williams. J. Chem. Soc. 1062 (1947). 21. J. I. Cunneen and F. Smith. J. Chem. Soc. 1146 (1948). 22. E. V. White. J. Am. Chem. Soc. 68, 272 (1946). 23. S. A. Black and G. G. S. Dutton. Unpublished r e s u l t s . 24. P. A. Levene and A. L. Raymond. J. B i o l . Chem. 102, 331 (1933). 25. R. Kuhn, I. Low and N. Trischman. Ber. j_0, 203 (1957). 26. H. H. Schlubach and P. Olters. Ann. _550, 140 (1942). 27. P. D. Bragg and L. Hough. J. Chem. Soc. 4347 (1957). 28. M. Guernet, A. Jurado-Soler and P. Malangeau. B u l l . Soc. chim. (France) 1183 (1963). 29. D. J. B e l l . J. Chem. Soc. 473 (1944). 30. G. W. Hay, B. A. Lewis and F. Smith. J. Chromatog. 11, 479 (1963). 31. R. Schaffer and H. S. I s b e l l . J. Am. Chem. Soc. 79, 3864 (1957). 32. M. E. Tate and C. T. Bishop. Can. J. Chem. 40, 1043 (1962), 33. 0. Svanberg and K. Sjoberg. Ber. j56, 863 (1923). 34. R. A. Laidlaw. J. Chem. Soc. 2941 (1952). 35. D. A. Applegarth, G. G. S. Dutton and Y. Tanaka. Can. J. Chem. 40, 2177 (1962). 

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