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Synthesis of disaccharides as reference compounds Berry, Joffre Mario 1974

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SYNTHESIS OF DISACCHARIDES AS REFERENCE COMPOUNDS BY JOFFRE MARIO BERRY B.S., University of Wisconsin, 1 9 6 8 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy i n the Department of CHEMISTRY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1 9 7 ^ In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th i s thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th i s thes is for f inanc ia l gain sha l l not be allowed without my written permission. Department of Chemistry The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date a p r i l 2 5. 197 -^i ABSTRACT A multistep synthesis of a monoraethylated beta linked d i -saccharide of b i o l o g i c a l s i g n ificance, 6-O-(4-O-methyl-0-D-glucopyranosyl)-D-galactose i s described f o r the f i r s t time. A key intermediate i n t h i s synthesis was 4-0-methyl-D-glucose, which was obtained by a synthetic route involving open chain carbohydrate derivatives, and by i s o l a t i o n from mesquite gum, a polysaccharide that contains ^-O-methyl-D-glucose as the uronic a c i d . Condensation of the derived 2 , 3 , 6 - t r i - 0 - a c e t y l - 4 - 0 - m e t h y l -CL-D-glucopyranosyl bromide with 1,2»3»^-di-O-isopropylidene-a-D-galactopyranose and subsequent removal of the blocking groups afforded 6-O-(4-O-methyl-0-D-glucopyranosyl)-D-galactose i n 55% y i e l d . The synthesis of alpha linked glucosides using 0-D-gluco-pyranosyl bromides having a non-participating benzyl group at C-2 has been investigated. Two of these bromides were used i n glycoside forming reactions, 2-0-benzyl-3,4,6-tri-0-p_-nitrobenzoyl -P-D-gluco-pyranosyl bromide, and 2,3-di-0-benzyl-^,6-di-0-p_-nitrobenzoyl-p-D-glucopyranosyl bromide. The preparation of a glucosyl bromide having a non-participating 2,3-carbonate function was attempted, but was unsuccessful. Isomaltose and 6-0-a-D-glucopyranosyl-D-galactose were prepared using the 2,3-dibenzyl compound. Isomaltose, 6-0-a-D-glucopyranosyl-D-mannose and 4-0-cu D-glucopyranosyl -L-rhamnose were synthesized using the 2-benzyl compound. The l a t t e r disaccharide represents the f i r s t a-linked glucoside containing L-rhamnose to be synthesized. The synthesis of several methylated sugars useful f o r the analysis of polysaccharide structures i s described. 73 -O-Methyl-D-galactose was prepared i n a multistep synthesis involving l , 2 i 5,6-di -O-isopropylidene-a -D-galactofuranose. F i n a l l y , a new synthesis of 3 » ^ » 6 - t r i - O - m e t h y l - D - g l u c o s e , D-galactose and D -mannose i s described. i i i TABLE OF CONTENTS INTRODUCTION 1 RESULTS AND DISCUSSION 9 1, Model syntheses of g-D-glucosides A. 6-0-0-D-Glucopyranosyl-D-galactose- 18 B. 3-0-3-D-Glucopyranosyl-D-mannose* 20 C. 6-0-(4-0-Methyl-£-D-glueopyranosyl)-D-galactose 25 a) Synthesis of 4-0-methyl-D-glucose ... 25 b) I s o l a t i o n of 4-0-methyl-D-glucose ; from mesquite gum 30 c) 2,3,4-Tri-0-ac etyl-4-0-methyl-a-D-glucopyranosyl bromide 35 d) 1,213,4-Di-j|- isopropyl idene- 6-0- (4-0-methyl-0-D-gluc opyrano syl) - a- D-galactopyranose 36 e) 6-0-(4-O-Methyl-0-D-glue opyrano s y l ) -D-galactose 39 i ) Methylation 40 i i ) Periodate oxidation 4-5 i i i ) Disaccharide a l d i t o l 4-9 2. Syntheses of a-D-glucosides A. Attempted use of a non-participating carbo-nate group , 52 B. Using 2,3-Di-0-benzyl-4,6-di-0-p_-nitro-benzoyl-0-D-glucopyranosyl bromide ...... 59 a) 6-0-a-D-Glucopyranosyl-D-glucose .... 64 b) 6-0-a-D-Glucopyranosyl-D-galactose .. 66 " i v C. Using 2-0-benzyl-3» i*'»6-tri-0-2-nitro-benzoyl-I-D-glucopyranosyl bromide .... 67 a) 6-0-a-D-Glucopyranosyl-D-glucose .. 71 b) 6-0-a-D-Glucopyranosyl-D-mannose .. 76 c) 4-0-a-D-Glucopyranosyl-L-rhamno-pyranose 83 i ) Methylation 9 3 i i ) Periodate oxidation 9 8 CONCLUSIONS 102 EXPERIMENTAL '. 10k General conditions 10k Preparation of 6-0-g-D-glucopyranosyl-D-galactose 0-D-Glucopyranose pentaacetate 7 106 a-D-Glucopyranose pentaacetate 8 107 Tetra-O-acetyl-a-D-glucopyranosyl bromide 2 1°? 1,213,^-Di-0-isopropylidene-a-D-galactopy-ranose 10 110 1,2«3,k-Di-0-isopropylidene-6-0-(tetra-0-. , acetyl-g-D-glucopyranosyl)-a-D-galactopy-ranose 71 • I l l 6-0 -P-D-Glucopyranosyl-D-galactose 13. 112 Synthesis of 3-0-0-D-glucopyranosyl-D-mannose , ... Methyl kt6-0-benzylidene-a-D-mannopyranoside Ik 112 3-0- and 2-0-0-D-glucopyranosyl-D-mannose 21.22 113 Synthesis of 6-O-(4-O-methyl-0-D-glucopyranosyl-D-galactose D-Glucose d i e t h y l t h i o a c e t a l 23. 119 Di-O-isopropylidene-D-glucose d i e t h y l thioacetals 2k 120 2,3»5*6-Di-0-isopropylidene D-glucose dimethyl acetals (mixture) 2j5_ 120 2,315,6-Di-0-isopropylidene-4-p_-nitro-benzoyl-D-glucose dimethyl acetal 26 ........ 121 2,3«5,6-Di-0-isopropylidene D-glucose dimethyl acetal 2J£ 121 2,3«5,6-Di-0-isopropylidene-^-O-methyl D-glucose dimethyl acetal 2£ 122 i ) By Purdie methylation 122 i i ) By Hakomori methylation 1 2 3 4-0-Methyl-D-glucose 28 1 2 5 i ) 4-0-Methyl-D-arabino-hexose phenylo-sazone 2£ ••• • ••• 1 2 5 1,2,3,6-Tetra-0-ac etyl-4-0-methyl-3-D-glucopyranose JO ..... 1 2 5 k-O-Methyl-D-glucose from mesquite gum i) P a r t i a l hydrolysis of mesquite gum ... 126 i i ) Methanolysis of the polysaccharide. Methyl 4-0-methyl-D-glucuronic acid methyl ester j22 • 1 2 7 i i i ) Methyl 4-0-methyl-D-glucose ^± 128 iv) 4-0-Methyl-D-glucose 28 128 1,213,^-Di-0-isopropylidene-6-0-(4-0-methyl-g-D-glucopyranosyl)-a-D-galactopyranose ^8 .. 1 2 9 6-0-(k-0-Methyl-£-D-glue opyranosyl)-D-galactose j[2 131 i ) p.-Nitroanilide 4 0 1 3 3 i i ) p_-Nitrobenzoate ••• • i i i ) Reaction with g-glucosidase 1 3 ^ iv) Methylation 1 3 ^ v) Periodate oxidation 1 3 5 v i ) Methyl 2,3,^-tri-0-methyl-D-galactoside k% 137 6-0-(4-0-Methyl-P-D-glucopyranosyl)-D-g a l a c t i t o l 4£ Attempted synthesis of l , 4 , 6 - t r i - 0 - a c e t y l -D-glucopyranose 2,3-carbonate Methyl 4,6-0-benzylidene-a-D-glucopyrano-side 52 Methyl k$6-0-benzylidene-a-D-glucopyrano-side 2,3-carbonate 60 Acetolysis of methyl 4,6-0-benzylidene-a-D-glueopyranoside 2,3-carbonate 6 0 .......... Syntheses by 2,3-dibenzyl method A. Isomaltose : Methyl 2,3-di-0-benzyl-4,6-0-benzylidene-a-D-glucopyranoside 66 Methyl 2,3-di-O-benzyl-a-D-glucopyranoside 6 7 . l,4,6-Tri-p-acetyl-2,3-di-0-benzyl-a-D-glu-copyranose" 68 .7 2,3-Di-O-benzyl-a-D-glucose 6£ . 2,3-Di-O-benzyl-l, 4,6-tri-0-£-nitro-benzoyl-0-D-glucopyranose 29. • • • • • • • • • • 2,3-Di-0-benzyl-4,6-0-2-nitrobenzoyl -P-D-glucopyranosyl bromide £1 l»2,3,4-Tetra-0-acetyl-6-0-trityl-e-D-gluco-pyranose 22 • • 1,2,3»^-Tetra-O-acetyl-0-D-glucopyranose 71 6-0-a-D-Glucopyranosyl-D-glucose (isomaltose) 2 Z B, 6-0-a-D-glucopyranosyl-D-galactose 80 ....... Syntheses by the 2-benzyl method A.Isomaltose 3,4,6-Tri-O-acetyl-l-deoxy-l-piperidino-g-D-glucopyranose 81 ., v i i 3,4,6-Tri-0-acetyl-2-0-benzyl-l-deoxy£. 1 - piperidino-£-D-glucopyranose 82 1 5 2 2- 0-Benzyl-D-glucose 84 1 5 3 2-0-Benzyl-l,3t^#6-tetra-0-p_-nitrobenzoyl-a-D-glueopyranose 8 £ 1 5 3 2-0-Benzyl-3, 4, 6-tri-O-p_-nitrobenzoyl-0-D-glucopyranosyl bromide 8 8 1 5 4 6-0-(2-0-Benzyl-a-D-glucopyranosyl)-D-glucose £ 1 1 5 ^ 1, 2 , 3 , 4 - T e t r a - O - a c e t y l - 6 - 0 - ( 2 - 0 - b e n z y l - 3 , 4 , 6 -tri-O-ac etyl-a-D-gluc opyrano syl)-D-gluc opy-ranose £ 2 1 5 6 6-0-a-D-Glucopyranosyl-D-glucose (i s o -maltose) 22 •••••••• 1 5 6 0-Isomaltose octaacetate £ 8 ••• • 1 5 7 B. 6-0-a-D-Glucopyranosyl-D-mannose ... J , .v •>.-.« 1.2,3,4-Tetra-O-ac etyl-6-0-trityl-P-D-manno-pyranose 21 1 5 7 1, 2,3,4-Tetra-Oj-acetyl-0-D-mannopyranose £ 4 1 5 8 6-0-(2-0-Benzyl-a-D-glucopyranosyl)-D-mannose £ 6 1 5 8 1 » 2 , 3 » 4 - T e t r a - 0 - a c e t y l - 6 - 0 - ( 2 - 0 - b e n z y l -3 , 4 , 5 - t r i - 0 - a c etyl-a-D-glucopyranosyl)-D-mannopyranose £ 2 1 5 9 6-0-a-D-Glucopyranosyl-D-mannose £ 8 1 6 0 112,3.4-Tetra-O-ac e t y l - 6 - 0 - ( 2 , 3 , 4 , 6 - t e t r a - 0 -ac etyl-a-D-glue opyrano syl7-D-mannopyranose £ £ , 1 6 0 C. 4-0-a-D-glucopyranosyl-L-rhamnopyranose Methyl a-L-rhamnopyrand>side 1 0 0 1 6 0 Methyl 2,3-0-isopropylidene-a-L-rhamno-pyranoside 1 0 1 l 6 l Methyl 4 - 0 - ( 2 - 0 - b e n z y l - 3 , 4 , 6 - t r i - 0 - p _ -nitrobenzoyl-alD-glucopyranosyl)-2,3-0-v i i i isopropylidene-a-L-rhamnopyranoside 103 164 Methyl- 4 - 0 - ( 2-0-benzyl-a-D-glucopyranosyl) -2 ,3 -0 -isopropylidene-a-L-rhamnopyranoside 105 165 Methyl 4 - 0-a-D-glueopyranosyl-2 , 3 - 0-isopro-pylidene-a-L-rhamnopyranoside 107 166 Methyl 4-0-a-D-glucopyranosyl-a-L-rhamno-pyranoside 111 167 i ) Methylation 171 i i ) Periodate oxidation 173 Methyl 2 , 3 - d i - 0 - a c e t y l - 4 - 0 - ( 2 , 3 , 4 , 6 - t e t r a - 0 - , , ac etyl-a-D- glue opyrano syl7-ct- L- rhamnopyrano -side 112 168 1 , 2 , 3 i T r i - 0 - a c e t y l - 4 - 0 - ( 2 . 3 , 4 , 6 - t e t r a - 0 -ac etyl-a-D-glucopyrano syl)-L-rhamno-pyranose 113 169 4-0-a-D-Glue opyrano syl-L-rhamnopyranose 114 I69 i ) T r i m e t h y l s i l y l derivative • 170 i i ) Enzymatic hydrolysis • 170 4-0-a-D-Glucopyranosyl-L-rhamnitol 11| 170 APPENDIX I Proton magnetic resonance spectroscopy of the t r i m e t h y l s i l y l ethers of some disaccharide derivatives 175 APPENDIX II Syntheses of monosaccharide esters having one free hydroxyl group I83 1 » 31^ » 6-Tetra-O-acetyl-D-glucose, D-galactose and D-mannose 123t 124 , 125, 184 Tri - 0 -acetyl-L-rhamnose 1 2 6 i 127 186 ix Methyl 4-0-acetyl-O-benzoyl-a-L-rhamnopyranoside 129 • 188 APPENDIX III Synthesis of 3-0-methyl-D-galactose 190 a) 1,215»6-Di-O- isopropylidener-a-D-glucofuranose 1J0 192 b) l,2i5,6-Di-0-isopropylidene-3-0-tosyl-a-D-glueofuranose 1^1 192 c) 3-Deoxy-l,2i5,6-di-0-isopropylidene-a-D-xylo-hex-3-enofuranose 13.2 193 d) 1,215» 6-Di-O-isopropylidene-a-D-galactofuranose 133 •"• •. 19^ e) 1,2»5,6-Di-O-isopropylidene-3-0-methyl-a-D-galactofuranose 134" 19^ f) 3-0-Methyl-D-galactose 122 195 APPENDIX IV Synthesis of 3,4,6-tri-O-methyl-D-glucose, D-galactose and D-mannose • 196 a) Using the O-(l-methoxyethyl) group ....... 200 b) Using the t-butyl ether 201 c) Using the p_-toluene sulfonate group 202 d) Using the tetrahydropyranyl ether ...... 202 \ i ) 3i^#6-Tri-0-methyl-D-glucose 13_£ .... 204 i i ) 3,4,6-Tri-O-methyl-D-mannose 142 .... 206 i i i ) 3.^6-Tri-O-methyl-D-galactose l4j> .. 207 BIBLIOGRAPHY X LIST OF FIGURES Figure P age 1. Disaccharides chemically synthesized 3 2. P.M.R. Spectrum (CHCl^) of compound J3.0 31 3. P.M.R. Spectrum (DgO) of compound 49 ............ 51 4. P.M.R. Spectrum (CDCl^) of compound 6% 5 6 5. P.M.R. Spectrum (D 20, 9 0 ° ) of compound JZ 7 5 6. P.M.R. Spectrum (D 20) of compound £8 ?8 7. P.M.R. Spectrum (DgO) of compound 111 9 2 8. P.M.R. Spectrum (CDCl^) of compound 112 9 6 9. P.M.R. Spectra of 111, 115_, 112 a n d P-linked analogs • • 9 7 10. P.M.R. Spectrum (CClj.) of the t r i m e t h y l s i l y l ether of compound J 3 J 178 11. P.M.R. Spectrum (OAi/) of the t r i m e t h y l s i l y l ether of compound JM? 102 179 12. P.M.R. Spectrum (CAl,) of the t r i m e t h y l s i l y l ether of compound ° ° 108 180 1 3 . P...M.R. Spectrum (CC1|.) of the t r i m e t h y l s i l y l ether of compound 12 181 14. P a r t i a l P.M.R. Spectrum of compound 12 A (CCl^) and B (C 6H 6) 182 x i LIST OF SCHEMES Scheme page 1 . Side reactions i n disaccharide synthesis 1 5 2. 6-0-0-D-Glucopyranosyl-D-galactose 1 9 3. 3- and 2-0-D-Glucopyranosyl-D-mannose .......... 2 2 4. Synthesis of 4-0-methyl-D-glucose .............. 26 5. I s o l a t i o n of 4-0-methyl-D-glucose from mesquite gum .... 32 6. Synthesis of 6-O-(4-O-methyl-0-D-glucopyranosyl)-D-galactose 37 7. Methylation of 6-0-(4-0-methyl-g-D-glucopyrano-syl)-D-galactose 7 42 8. Preparation of 2,3,4-tri-O-methyl-D-galactose .. 44 9. Periodate oxidation of 6-O-(4-O-methyl-0-D-glucopyranosyl)-D-galactose 47 1 0 . A c e t o l y s i s of methyl 4,6-0-benzylidene-a-D-glucopyranoside 2,3-carbonate 54 1 1 . Mechanism of the a c e t o l y s i s reaction ........... 58 12. 2,3-Di-0-benzyl-4,6-di-0-jj-nitrobenzoyl-0-D-glucopyranosyl bromide 61 1 3 . Synthesis of isomaltose and 6-0-a-D-gluco-pyranosyl-D-galactose by the 2,3-dibenzyl method 6 5 14. 2-0-Benzyl-3,4,6-tri-0-rj-nitrobenzoyl-p-D-glucopyranosyl bromide ......................... 68 1 5 . Synthesis of isomaltose and 6-0-cc-D-gluco-pyranosyl-D-mannose by the 2-benzyl method ..... 72 16. Synthesis of 4-0-a-D-glucopyranosyl-L-rhamnose • • 86 17. Methylation of 4-0-a-D-glucopyranosyl-L-rhamnose 95 18. Periodate oxidation of 4-0-a-D-glucopyranosyl-L-rhamnose 99 X l l 19. Synthesis of 3-0-methyl-D-galactose 191 20. Synthesis of 3,4,6-tri-0-methyl-D-glucose 199 •• • • XI11 ACKNOWLEDGEMENTS I wish to express my sincere gratitude to Dr. G.G.S. Dutton fo r his excellent guidance, assistance and patience during the course of t h i s work. I also wish to express my appreciation to the members of Dr. Dutton's laboratory f o r t h e i r stimulating discussions and suggestions, and to the members of Dr. Rosenthal's and Dr. Hal l ' s groups f o r t h e i r i n t e r e s t i n g seminars and discussions. The a s s i s -tance provided by Dr. Matthew Yuen-Min Choy during the synthesis of the 3»4,6-trimethyl hexoses i s g r a t e f u l l y acknowledged. The g i f t of seed c r y s t a l s or chromatographic standards from the following persons i s much appreciatedi G.M. Bebault, Dr. C T . Bishop, Dr. A. Gauhe, Dr. K.B. Gibney, Dr. G. K e i l i c h , Dr. B.H. Koeppen, Dr. E. Lee, Dr. K.N. Slessor, R.H. Walker and M.T. Yang. This work would have been much more d i f f i c u l t had i t not been for the understanding and encouragement of my wife Jean, to whom I dedicate t h i s t h e s i s . Her e f f o r t s i n the preparation and typing of t h i s manuscript are also g r a t e f u l l y acknowledged. I also wish to express my gratitude to the University of B r i t i s h Columbia f o r the award of the MacMillan Bloedel Limited Scholarship and f o r the B r i t i s h Columbia Sugar Refining Company Scholarship. F i n a l l y , I wish to thank Dr. N.S. Thompson, Inst i t u t e of Paper Chemistry, Appleton, Wisconsin, f o r agreeing to be the external examiner. 1 INTRODUCTION Complex polysaccharides are found i n a wide var i e t y of natural systems where they serve many diverse functions. For example, they may be of s t r u c t u r a l importance, serve as energy reserves, or be responsible f o r antigenic s p e c i f i c i t y . One of the in t e r e s t s i n t h i s laboratory i s the immuno-chemistry of the capsular polysaccharides from K l e b s i e l l a and i t i s with t h i s background that the synthesis of some of the disaccharides i n t h i s thesis i s discussed. The need f o r disaccharides as model compounds ;> In the study of complex polysaccharides i n b i o l o g i c a l systems, i t i s often necessary to have in d i v i d u a l disaccharide or other oligosaccharide units of unambiguous structure: Thus, as part of the s t r u c t u r a l elucidation of polysaccharide struc-tures, - i t i s customary to subject the polysaccharide to par-t i a l acid hydrolysis, an operation which cleaves the poly-saccharide at the more l a b i l e linkages giving oligosac-charides of d i f f e r i n g lengths. I d e n t i f i c a t i o n of these d i , trisaccharides, etc. o f f e r s a convenient means of obtaining information on the sequence of the sugars i n the o r i g i n a l poly-saccharide. In investigations related to the antigenic a c t i v i t y of capsular and c e l l wall polysaccharides i n bacteria, the ques-t i o n a r i s e s as to what part of the polymer i s responsible f o r the antigenic a c t i v i t y . This can be accomplished by serologi-c a l s p e c i f i c i t y and i n h i b i t i o n studies using mono, d i , and trisaccharide units of known structure (1,2,3,4). 2 Disaccharides can also he used i n enzyme s p e c i f i c i t y studies and, i n the case of c e r t a i n neutral and nitrogen con-t a i n i n g derivatives, i n investigations of a n t i b i o t i c s con-ta i n i n g d i - and trisaccharides ( 5 * 6 , 7 , 8 , 9 ) . The technique of nuclear magnetic resonance as applied to o l i g o - and polysaccharide structures i s useful for obtaining information mainly on the anomeric linkage (alpha or beta) of the sugars involved, but only when well defined repeating units are present. This technique however, cannot be used f o r un-ambiguously demonstrating whether the linkages are 1-2, 1-3, etc. because the spectra are usually very complex and not enough background knowledge i s a v a i l a b l e . Thus, nuclear mag-netic resonance studies of oligosaccharides of d i f f e r e n t sizes may eventually lead to the use of t h i s technique for the l i n k -age analysis and sequencing of complex polysaccharides. The same can be said for other instrumental methods, such as mass spectrometry. Contrary to what might be expected, r e l a t i v e l y few d i s -accharides have been chemically synthesized. This i s i l l u s -trated i n Figure 1 (10), which shows the neutral disaccharides chemically synthesized up to the present time, but not those prepared i n t h i s laboratory. Inspection of t h i s Figure w i l l show that mostly glucose and galactose disaccharides have been made, but few disaccharides of other sugars have been prepared. H i s t o r i c a l background Synthetic 1,2-trans glycopyranosides (0-D-glucopyrano-sides, a-D-mannopyranosides) have been known for a long time. 3 DISACCHARIDES CHEMICALLY SYNTHESISED W o % \ ARABINOSE TO LU CO o CD a CC 8 LU CO O . _J >-a X 8 p GALACTOSE TO p GLUCOSE TD MANNOSE LU CO O ( J a U_ 6 . 1 RHAMNOSE ' to i * i 1-2 A R A B I N O S ^ 1*4 1*5 1*6 X X • X X l - l 1*2 RIBOS? 1*4 1*5 1*6 X X X 1*1 1*2 XYLOSE* 1*4 1*5 1*6 X X X X X X X X X X X X X X X X X X U l GALACTOSE 5 • 1*4 U 5 1*6 X X X X X X X X X X X X X X X X X X X X X X X X X 1*1 1*2 GLUCOS'E 3 1*4 1*5 1*6 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 1*1 1*2 MANNOSE 1*4 1*5 1*6 X X X X X X X X X X 1*1 1*2 FUCOSS 1*4 1*5 l-,f? X X X 1*1 1*2 RHAMNC&E 1*4 1*5 X X 1*6 X X Figure 1 4 They are usually made hy the Koenigs-Knorr reaction ( 1 1 ) , i n which a f u l l y e s t e r i f i e d glycosyl halide i s condensed i n the presence of s i l v e r s a l t s with a suitably blocked monosac-charide unit which has one free hydroxyl group. Although these condensations give on some occasions a small amount of 1 , 2 -c i s glycosides (a-D-glueopyranosides, 0-D-mannopyranosides), the trans product i s u s u a l l y predominant. This i s due to part-i c i p a t i o n of the ester group at C-2 with C - l , which forces the incoming nucleophile to attack from a trans p o s i t i o n at C - l . Because of t h i s stereoselective preference f o r the trans products, the synthesis of the c i s anomers, many of which are found i n b i o l o g i c a l systems, has been a much more d i f f i c u l t task. Although using mercury s a l t s as condensing agents i n the Koenigs-Knorr reaction gives on some occasions the c i s anomers ( 1 2 , 1 3 ) , the trans products are usually found i n l a r -ger proportions. Several workers found that s u b s t i t u t i o n of the ester group at C-2 of the f u l l y e s t e r i f i e d glycosyl halide by a non-participating group increased s u b s t a n t i a l l y the y i e l d of c i s glycosides obtained. Thus, a mannosyl bromide with a 2 , 3 carbonate function gives the 0-D-mannopyranosides pre-f e r e n t i a l l y (14). However, t h i s was not the case with a non-p a r t i c i p a t i n g benzyl at C-2 of the mannosyl residue, which gives the alpha anomer. The 2,3 carbonate method has not been used f o r making glucosides. Isomaltose (6-0-a-D-glucopyranosyl-D-glucose) has been synthesized v i a glucosyl chlorides having non-participating n i t r o ( 1 5 ) and £-toluenesulfonyl ( 1 6 ) groups at C-2, but these methods have not been widely used probably because of the d i f -f i c u l t i e s involved i n the preparation of the chlorides (17). S i m i l a r l y , 3,4,6-tri-O-acetyl-2-0-benzyl-a-D-glucopyrano s y l bromide gave the methyl 0-D-glucoside when treated with sodium methoxide (18). Although 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl chloride (17) and the corresponding galacto compound (19) gave mostly 1,2-cis products, large amounts of the unwanted 1,2-trans glycosides were s t i l l obtained. These and other investigations provided ways toward the synthesis of 1,2-cis glycosides, but the condition of having a non-participating group at C-2 of the glycosyl halide, a l -though successful i n some condensations, gave i n others, most l y the trans product. Thus, the search f o r highly stereo-se l e c t i v e methods continues, e s p e c i a l l y f o r procedures which would be applicable to a wide v a r i e t y of sugars. Recently, Lemieux and co-workers (20) synthesized alpha linked disaccharides v i a 3t4,6-tri-0-acetyl-2-deoxy-2-nitroso a-D-glueopyranosyl chloride and the corresponding galacto compound, i n condensations which give the a-D-configuration s t e r e o s e l e c t i v e l y . Conversion of the r e s u l t i n g 2-oximino gly eoside to the corresponding 2-keto compound and subsequent re duction gave alpha linked disaccharides which were epimeric at carbon 2 of the non-reducing end. This method was used by Miyai and Jeanloz (21) f o r the synthesis of a disaccharide, but these authors found that the unwanted beta linked (1,2-trans) product was predominant, which indicates that the method i s not general. Lemieux and Hendriks have also synthesized a-D-glucopy-6 ranosides v i a tetra-O-benzyl-a-D-glucopyranosyl bromide, us-ing tetraethylammonium bromide and ethyldiisopropylamine as condensing agents. This method i s under further investiga-tion., (22). In 1969, Ishikawa and Fletcher (23) published a study on the methanolysis of p_-nitrobenzoyl-D-glucopyranosyl bromides having a non-participating benzyl group at C-2. In addition to f u l f i l l i n g the condition of non-participation, these bro-mides were e f f e c t i v e l y s t a b i l i z e d by the p_-nitrobenzoate groups, making possible f o r the f i r s t time, the synthesis of stable P-D-glueopyranosyl bromides. Treatment of these bro-mides with methanol gave a-D-glucosides with a high degree of s t e r e o s e l e c t i v i t y , the rate of reaction of the beta bromides being much fa s t e r than the corresponding alpha ones. At t h i s time these bromides were not tested f o r disaccharide synthe-s i s , but the p o s s i b i l i t y of a highly•stereoselective general method fo r the synthesis of 1,2-cis disaccharides was opened. A p o t e n t i a l l y v e r s a t i l e synthesis of a-D-glucopyrano-sides has been reported by F e r r i e r and co-workers (24); In t h i s method, a phenyl 1-thioglucopyranoside derivative i s treated with an alcohol i n the presence of a mercury s a l t to give the O-glycoside with inversion of configuration. A der-i v a t i v e of the disaccharide 6-0-a-D-glucopyranosyl-D-galac-tose was prepared i n t h i s manner. Present study The synthesis of disaccharides i s the main subject of t h i s t h e s i s . The preparation f i r s t described, i s that of a 7 known disaccharide 6-0-a-D-glucopyranosyl-e-D-galaetose, which was undertaken i n order to test some of the older l i t -erature methods. This i s followed by an attempted but un-successful synthesis of 3-£-3-D-glueopyranosyl-D-mannose, which i s found i n bac t e r i a . Later, a multistep synthesis of an otherwise inaccessible monomethyl beta linked disaccharide of b i o l o g i c a l importance, 6-0-(4-0-methyl-3-D-glueopyranosyl)-D-galactose, i s described f o r the f i r s t time. F i n a l l y , the synthesis of a-D-glucosides i s investigated, using mainly the newly discovered beta bromides of Ishikawa and Fletcher. In addition, the possible use i n the synthesis of a-D-glucosides of a glu c o s y l bromide having a 2,3 carbonate group and the problems encountered, i s discussed. Four alpha linked d i s -accharides are synthesized» isomaltose, 6-0-a-D-glucopyranosyl-D-galactose, 6-0-a-D-glucopyranosyl-D-mannose, and 4-0-a-D-glucopyranosyl-L-rhamnose. The f i r s t two have been synthe-sized before by other methods, but were chosen i n order to test the generality of the methods f o r actual disaccharide syntheses. 6-0-a-D-Glucopyranosyl-D-mannose has not been described as f a r as i s known, although a 2 - t r i c h l o r o - a c e t a t e derivative was is o l a t e d i n low y i e l d ( 2 5 ) . F i n a l l y , the f i r s t synthesis of an alpha linked glucoside of rhamnose, 4-0-a-D-glucopyranosyl-L-rhamnose, i s presented. This synthesis re-f l e c t s the current interest i n our laboratory i n the struc-tures of capsular polysaccharides of K l e b s i e l l a , many of which are now under inv e s t i g a t i o n , and some of which may contain t h i s alpha linked disaccharide unit. 8 One year a f t e r t h i s program on the synthesis of a-D-glucosides was started, our e f f o r t s were concentrated on the possible use of two of the beta bromides, namely, 2-0-benzyl-3,4,6-tri-0-p_-nitrobenzoyl -e-D-glucopyranosyl bromide and 2,3-di-0-benzyl-4,6-di-0-p_-nitrobenzoyl-g-D-glucopyranosyl bromide. At t h i s point, Flowers (26) published the synthesis of 6-0-a-D-glucopyranosyl-D-galactose, on which we were also working, v i a the 2-benzyl compound. Although t h i s method for the synthesis of 1,2-cis disaccharides was no longer novel, i t was decided nevertheless to continue the synthesis, using the 2,3-dibenzyl compound fo r t h i s p a r t i c u l a r disaccharide. Re-cently, Frechet and Schuerch (27) published the use i n glu-coside forming reactions, of s t i l l another of these bromides, a 2,3,4-tribenzyl bromide with d i f f e r e n t substituents at C-6, Their study, which i s concerned with stereoelectronic de-pendencies, does not overlap with our e f f o r t . The appendix to t h i s thesis describes the synthesis of several methylated hexoses useful f o r the s t r u c t u r a l analysis of capsular polysaccharides of K l e b s i e l l a , many of which con-t a i n 1-2 linkages. Thus, 3»^»6-trimethyl hexoses are syn-thesized s t a r t i n g from intermediates which i n the future may be used f o r the synthesis of 1-2 l i n k e d disaccharides. In addition, a multistep synthesis of 3-0-methyl-D-galactose i s described. 9 RESULTS AND DISCUSSION The synthesis of disaccharides has received the attention of many workers fo r several decades. Early attempts can be divided into two broad categories, namely, using the free su-gars, and using blocked sugars. When a free sugar i s placed i n a concentrated acid solution, a mixture of d i , trisaccha-rides etc. i s formed, i,a process known as "acid reversion" (28, 2 9 ) . Since t h i s process gives complex mixtures, i t i s not a p r a c t i c a l system f o r making disaccharides. Acid re-version however, i s s t i l l of interest today i n connection with studies related to the acid hydrolysis of polysaccha-r i d e s . The enzymatic method, which i s more stereoselective than acid reversion, has also been known for a long time. The f i r s t synthesis of the disaccharide sucrose was reported by Hassid and co-workers i n 1 9 ^ 4 , who used the enzymes pro-duced by the organism Pseudomonas saccharophila ( 3 0 ) . Enzy-matic syntheses however, have not been used as general pre-parative methods p a r t l y because of the d i f f i c u l t y i n ob-taining pure enzymes, and because disaccharide synthesizing enzyme preparations may also contain disaccharide degrading enzymes. Enzymatic systems produced i n vivo by microorga-nisms usually contain a mixture of enzymes which gives r i s e to mixtures of oligosaccharides. The enzymatic synthesis and hydrolysis ( 3 1 , 3 2 ) of saccharides have been reviewed. Blocked sugars have also been used for a long time. They have the advantage that only one or two hydroxyl groups react at one time, therefore l i m i t i n g the v a r i e t y of products. 1 0 Some of these methods are l i s t e d b r i e f l y below. The dehydration method This procedure uses a dehydrating agent such as phosphorus pentoxide or zinc chloride on a p a r t i a l l y blocked sugar. 0 - 0 Trehalose was f i r s t synthesized i n t h i s manner, by dehydration of 2,3,4,6-tetra-O-acetyl-D-glucose with phosphorus pentoxide (33)« Additions to 1,2 anhydrides This involves the use of anhydrides such as 3 » 4 , 6 - t r i - 0 - a c e t y l -1,2-anhydro-D-glucose, which was discovered by B r i g l ( 3 4 ) i n 1 9 2 2 . This compound was used by Lemieux and Huber i n 1 9 5 3 and 1 9 5 6 i n the f i r s t chemical synthesis of sucrose ( 3 5 K This was accompli-shed by condensation of B r i g l ' s anhydride with l , 3 , 4 , 6 - t e t r a - 0 -acetyl-D-fructose, which gave sucrose i n about 6% y i e l d . A l t e r a t i o n of a disaccharide into a d i f f e r e n t disaccharide This method has been used by Lindberg and co-workers ( 3 6 ) , who inverted the glyc o s i d i c linkage of gentiobiose octaacetate into the isomaltose analog under the influence of titanium t e t r a -c h l o r ide. This inversion only works f o r 1-6 linkages. These same workers have recently reported ( 3 7 ) the epimerization of a glucosyl disaccharide into a mannosyl one, a f t e r several steps. These and other methods have been reviewed by Stanek and co-workers ( 2 9 ) . It s u f f i c e s to say that because t h e i r applications have been to rather s p e c i f i c cases, they have not been used as general methods of disaccharide synthesis. The Koenigs-Knorr reaction This method ( 3 8 ) has been the most widely used because of i t s generality and wide a p p l i c a b i l i t y to glycoside synthesis. With 11 many important modifications, i t continues to be the most general method of disaccharide synthesis available today. The reaction involves the condensation of a f u l l y e s t e r i f i e d glycosyl halide with a p a r t i a l l y blocked carbohydrate having one (sometimes two) alcohol functions ..free. S i l v e r s a l t s (oxide or carbonate) are used as acid acceptor f o r the l i b e r a t e d hydrogen halide. Con-densations i n which s i l v e r oxide i s used, give water as a by-product, thus a drying agent (usually D r i e r i t e (39, 40, 41)) must be added to the reaction. Other agents which have been used assdriving force f o r the reaction include zinc chloride, phosphorus pentoxide (42,43), quinoline (44), and calcium chlor-ide (45). The H e l f e r i c h modification (46) of the Koenigs-Knorr reaction uses mercury s a l t s (cyanide and bromide) i n polar solvents such as nitromethane and a c e t o n i t r i l e . These acid acceptors have the advantage of d i s s o l v i n g i n the solvent, making the reaction homo-geneous. Mercuric oxide has also been used, but i t was replaced by the more e f f i c i e n t cyanide and bromide. Other modifications i n glycoside forming reactions include the use of c a t a l y t i c amounts of s i l v e r perchlorate i n conjunc-t i o n with s i l v e r carbonate or oxide (47), which was found to i n -crease the y i e l d of disaccharide. A study on the reaction of gly-cosyl chlorides with s i l v e r perchlorate has been published re-cently, (48). Lately, organic s i l v e r s a l t s were found to react with the glycosyl halides ( 5 0 ) giving unwanted products. Cad-mium carbonate has been used ( 5 1 ) as acid acceptor i n the synthe-s i s of st e r o i d glycosides. One of the main features of the Koenigs-Knorr method i s i t s 12 high degree of s t e r e o s e l e c t i v i t y , which r e s u l t s i n the formation of 1 , 2-trans g l y c o s i d e s almost e x c l u s i v e l y . Thus, bromides of the a-D-gluco c o n f i g u r a t i o n give r i s e to 0-D-glucosides, w h i l e bromides of the a-D-manno c o n f i g u r a t i o n g i v e r i s e to a-D-manno-s i d e s i a-D-manno a-D-mannoside T h i s s t e r e o s e l e c t i v e formation of the 1 , 2-trans product i s be-l i e v e d to be caused by p a r t i c i p a t i o n (52,53.54) of the e s t e r group ( u s u a l l y acetate o r benzoate) at C-2 w i t h C - l as the ha l o -gen atom departs. The incoming a l c o h o l then a t t a c k s from the l e a s t hindered s i d e , which i s t r a n s t o the e s t e r group at C-2. T h i s i s shown below f o r the D-gluco c o n f i g u r a t i o m 13 £ - D - g l u c o s i d e The s t e r e o s e l e c t i v e s y n t h e s i s o f g l y c o s i d e s o f t h e g - D - g l u c o o r t h e a-D-manno c o n f i g u r a t i o n c a n t h e r e f o r e be c o n v e n i e n t l y a c c o m p l i s h e d u s i n g t h i s r e a c t i o n , p r o v i d e d t h e g l y c o s y l b r o -m i d e a n d a l c o h o l i n t e r m e d i a t e s a r e a v a i l a b l e , a n d p r o v i d e d t h a t t h e y c o n d e n s e , w h i c h i s n o t a l w a y s t h e c a s e . T h i s s t e r e o -s e l e c t i v e f o r m a t i o n o f t h e t r a n s p r o d u c t h o w e v e r , h a s made t h e s y n t h e s i s o f t h e 1 , 2 - c i s g l y c o s i d e s a v e r y d i f f i c u l t p r o b l e m . I t i s i n t h i s a r e a t h a t much w o r k i s b e i n g done t o d a y i n many l a b o r a t o r i e s . The s y n t h e s i s o f 1 , 2 - c i s g l y c o s i d e s w i l l b e d i s c u s s e d i n g r e a t e r d e t a i l i n c o n n e c t i o n w i t h o u r e f f o r t i n t h i s l a b o r a t o r y i n t h e s y n t h e s i s o f r x - D - g l u c o p y r a n o s i d e s . T h e s e c o n d e n s a t i o n r e a c t i o n s v e r y o f t e n g i v e l o w y i e l d s o f d i s a c c h a r i d e p r o d u c t s . T h i s d e p e n d s i n p a r t o n t h e r e a c -t i v i t y o f t h e a l c o h o l u s e d , a c y c l i c a l c o h o l s s u c h a s m e t h a n o l a n d e t h a n o l b e i n g much more r e a c t i v e t h a n c a r b o h y d r a t e a l -14 cohols, whieh are usually quite s t e r i c a l l y hindered. P r i -mary alcohol groups i n carbohydrates tend to be more reac-t i v e than secondary ones, although some primary ones (e.g. l , 2 i 3 » 4-di - 0-isopropylidene - c t-D-galactopyranose) may be comparable to some secondary ones (e.g. I , 2 i 5 » 6-di - 0-iso-propylidene -ct-D-galactofuranose) i n r e a c t i v i t y . Equatorial secondary alcohols are i n general more reactive than a x i a l ones. The nature of the sugar halide used i s also important i n determining the course of the reaction. Chlorides are more stable than bromides, but iodides are unstable and f l u -orides do not react under most condensation conditions. The bromides are therefore the best choice, es p e c i a l l y when re-l a t i v e l y unreactive alcohols are used. Iodides can be gen-erated i n s i t u from the bromides (55»56) by addition of iodine c r y s t a l s to the reaction medium. The success of a condensation reaction w i l l also depend on the amount of side products obtained. How some of these side products may arise are summarized i n Scheme 1, i n which only positions 1 and 2 of the pyranose r i n g are considered. The side reactions may be considered to derive from the ace-toxonium intermediate 1, i n which attack by the alcohol at the anomeric center A w i l l r e s u l t i n disaccharide formation, but attack by the alcohol at B w i l l r e s u l t i n the formation of two isomeric orthoesters 2. P e r l i n has reported the iso-l a t i o n of orthoesters from condensation reactions (57). A new method of glycosylation based on the deliberate formation Scheme 1 o f o r t h o e s t e r i n t e r m e d i a t e s h a s b e e n d e s c r i b e d b y K o c h e t k o v a n d c o - w o r k e r s ( 5 8 , 5 9 ) , who o b t a i n e d 1 , 2 - t r a n s g l y c o s i d e s s t e r e o s e l e c t i v e l y . S i m i l a r l y , i n c o n d e n s a t i o n s w h e r e m e r c u r i c c y a n i d e i s u s e d , a t t a c k b y c y a n i d e o n A w i l l g i v e a g l y c o s y l c y a n i d e 2t w h i l e a t t a c k a t B w i l l r e s u l t i n i s o m e r i c compounds o f s t r u c t u r e 4. C o x o n a n d F l e t c h e r h a v e r e p o r t e d t h e i s o l a t i o n 1 6 of a glycosyl cyanide and compounds of structure 4 from the reaction of 2,3,4,6-tetra-O-aeetyl-a-D-glucopyranosyl bromide with mercuric cyanide i n nitromethane ( 6 0 ) . When s i l v e r oxide i s used as an acid acceptor, water i s given o f f as a byproduct of the condensation reaction, there-fore drying agents must be used to remove the water. Even with e f f i c i e n t drying agents however, a water molecule may react with the sugar before i t reacts with the desiccant. This competitive reaction w i l l increase the amount of side products i n the case of the more reactive bromides. Water can react d i r e c t l y with the bromide to give a 1-hydroxy sugar j>, or may react with the acetoxonium intermediate 1 to give either a 1-hydroxy or a 2-hydroxy compound (j> and 6 r e s p e c t i v e l y ) . The i s o l a t i o n of 2,3,4,6-tetra-O-acetyl-D-glucose from the reaction of the corresponding bromide with s i l v e r oxide has been documented by Goldschmid and P e r l i n ( 5 6 ) . Acetate migration of intermediates such as j5 and 6 can occur under c e r t a i n conditions, as observed (p. 70 ) dur-ing the benzylation of 1,3,4,6-tetra-O-acetyl-a-D-glucopy-ranose i n the presence of s i l v e r oxide. In p r i n c i p l e , these hydroxy compounds can i n turn react with the bromide to give unwanted disaccharides. I t i s because of these and other side products that ty-p i c a l condensation reactions give multicomponent mixtures r e s u l t i n g i n lower y i e l d s of disaccharide. Since each con-densation reaction w i l l vary depending on the halide, hydro-x y l i c component, etc., i t i s therefore desirable to t r y a v a r i e t y of d i f f e r e n t conditions f o r each disaccharide and choose the ones which give the best r e s u l t s . 18 Model synthesis of g-D-glucopyranosides A. 6-0- j3-D- Glue opyrano syl-D- galac t o se The disaccharide l,2t3,4-di-0-isopropylidene-6-0-(tetra-0-acetyl-j3-D-glucopyranosyl)-a-D-galactopyranose 11 was pre-pared (Scheme 2) e s s e n t i a l l y as described by Freudenberg (61). This disaccharide served as a model f o r testing some of the conditions used i n the attempted, stereoselective synthesis of 3-0-0-D-glucopyranosyl-D-mannose, to be described l a t e r . Com-pound 11 was obtained by condensation of tetra-O-acetyl-a-D-gluc opyrano s y l bromide 9_ with l,2i3,4-di-0-isopropylidene-a-D-galactopyranose 10 i n carbon tetrachloride, using s i l v e r oxide as an acid acceptor. The condensation gave a mixture of com-ponents i n which the disaccharide was one of the major pro-ducts. This c r y s t a l l i z e d a f t e r t r i t u r a t i o n with ethanol. The p.m.r. spectrum of the product indicated the presence of four acetates and two isopropylidene groups, consistent with the structure 11. The melting point of the disaccharide (139-l 4 l G ) was close to the l i t e r a t u r e value (141°). Deacetyla-t i o n (sodium methoxide) of 11 gave the corresponding I,2i3,4-di-O-isopropylidene-6-O-0-D-glucopyranosyl-D-galactose 12, which on subsequent treatment with d i l u t e acid afforded the free disaccharide 6-0-0-D-glucopyranosyl-D-galactose 13_ as a syrup. Bromide £ was prepared by brominating penta-O-acetyl-D-glucopyranose (usually the beta anomer) with hydrogen bro-mide i n acetic acid. When large amounts of bromide were pre-Scheme 2 20 pared, the methods of Lemieux ( 6 2 ) and Dale ( 6 3 ) were used, both s t a r t i n g from glucose. The method of Lemieux, i n which the generation of the pentaacetate i n s i t u i s followed by bromination with phosphorus tribromide and water, gave about 75% y i e l d of product. The method of Dale, which uses hydro-gen bromide i n acetic anhydride f o r the simultaneous acety-l a t i o n and bromination of glucose, usually gave 40-50$ of the bromide, due to extensive hydrolysis of the product. This was improved by conducting the reaction under anhydrous con-di t i o n s and by preventing the reaction mixture from getting too hot, y i e l d s of ?0-80$ being obtained i n t h i s manner. The isopropylidene compound 10 was prepared by acetala-t i o n of D-galactose as described by Raymond and Schroeder (64). B. 3-0-0-D-Glucopyranosyl-D-mannose This disaccharide has been isol a t e d from several plant polysaccharides ( 6 5 ) . In our laboratory, i t has recently been i s o l a t e d from the p a r t i a l hydrolysis of the capsular polysaccharide from K l e b s i e l l a type 5 (66), a f i n d i n g which prompted us to attempt the synthesis of the disaccharide from two r e a d i l y available intermediates, namely tetra-0-acetyl-a-D-gluc opyrano s y l bromide £ and methyl 4,6-0-benzylidene-a-D-mannopyranoside 14. Although compound 14 has two free hy-droxyl groups which can condense with the glycosyl bromide, i t was believed that the 3-hydroxyl function, which i s equa-t o r i a l , would condense much more re a d i l y than the 2-hydroxyl function which i s a x i a l . It was therefore expected that the 2 1 1 - 3 linked disaccharide would predominate. A s i m i l a r ap-proach has been used by Flowers ( 6 7 ) i n the condensation of tetra-O-acetyl-a-D-glucopyranosyl bromide and benzyl 2 , 6 - d i -O-acetyl-0-D-galactopyranoside. This l a s t intermediate also has two free hydroxyl groups, the 3-hydroxyl which i s equa-t o r i a l , and the 4-hydroxyl which i s a x i a l and rather unreac-t i v e . As expected, the condensation reaction of these two intermediates gave the 1-3 linked disaccharide i n good y i e l d . Tetra-O-acetyl-a-D-glueopyranosyl bromide £ was reacted (Scheme 3 ) with methyl 4,6-0-benzylidene-ct-D-mannopyranoside 14fusing several reaction conditions. The best conditions were those i n which s i l v e r oxide was used as the acid ac-ceptor and a c a t a l y t i c amount of iodine was added to the reac-t i o n . The f u l l y blocked disaccharides methyl 4,6-0-benzyli-dene-2-0-(tetra-O-acetyl-D-glucopyranosyl)-a-D-mannopyranoside 1 5 and methyl 4,6-0-benzylidene-3-0-(tetra-0-acetyl-D-gluco-pyranosyl)-a-D-mannopyranoside 1 6 which resulted, could not be separated from the byproducts by chromatography because of overlapping of several of the components. This condition was improved by deacetylating the crude mixture, which f a c i l i -tated the separation between the p a r t i a l l y blocked intermed-iates methyl 4,6-0-benzylidene-2 and 3-0-D-glucopyranosyl-a-D-mannopyranoside 12 and 18, and other impurities. It was found however, that during column or t h i n layer chromato-graphic p u r i f i c a t i o n of the mixture, the l a b i l e benzylidene group was eliminated making the separation d i f f i c u l t . This e f f e c t was minimized by keeping the mixture s l i g h t l y basic. Scheme 3 2 3 A s p i n a l l and co-workers (68) have shown that a methyl glycoside i n a disaccharide can be removed r e a d i l y by ace-t o l y s i s without cleaving the glycosidic linkage i n the d i s -accharide. These conditions (2$ s u l f u r i c acid i n acetic anhy-dride) were used to remove the methyl group from the f u l l y blocked disaccharides 3J5 and 16 and also from the p a r t i a l l y blocked compounds 17 and 18. Milder a c e t o l y s i s conditions (1$ and s u l f u r i c acid i n acetic anhydride) also removed the methyl glycosides without cleaving the glyco s i d i c l i n k of the disaccharides. In the case of the disaccharides i n question, a c e t o l y s i s removed both the methyl glycoside and the l a b i l e benzylidene group. The freed hydroxyl groups are immediately acetylated, giving a mixture containing the f u l l y acetylated disaccharides 1£ and 20. Deacetylation of the products from acetolysis and sub-sequent analysis by paper chromatography gave a mixture con-t a i n i n g monosaccharides, two disaccharides 21 and 22, and s l o -wer moving components which included a tr i s a c c h a r i d e . The two disaccharides were estimated to be i n a r a t i o of about 3«2, i n d i c a t i n g that there was some s t e r e o s e l e c t i v i t y i n the condensation reaction, but not as much as was expected. This r a t i o did not change su b s t a n t i a l l y by changing solvent, reaction temperature, or condensing agent. As was mentioned e a r l i e r , chromatographic separation of the f u l l y blocked disaccharides i n the condensation mixture as well as the deacetylated mixture was d i f f i c u l t . However, the separation of the two free disaccharides from other im-2 4 p u r i t i e s c o u l d be c a r r i e d o u t b y p a p e r c h r o m a t o g r a p h y , b u t o n l y s m a l l a m o unts c o u l d b e c h r o m a t o g r a p h e d i n o r d e r t o p r e -v e n t o v e r l a p p i n g b e t w e e n t h e two d i s a c c h a r i d e s . T h i s r a t h e r t e d i o u s s e p a r a t i o n made t h e o v e r a l l y i e l d o f d i s a c c h a r i d e v e r y l o w a n d t h e a t t e m p t was a b a n d o n e d . A l f r e d s o n a n d c o - w o r k e r s ( 6 9 ) h a v e r e c e n t l y d e s c r i b e d t h e s y n t h e s i s o f 3 - 0 - 3 - D - g l u e o p y r a n o s y l - D - m a n n o s e . T h e i r s y n -t h e s i s i n v o l v e d t h e c o n d e n s a t i o n o f t e t r a - O - a c e t y l - a - D - g l u c o -p y r a n o s y l b r o m i d e £ w i t h m e t h y l 2 - 0 - b e n z y l - 4 , 6 - 0 - b e n z y l i d e n e -c t - D - m a n n o p y r a n o s i d e i n a r e a c t i o n w h i c h g i v e s t h e 1-3 l i n k e d g l y c o s i d e a s t h e o n l y d i s a c c h a r i d e . The k e y i n t e r m e d i a t e u s e d b y t h e s e w o r k e r s was o b t a i n e d b y s e l e c t i v e b e n z y l a t i o n o f m e t h y l 4 , 6 - 0 - b e n z y l i d e n e - a - D - m a n n o p y r a n o s i d e w i t h one m o l e o f b e n z y l b r o m i d e i n t h e p r e s e n c e o f s i l v e r o x i d e i n d i m e t h y l -f o r m a m i d e , a r e a c t i o n w h i c h g a v e t h e 2 - 0 - b e n z y l compound p r e -f e r e n t i a l l y , and w h i c h a l s o c r y s t a l l i z e d . T h e i r r e a c t i o n g a v e p r e f e r e n t i a l b e n z y l a t i o n a t t h e 2 - h y d r o x y l , n o t 3» a s was e x p e c t e d f o r o u r c o n d e n s a t i o n r e a c t i o n . A s m e n t i o n e d e a r l i e r , t h e a x i a l ( 2 - h y d r o x y l ) p o s i t i o n was e x p e c t e d t o be l e s s r e a c t i v e t h a n t h e e q u a t o r i a l ( 3 - h y d r o x y l ) o n e , i n a p a r a l l e l s i t u a t i o n t o t h e a x i a l ( 4 - h y d r o x y l ) a n d e q u a t o r i a l ( 3 - h y d r o x y l ) p o s i t i o n s i n g a l a c t o s e . The p r e s e n c e o f t h e b u l k y b e n z y l i d e n e g r o u p a t p o s i t i o n s 4 a n d 6 o f t h e manno-p y r a n o s e u n i t c o u l d make t h e 3 - h y d r o x y l r e l a t i v e l y h i n d e r e d w i t h r e s p e c t t o t h e 2 - h y d r o x y l , m a k i n g t h e 2 - h y d r o x y l more a v a i l a b l e . I t i s a l s o p o s s i b l e t h a t t h e s o l v e n t u s e d ( d i -m e t h y l f o r m a m i d e ) m a y h a v e some i n f l u e n c e o n t h e s t e r e o s e l e c -2 5 t i v i t y of the reaction. C. 6-0-(4-O-Methyl-0-D-glucopyranosyl)-D-galactose In studying the structure of aci d i c polysaccharides i t i s often more convenient to carry out fragmentation a f t e r the uronic acid function has been reduced, a transformation which i s r e a d i l y achieved with diborane ( 7 0 ) or complex hydrides ( 7 1 , 7 2 ) . S i m i l a r l y , an aldobiouronic acid which has been iso -l ated from the p a r t i a l hydrolysis of a polysaccharide may be more r e a d i l y characterized as the derived neutral disac-charide ( i . e . 7 3 . 7 4 ) . The disaccharide 6-O-(4-O-methyl-0-D-glucopyranosyl)-D-galactose i s found as the uronic acid i n many plant gums ( 6 5 , 7 5 . 7 6 ) . The synthesis of t h i s disaccharide, which has not been described previously, was undertaken i n part to provide a model compound which would f a c i l i t a t e the s t r u c t u r a l deter-mination of polysaccharides containing t h i s unit. (a) Synthesis of 4-0-methyl-D-glucose A key intermediate i n the synthesis was 4-0-methyl-D-glu-cose, which was prepared as described i n Scheme 4. D-Glucose was treated with ethanethiol i n the presence of concentrated hydrochloric acid e s s e n t i a l l y as described i n the l i t e r a t u r e ( 7 7 . 7 8 ) . The r e s u l t i n g D-glucose d i e t h y l t h i o a c e t a l 2 ^ , an ac y c l i c compound, had a melting point (124-125°) somewhat high-er than the corresponding l i t e r a t u r e ( 7 7 ) value ( 1 1 9 - 1 2 0 ° ) , but i t s p.m.r. spectrum indicated the presence of two ethyl groups i n accordance with the structure of 2 3 . Scheme 4 E t S \ _ ^ S E t EtSH HC1 HO-D-glucose -OH acetone -OH 1 OH —OH H 22 E t S ^ / S E t H 0 3 H HgCl~ H CdCO? M 3 — — — — > H 3 "MeOH > OH — O ^ C H 3 24 CH O^^-ocH "OH 3 p-nitro-benzoyl chloride, ( mixture) J CH 3O^J3CH 3 CH CH CH 3 O O C H 3 NaOH CH CH--> 3 -OR R = P - N 0 2 C 6 ° 4 o i . C H 3 ( K ^ O C H 3 CH, CH, -OH H —OCH 3 3 * N CH30 NaH/DMSO 0H.I -OH 3 H 3 H 22 3 4-0-methyl-D-glucose 28 2 7 The d i e t h y l t h i o a c e t a l 2 ^ was t r e a t e d w i t h a c e t o n e i n t h e p r e s e n c e o f a c a t a l y t i c amount o f c o n c e n t r a t e d s u l f u r i c a c i d , g i v i n g a m i x t u r e o f i s o p r o p y l i d e n e compounds w h i c h i n -c l u d e d 2, 3 « ' 5 . 6 - d i - 0 - i s o p r o p y l i d e n e - D - g l u c o s e d i e t h y l t h i o a c e -t a l 2k. The o t h e r p r o d u c t s o f t h i s r e a c t i o n p r o b a b l y i n -c l u d e t h e 3 » 4 i 5 » 6 d i a c e t a l a n d m o n o a c e t a l s . A l t h o u g h i t i s p o s s i b l e t o u s e t h e i s o p r o p y l i d e n e d i e t h y l t h i o a c e t a l ( w i t h t h e 4 - h y d r o x y l f r e e ) f o r t h e s u b s e q u e n t m e t h y l a t i o n s t e p , t h e O - a c e t a l i s p r e f e r a b l e b e c a u s e i t i s e a s i l y h y d r o l y z a b l e i n d i l u t e a c i d w h i l e t h e S - a c e t a l i s n o t . C u r t i s a n d J o n e s ( 7 7 ) u s e d t h e a c y c l i c 2 , 3 « 5 » 6 - d i - 0 - i s o p r o p y l i d e n e - D - g l u c o s e d i e t h y l a c e t a l f o r t h e s y n t h e s i s o f l a c t o s e . R a t h e r t h a n u s i n g t h i s O - a c e t a l , i t was d e c i d e d t h a t t h e c o r r e s p o n d i n g d i m e t h y l a c e -t a l w o u l d be more a p p r o p r i a t e b e c a u s e t h i s i n t e r m e d i a t e c o u l d be e a s i l y p u r i f i e d v i a t h e c r y s t a l l i n e 4 - 0 - p _ - n i t r o b e n z o a t e , a known compound ( 7 9 ) . T h u s t h e m i x t u r e c o n t a i n i n g 2 , 3 t 5 . 6 - d i -O - i s o p r o p y l i d e n e - D - g l u c o s e d i e t h y l t h i o a c e t a l 2k was c o n v e r -t e d t o t h e c o r r e s p o n d i n g d i m e t h y l a c e t a l s 2J5 b y t r e a t m e n t o f t h e m i x t u r e w i t h m e r c u r y and cadmium s a l t s . The r e s u l t i n g s y r u p , w h i c h c o n t a i n e d 2 , 3 i 5 , 6 - d i - 0 - i s o p r o p y l i d e n e - D - g l u c o s e d i m e t h y l a c e t a l 2j5 and o t h e r p r o d u c t s , was t r e a t e d w i t h p_-n i t r o b e n z o y l c h l o r i d e i n p y r i d i n e t o g i v e c r y s t a l l i n e 2 , 3 i 5 , 6 -d i - 0 - i s o p r o p y l i d e n e - 4 - 0 - £ - n i t r o b e n z o y l - D - g l u c o s e d i m e t h y l a c e t a l 2 6 . The m e l t i n g p o i n t o f 2 6 ( 1 0 3 - 1 0 5 ° ) was somewhat l o w e r t h a n t h e l i t e r a t u r e v a l u e ( 1 0 6 - 1 0 7 ° ) f o r t h i s com-p o u n d . The p.m.r. s p e c t r u m o f 2 6 i n c l u d e d two i s o p r o p y l i d e n e g r o u p s , two m e t h o x y s i g n a l s , and t h e j j - n i t r o b e n z o a t e g r o u p . 28 The p_-nitrobenzoate 2 6 was d e - e s t e r i f i e d under basic conditions to give pure 2,3i5,6-di-0-isopropylidene-D-glucose dimethyl acetal 2j> as a syrup which on p.m.r. showed two me-thoxy signals around 6.6 and four methyl signals around 8 . 7 , corresponding to the two isopropylidene groups. This acetal slowly hydrolyzed on standing under neutral conditions, but not when stored under s l i g h t l y basic conditions. The acetal 2J> was methylated by the method of Purdie ( 8 0 ) and also by the method of Hakomori (81) to the corres-ponding 4-0-methyl derivative 2 £ . Methylation by the Purdie method required several successive treatments under reflux f o r 1-2 days with methyl iodide and s i l v e r oxide i n order to get complete reaction. This d i f f i c u l t y i n methylating 2j> was somewhat surprising since i t was expected that t h i s a c y c l i c structure should have been f a i r l y reactive r e l a t i v e to a r i n g structure. In a t y p i c a l series of Purdie methylations, 50% reaction was obtained at the end of the f i r s t methylation, 8 0 $ at the end of the t h i r d , and over 9 0 $ at the end of the fourth. Freshly prepared s i l v e r oxide was used, since the commercially available product was l e s s active and many more methylations were required i n order to get complete reaction. Although the product obtained aft e r several Purdie methylations was quite clean and not much degradation resulted, the time required (1-2 weeks depending on the s i l v e r oxide used) made t h i s method l e s s p r a c t i c a l than the Hakomori met-hod. The methylation method described by Hakomori ( 8 l ) f o r 2 9 the methylation of polysaccharides converts the carbohydrate alcohol into the corresponding sodium s a l t by reaction with methylsulfinylanion (82), a strong base. Reaction of the sodium s a l t with methyl iodide r e s u l t s i n the formation of the methyl ether qu a n t i t a t i v e l y . The method has the advan-tage that methylation can be ca r r i e d out by one single t r e a t -ment and i n a much shorter time than required by other met'r hods. 2,3«5»6-Di-O-isopropylidene-D-glucose dimethyl acetal ( 2 5 ) i n methyl sulfoxide was reacted with methylsulfinylanion f o r 4-5 minutes at room temperature, followed by the dropwise ad-d i t i o n of methyl iodide. Reaction of the sugar with the anion for 2-3 hours gave more degradation products. The methylated product 2 7 was best iso l a t e d by extraction of the methyl s u l -foxide sol u t i o n with petroleum ether or hexane. Concentra-t i o n of the extracts then gave the syrup 2 £ contaminated with small amounts of methyl sulfoxide. This methyl sulfoxide con-taminant could not be removed without passing the mixture through a s i l i c a gel column. The mixture was therefore hy-drolyzed without further p u r i f i c a t i o n i n .025M s u l f u r i c acid at 6 0 ° , which removed a l l the acetal functions to give 4 - 0 -methyl-D-glucose 28 as an impure syrup. Paper chromatographic analysis of 2 8 showed e s s e n t i a l l y one carbohydrate spot, corresponding to 4-0-methyl-D-glucose. In addition, a small amount of degradation products was pre-sent. No glucose was detected, i n d i c a t i n g that the methyla-t i o n step gave a complete reaction. However, 4-0-methyl-D-3 0 glucose obtained a f t e r several Purdie methylations of 25_ and subsequent hydrolysis showed a small amount of glucose on paper chromatography. A portion of 4-0-methyl-D-glucose was treated with phenyl-hydrazine and a c a t a l y t i c amount of acetic acid. The r e s u l t -ing phenylozazone 2£, a known compound ( 7 9 . 8 4 ) , gave a melting point i d e n t i c a l to the l i t e r a t u r e value. Acetylation of 4-0-methyl-D-glucose 28 with acetic anhy-dride and sodium acetate gave c r y s t a l l i n e 1,3.4,6-tetra-0-acetyl-4-0-methyl -e-D-glucopyranose J3_0. This compound has also been obtained i n the l i t e r a t u r e ( 8 5 , 8 6 ) by methylation of methyl 2,3,4-tri-O-acetyl -g-D-glucopyranoside, i n which the 4-acetate migrated to p o s i t i o n 6 during the methylation. The proton magnetic resonance spectrum (Figure 2) of 30 showed four acetate groups and one methoxy si g n a l . The coup-l i n g constant for. the low f i e l d anomeric hydrogen was large ( 8 . 0 H ), which indicates ( 8 3 ) that the beta acetate was ob-z tained. (b) I s o l a t i o n of 4-0-methyl-D-glucose from mesquite gum An al t e r n a t i v e method fo r obtaining 4-0-methyl-D-glucose was by i s o l a t i o n from a natural source. This source was mes-quite gum, a polysaccharide that has a galactan core contain-ing 4-0-methyl-D-glucuronic acid. Arabinofuranose side chains are linked to the galactose units by 1-2, 1-3 and 1-4 linkages ( 7 5 ) . The i s o l a t i o n procedure (Scheme 5 ) involved f i r s t the par-31 Scheme: 5 Mesquite gum 1. .mild E + 2. d i a l y s i s polysaccharide core 31 HCl/MeOH IIAIH^/THP 1. AcoO-pyr. 2. NaDCH^ 33 t i a l hydrolysis of the gum with d i l u t e acid as described by White ( 8 7 ) . This removed the more l a b i l e arabinofuranose side chains without cleaving the polysaccharide core. Sub-sequent d i a l y s i s of the hydrolysis mixture then gave a poly-saccharide J l which was recovered by p r e c i p i t a t i o n from met-hanol • The polysaccharide J l was subjected to methanolysis i n 7 $ methanolic hydrogen chloride, which cleaved the glycosidic linkages and gave the monosaccharide methyl glycosides at the same time. In addition, the uronic acid was e s t e r i f i e d , giving a mixture containing methyl galactoside J3J2 and methyl 4-0-methyl-D-glucuronic acid methyl ester J J . In addition, a small amount of methyl arabinoside was obtained, r e s u l t i n g from incomplete hydrolysis of the arabinose side chains. Methanolysis under reflux for I8h of the polysaccharide J l was incomplete as shown by paper chromatography, which i n d i -cated a large amount of material of low mobility character-i s t i c of oligosaccharides. Reflux f o r an additional 24h did not cleave the oligosaccharides completely. The methyl ester 33 was then separated from the water soluble components by chloroform extraction of the mixture. Methyl 4-0-methyl-D-glucuronic acid methyl ester _3_2 w a s reduced with l i t h i u m aluminum hydride i n tetrahydrofuran to give the corresponding methyl 4-0-methyl-D-glucoside _3_4. In order to separate the product ^ 4 from the aluminum s a l t s , the mixture was acetylated following workup. The acetylated pro-duct 21 w a s "then extracted with chloroform. Deacetylation 34 of 21 i - n the presence of base afforded methyl 4-0-methyl-D-glucoside Tjl as a syrup. Hydrolysis of the methyl glycoside 34 i n 0.5M s u l f u r i c acid at 1 0 0 ° gave a mixture which on pa-per chromatography showed a large proportion of 4-0-methyl-D-glucose 28 and a smaller proportion of galactose and other impurities. Paper chromatographic mobility of 28 was iden-t i c a l to the synthetic material previously obtained. The mixture was best p u r i f i e d by acetylation, which afforded cry-s t a l l i n e 1,2,3» 6-tetra-0-acetyl-4-0-methyl-$-D-glucopyranose 30 i n about 505^ y i e l d from the methyl glucoside _3_4. The mel-ti n g j p o i n t and proton magnetic resonance spectrum of JO were as previously described. The main d i f f i c u l t i e s encountered i n the i s o l a t i o n of 4-0-methyl-D-glucose from mesquite gum were the incomplete methanolysis of the polysaccharide, which resulted i n the loss of material, and d i f f i c u l t y i n recovering methyl 4 - 0 -methyl-D-glucoside _3_4 following reduction with lithium a l -uminum hydride. This l a t t e r step contributed i n part to the lowered y i e l d on account of binding of the carbohydrate on the aluminum s a l t s . In addition, chloroform extraction of the mixture containing the reduced-acetylated product from d i l u t e hydrochloric acid often resulted i n the formation of emulsions. The o v e r a l l y i e l d of 4-0-methyl-D-glucose ob-tained from 2 0 0 g,: of mesquite gum was about 3 g. In a separate experiment, the polysaccharide _3_i obtained a f t e r hydrolysis of the arabinose side chains from the gum, was subjected to p a r t i a l hydrolysis i n . 05M s u l f u r i c acid at 100° using the automated d i a l y s i s machine described by Ga-lanos and co-workers (88) and used on many occasions i n our laboratory for the p a r t i a l hydrolysis of capsular polysac-charides from K l e b s i e l l a (89,90). The purpose for t h i s ex-periment was to i s o l a t e the disaccharide 6-0-(4-O-methyl-0-D-glucuronosyl)-D-galactose, which i s part of the galactan backbone. However, the mixture which was obtained was com-plex and e f f o r t s to separate the components by paper chroma-tography were not successful. (c) 2,3,4-Tri-0-acetyl-4-0-methyl-a-D-glueopyranosyl bromide 1,2,3,6-Tetra-O-acetyl-4-O-methyl-0-D-glucopyranose JO was converted to the corresponding 2,3,6-tri-0-acetyl-4-0-methyl-a-D-glucopyranosyl bromide ^6 by treatment with satu-rated hydrogen bromide i n acetic acid. Preliminary experi-ments f o r t h i s bromination indicated that the reaction could be almost completed within 30 minutes at room temperature, but a small amount of unreacted tetraacetate would remain. Reaction f o r 45 minutes gave complete bromination accompa-nied by a small amount of degradation products, while l-2h gave increased amounts of degradation products. The reaction conducted for 45 minutes was f i n a l l y used f o r preparative purposes. The r e s u l t i n g bromide ^6 was obtained as a syrup. It was found to be quite unstable and would e a s i l y decompose from one day to the next, or even on the rotary evaporator i f temperatures higher than 35° were used. The bromide was 3 6 therefore used immediately a f t e r preparation. The high o p t i c a l r o t a t i o n ( + 1 9 0 ° i n chloroform) of bro-mide ^ 6 indicated that the a-D-anomer was obtained, i n agreement with the known preference of sugar bromides f o r the a-D-configuration, i n which the "anomeric e f f e c t " (91) makes the 0-D-anomer r e l a t i v e l y unstable with respect to the a-D-anomer. It i s i n t e r e s t i n g to note, that the o p t i c a l r o t a t i o n of 2§. i s quite close to the o p t i c a l rotation of the well known tetra-O-acetyl-a-D-glucopyranosyl bromide, a c r y s t a l l i n e com-pound ( 6 2 ) that has [oO D +198° i n chloroform. Considering that J3_6 i s a syrup, the rotations of the two bromides are probably closer, i n d i c a t i n g that substitution at p o s i t i o n 4 makes prac-t i c a l l y no difference to the o p t i c a l r o t a t i o n . (d) 1,213»4-Di-O-isopropylidene-6-0-(4-0-methyl - g-D-glue opy-anosyl)-a-D-galactopyranose In preliminary condensations l , 2 » 3 , 4 - d i - 0 - i s o p r o p y l i -dene-a-D-galactopyranose 1 0 was reacted with excess 2 , 3 , 6 - t r i -0-acetyl-4-0-methyl-a-D-glucopyranosyl bromide J3_6 (Scheme 6 ) using one mole of mercuric cyanide per mole of bromide. Ace-t o n i t r i l e or nitromethane was used as solvent. Although these test condensations gave a considerable amount of the desired disaccharide, inspection by t h i n layer chromatography indicated that these condensations also gave a substantial amount of byproducts. The acid acceptor was then changed from mercuric cyanide to f r e s h l y prepared s i l v e r oxide, which gave a higher proportion of disaccharide. Dry carbon t e t r a -Scheme 6 37 3 8 chloride and benzene were found to be suitable solvents, but absolute chloroform was somewhat better. S i l v e r carbonate (with chloroform as solvent) was also tested as acid acceptor, but the reaction was sluggish. S i l v e r oxide i n chloroform was therefore used f o r condensations on a preparative scale, using D r i e r i t e as an i n t e r n a l drying agent to adsorb the water which i s produced during the reaction. When one mole of bromide per mole of isopropylidene compound 1 0 was used, the reaction did not go to completion, some 1 0 remaining un-reacted. It was therefore necessary to use a 3 0 - 5 0 $ excess bromide, but even so, a small amount of 1 0 did not react. As was mentioned e a r l i e r , i f s i l v e r oxide i s used as conden-sing agent, water produced i n the reaction may react with the bromide before i t reacts with the drying agent, e s p e c i a l l y when r e l a t i v e l y reactive bromides are used. In the above condensation reaction, the major byproduct had the same R^ value as that produced when bromide J36 was overspotted with aqueous s i l v e r n i t r a t e on a t h i n layer plate'before devel-opment. The byproduct was not i s o l a t e d , but was presumed to be 2,3,6-tri-0-acetyl-4-0-methyl-D-glucose, which i s the pro-duct obtained when ^ 6 i s reacted with aqueous s i l v e r n i t r a t e . A few milligrams of the r e s u l t i n g f u l l y blocked disa-ccharide 2iL w a s i s o l a t e d by preparative t h i n layer chromato-graphy and the :proton magnetic resonance spectrum obtained confirmed that i t was i n fact the disaccharide as indicated by the presence of the 0-methyl, acetates, and isopropylidene groups. Although J3Z could be p u r i f i e d with some d i f f i c u l t y by 39 column or t h i n layer chromatography, a better separation was obtained a f t e r deacetylation to 1,2«3,4-di-0-isopropylidene-6-0 - (4 - 0 - me thy 1 - $ - D- g l uc o py r ano syl) - a- D- gal ac t opyrano s e 38» In any case, chromatographic p u r i f i c a t i o n of 2§ w a s neces-sary because t h i s compound c r y s t a l l i z e d . The o v e r a l l y i e l d of the c r y s t a l l i n e intermediate _3_8 was 6 2 $ based on 10. The proton magnetic resonance spectrum of J38 showed the expected groups, but the anomeric hydrogens were p a r t i a l l y hidden. The beta linkage was therefore better established as the disaccharide a l d i t o l , to be discussed l a t e r . (e) 6-O-(4-O-Methyl -0-D-glucopyranosyl)-D-galactose The isopropylidene groups i n l , 2 i 3 * 4 - d i - 0 - i s o p r o p y l i -dene-6-0-(4-0-methyl-£-D-glucopyranosyl)-D-galactose J8 were hydrolyzed using d i l u t e acid under several conditions. The main consideration i n choosing the hydrolytic conditions was that the isopropylidene groups be removed without cleaving the g l y c o s i d i c linkage. Of the conditions tested, heating of 38 i n 0.03M s u l f u r i c acid on a steam bath f o r 40 minutes was found to be the best, because e s s e n t i a l l y a l l the acetal was removed while l i t t l e glycoside cleavage resulted. The other conditions tested gave either incomplete acetal hydrolysis or too much glycoside cleavage. The free disaccharide 6-0-(4-O-methyl-0-D-glueopyranosyl)-D-galactose 22 w a s obtained as a syrup which gave [ a ] ^ + 4 ° i n water. Paper chromatographic analysis showed that i n addition to 22» a small amount of 4-0-methyl-D-glucose and galactose 4 0 were present. In order to obtain a good sample fo r micro-analysis, part of the mixture was p u r i f i e d by column chroma-tography using a Dowex 50W X 2 (Li+) column ( 9 2 ) . The pure disaccharide obtained i n t h i s manner gave [ a ] ^ =0° i n water, consistent with the assignment of a 0-D-linkage. According to Hudson's rules of i s o r o t a t i o n ( 9 3 ) the calculated values f o r [ c f ] D are approximately + 9 3 ° and - 6 ° for the a-D- and #-D-(l-6) l i n k s respectively. Gas l i q u i d chromatographic analysis of the t r i m e t h y l s i l y l ether of the pure disaccharide gave 3 peaks. The faster, well resolved peak was believed to be due to the galactofur-anose residue, while the slower and not c l e a r l y resolved peaks were due to the alpha and beta forms of the galactopy-ranose residue. No s a t i s f a c t o r y c r y s t a l l i n e derivative of the disaccha-ride was found, although the p_-nitroanilide 40 and p_-nitro-benzoate 4 l gave amorphous s o l i d s with melting point ranges of 2 and 3 degrees respectively. Methylation- In order to be c e r t a i n that a disaccharide obtained by synthetic means i s i n fact the expected one, i t i s often necessary to obtain an independent proof of i t s structure, although i n some cases, the structure of the syn-th e t i c disaccharide may be ascertained by the mode of synthe-s i s . The proof of structure should include f i r s t , the posi-t i o n of the linkage ( i . e . 1 - 2 , 1 - 3 , etc.), and second, the nature of the glycosidic form (alpha or beta). In addition, i n synthetic pathways which may re s u l t i n epimerization or 41 other transformations which give r i s e to unexpected sugars, the proof of structure should ascertain the nature of the two monosaccharide components. This insures that no unexpected transformations occured during the synthesis. The p o s i t i o n of the linkage can be demonstrated by me-t h y l a t i o n and periodate oxidation of the disaccharide. These two techniques, combined with gas chromatography and mass spectrometry, have been extensively used i n our laboratory for the s t r u c t u r a l analysis of polysaccharides. The Hakomori method of methylation, which was discussed i n connection with the methylation of the acetal 2j5, was the method chosen for the methylation of disaccharides because t h i s method gives a quantitative reaction a f t e r a single treatment. Methylation (Scheme 7) of 6-0-(4-0-methyl-g-D-glucopyranosyl)-D-galactose jQ_ gave the f u l l y methylated disaccharide 42 as a syrup. The reaction of J3£ with methyl-s u l f i n y l anion for 45 minutes to l h gave a minumum of de-gradation products. However, the addition of the methyl i o -dide had to be conducted very c a r e f u l l y , since extensive de-gradation would r e s u l t i f the temperature increased too much. The dropwise addition was therefore conducted at or below 20°. Compound 42 was p u r i f i e d by preparative t h i n layer chro-matography and the infrared spectrum of the r e s u l t i n g syrup showed no hydroxyl absorptions, in d i c a t i n g that methylation was complete. The next step i n the proof of structure consisted i n the methanolysis and hydrolysis of the f u l l y methylated disac-Scheme 7 39 I 1. NaH/DMSO g . l . c . analysis charide 42 and i d e n t i f i c a t i o n of the r e s u l t i n g fragments. Methanolysis of 42 and subsequent analysis by gas-liquid chromatography gave methyl 2,3,4,6-tetra-0-methyl-D-gluco-side 44 and methyl 2,3,4-tri-O-methyl-D-galactoside 4j>, frag-ments which are c h a r a c t e r i s t i c of the 1-6 l i n k only. In ad-d i t i o n , a fragment corresponding to methyl 2,3,5-tri-0-methyl-D-galactofuranoside 46 was obtained. Fragment 46 i s expected to a r i s e from methanolysis of the galactofuranose structure 43 (Scheme 7). The formation of a high proportion of galacto-furanose isomer during methylation has been reported i n the l i t e r a t u r e (73.97.98). S i m i l a r l y , hydrolysis of 42 and subsequent examination by paper chromatography gave 2,3,4,6-tetra-O-methyl-D-glucose 47 and 2,3,4-tri-O-methyl-D-galactose 48, as compared to authen-t i c standards. In addition, a f a s t e r moving component (2,3,5-tri-O-methyl-D-galactose) was detected. The above methylation analysis required standards with which to make comparisons. Methyl 2,3,4,6-tetra-O-methyl-D-glucoside and 2,3,4,6-tetra-O-methyl-D-glucose, which can be obtained by methylation and hydrolysis of D-glucose, were available i n the laboratory from previous studies by other workers. However, methyl 2,3,4-tri-O-methyl-D-galactoside and 2,3,4-tri-O-methyl-D-galactose were not available and thus had to be prepared by synthesis. This was c a r r i e d out (Scheme 8) by benzylation of 1,2 i3,4-di-O-isopropylidene-a-D-galacto-pyranose 10 with benzyl chloride and potassium hydroxide,-us-ing toluene as solvent. Although t y p i c a l benzylation reac-tions i n the l i t e r a t u r e use a large excess of benzyl chloride 44 Scheme 8 1. NaH/DMSO 2. C H i 4 5 ( i n many c a s e s a s t h e s o l v e n t ) , t h e a b o v e b e n z y l a t i o n c o u l d be c o n d u c t e d u s i n g a b o u t 1.2 m o l e s o f t h i s r e a g e n t p e r m o l e o f s u g a r , w h i c h f a c i l i t a t e d t h e w o r k u p b y s h o r t e n i n g t h e s t e a m d i s t i l l a t i o n o f t h e e x c e s s r e a g e n t . T o l u e n e r a t h e r t h a n t h e u s u a l x y l e n e was u s e d a s s o l v e n t b e c a u s e o f t h e l o w -e r b o i l i n g p o i n t . The s t r u c t u r e o f t h e r e s u l t i n g 6 - 0 - b e n z y l -l , 2 i 3 , 4 - d i - O - i s o p r o p y l i d e n e - a - D - g a l a c t o p y r a n o s e j > l , a s y r u p , was c o n f i r m e d b y p r o t o n m a g n e t i c r e s o n a n c e s p e c t r o s c o p y . Compound J51 was t h e n s u b j e c t e d t o h y d r o l y s i s i n d i l u t e s u l f u r i c a c i d , a f f o r d i n g c r y s t a l l i n e 6 * 0 - b e n z y l - D - g a l a c t o s e 52. a known ( 9 9 ) compound. The m e l t i n g p o i n t o f £2 ( 9 6 - 9 8 ° ) was i d e n t i c a l t o t h e l i t e r a t u r e v a l u e . Compound j5_l was a l s o s u b j e c t e d t o m e t h a n o l y s i s t o g i v e m e t h y l 6 - 0 - b e n z y l - D - g a l a c t o s i d e 52.* a l s o c r y s t a l l i n e . Me-t h y l a t i o n o f 52 b y t h e m e thod o f H a k o m o r i (compound 52 r a t h e r t h a n 52 was m e t h y l a t e d s i n c e t h e l a t t e r , b e i n g a r e d u c i n g s u g -a r , was e x p e c t e d t o g i v e more d e g r a d a t i o n p r o d u c t s d u r i n g me-t h y l a t i o n ) , p u r i f i c a t i o n o f t h e r e s u l t i n g m e t h y l a t e d p r o -d u c t j>4 b y t h i n l a y e r c h r o m a t o g r a p h y a n d s u b s e q u e n t r e m o v a l o f t h e b e n z y l g r o u p b y h y d r o g e n o l y s i s g a v e m e t h y l 2 , 3 , 4 - t r i -O - m e t h y l - D - g a l a c t o s e ^ a s a s y r u p . 2 , 3 , 4 - T r i - O - m e t h y l - D - g a -l a c t o s e 4 £ was o b t a i n e d b y h y d r o l y s i s o f 4 5 w i t h G.5M s u l f u r -i c a c i d a t 1 0 0 ° . P e r i o d a t e o x i d a t i o n - P r o o f o f s t r u c t u r e b y p e r i o d a t e o x i -d a t i o n makes u s e o f t h e w e l l known r e a c t i v i t y o f v i c i n a l g l y -c o l s w i t h p e r i o d a t e ( 1 0 0 , 1 0 1 ) . S i n c e t h e number o f v i c i n a l g l y c o l s i n a n o l i g o s a c c h a r i d e w i l l d e p e n d o n t h e n a t u r e o f 46 the linkage ( 1 - 2 , 1 - 3 , etc.), the t o t a l uptake of periodate w i l l he diagnostic of the linkage. In cases i n which the up-take cannot distinguish between two possible linkages, the pro-ducts of the oxidation (formic acid, formaldehyde, etc.) w i l l usually d i s t i n g u i s h between the d i f f e r e n t p o s s i b i l i t i e s . 6-0-(4-0-Methyl -3-D-glucopyranosyl)-D-galactose J£ w a s oxidized as shown i n Scheme 9. In duplicate oxidations, the uptake of periodate became constant at 4.9 and 5.0 moles per mole of sugar respectively, which i s diagnostic of the 1-6 linkage. The formic acid produced i n the reaction was t i t r a -ted with d i l u t e sodium hydroxide and 4 moles of formic acid per mole of disaccharide were obtained. The polyaldehyde _5_5_ obtained a f t e r oxidation was reduced to the polyalcohol j>6 with sodium borohydride i n water. The polyalcohol was then hydrolyzed, giving 2-0-methyl-D-erythri-t o l %2_ and ethylene g l y c o l J?6. These two fragments, j>2 a n a  58 are diagnostic of the disaccharide J9. The i d e n t i t y of 57 and j>8 were ascertained by paper chromatography and by gas - l i q u i d chromatography of the v o l a t i l e t r i m e t h y l s i l y l ethers ( 1 0 2 ) . Standard 2-0-methyl-D-erythritol, which was required f o r comparison, was obtained by periodate oxidation and borohydride reduction of 4-0-methyl-D-glucose, (iso l a t e d from mesquite gum) as suggested by A s p i n a l l and Fairweather ( 7 3 ) . The uptake of periodate during the oxidation can be monitored by several t i t r a t i o n methods ( 1 0 3 ) . However, the oxidation of J3_9_ was not monitored using these methods Scheme 9 39 ' CHO CH3O CHO CHO NaBH, OH i 6 OH OH H -OH -OCH "OH 3 + -^-OH 'OH t r i m e t h y l s i l y l ethers g . l . c analys i -OH ^ 8 48 because not enough disaccharide was available to do so, and because i t would have been impractical to use a l l the a v a i l -able material f o r a structure proof which necesitates the destruction of the material. The spectrophotometric method (104), i n which the absorbance of periodate at 222.5nm i s followed, was therefore chosen to monitor the reaction. About lOmg of sugar was used f o r each oxidation. In order to obtain a more accurate determination, a Beer Lambert plot of absorbances vs. known concentration of per-iodate was obtained. The concentration of the unreacted periodate i n the reaction was therefore determined from i t s absorbance at 222.5nm. It should be noted that the absorbance of the oxidation mixture at 222.$xm includes a large contribution due to periodate and a smaller contribution due to iodate, a bypro-duct of the reaction. The contribution to the absorbance due to iodate i s usually ignored, but i n t h i s ease i t was f e l t that a correction was necessary because a small amount of sugar was being used. The correction was obtained by mak-ing a standard iodate solution having a concentration equivi-lent to the expected concentration of iodate i n the reaction (5 moles iodate per mole of sugar) and taking the absorbance of t h i s standard solution at 222,5nm. The absorbance ob-tained (0.040) was then rsubtracted from the absorbance of the oxidation mixture before extrapolation from the Beer-Lambert p l o t . It i s of int e r e s t to note that i n addition to fragments 4 9 57 and j>8 obtained a f t e r reduction and hydrolysis of the periodate oxidation mixture, gas-liquid chromatographic analy-s i s of the t r i m e t h y s i l y l ethers usually gave r i s e to other peaks of low i n t e n s i t y . No explanation for the presence of these extraneous peaks was found. However, they may be due i n part to overoxidation ( 1 0 0 ) or to acetals of glycolalde-hyde formed a f t e r the oxidation ( 1 0 5 ) . Disaccharide a l d i t o l - In order to f a c i l i t a t e the pro-cess of c r y s t a l l i z a t i o n , i t i s often convenient to reduce the disaccharide to the corresponding a l d i t o l , so that the mixture of alpha and beta anomers ( i n pyranose and furanose forms) at the reducing end are s i m p l i f i e d into one compo-nent. This transformation i s also very useful i n the l i n k -age analysis of polysaccharides because gas-liquid chromato-graphic analysis of the derivatized fragments w i l l give only one peak per sugar, whereas the reducing sugar can give up to 4 peaks. In addition, spectral; i d e n t i f i c a t i o n of the sugar i s f a c i l i t a t e d i n the reduced form. The syrup 22 w a s reduced with sodium borohydride i n water to give 6-O-(4-O-methyl-0-D-glucopyranosyl)-D-galacti-t o l ( i . e . l-0-(4-0-methyl - g-D-glucopyranosyl)-L-galactitol) 4 9 , a syrup which c r y s t a l l i z e d a f t e r standing f o r about one year. Acetylation of t h i s syrup i n the usual manner afforded 6-0-(4-0-methyl-g-D-glue opyrano syl)-D-galac t i t o l octaacetate ^ as a syrup. Injection of a sample of j50 on the gas chromatograph, gave one peak only. The sample 50 was c o l l e c t e d from the gas chromatograph using a small c a p i l a r y at the gas e x i t , as described by Gibney (9*0. The pure a l d i t o l acetate c r y s t a l l i z e d immediately and the co l l e c t e d sample was used to seed the bulk of the material. The proton magnetic resonance spectrum of the a l d i t o l acetate £0 showed the methoxy signal and eight acetates, but the anomeric hydrogen could not be distinguished c l e a r l y due to overlapping with the other r i n g hydrogens. On the other hand, the spectrum of the free a l d i t o l 4£ (Figure 3) showed c l e a r l y the H-l si g n a l at 5*53 having a coupling constant of 7.5 H_, which i s consistent with the 0-D-configuration (95»96). tr 5.0 6.0 " 7.0 Figure 3. P.M.R. Spectrum (DgO) of compound 4_9 t—1 5 2 Synthesis of ot-D-glucosides A. Attempted use of a non-participating carbonate group In order to investigate the p o s s i b i l i t y of using a glu-cosyl bromide having a non-participating 2,3 c y c l i c carbo-nate group for the synthesis of 1,2-cis glucosides, the preparation of 4,6-di-0-acetyl-2,3-0-carbonyl-a-D-glucopy-ranosyl bromide 6 2 was attempted. As was mentioned i n the introduction, Gorin and P e r l i n (14) obtained 1,2-cis man-nosides by condensations of a s i m i l a r bromide with alcohols, but to the knowledge of t h i s author, the synthesis of the glucosyl bromide 62 has not been reported i n the l i t e r a t u r e . The c y c l i c carbonates are usually obtained by reaction of phosgene with a suitable blocked sugar having two v i c i n a l alcohol functions, as for example, i n the preparation of methyl 5-0-benzyl-D-ribofuranoside 2,3 carbonate (106). A more convenient method has been described by Doane and co-workers (107), i n which the c y c l i c carbonate i s obtained by reaction of the sugar with ethyl chloroformate and t r i e t h y -lamine. Recently, c y c l i c carbonates have been obtained by t r a n s e s t e r i f i c a t i o n with ethylene carbonate (108). Acetolysis of methyl 4,6-0-benzylidene-a-D-glucopyra-noside 2,3-carbonate- Methyl a-D-glucopyranoside was treated with benzaldehyde and zinc chloride and methyl 4,6-O-benzylidene-a-D-glucopyranoside £2 was obtained. Addition of triethylamine and ethyl chloroformate to a benzene s o l -5 3 ution of j5£ as described by Doane ( 1 0 7 ) gave methyl 4,6-0-ben-zylidene-a-D-glucopyranoside 2,3-0-carbonate 6 0 i n good y i e l d (Scheme 1 0 ) . Instead of allowing the reaction to stand f o r l h as described by these workers, the mixture was worked up with-i n 1 0 minutes a f t e r the addition of triethylamine was com-pleted. This was necessary because when the reaction was a l -lowed to stand f o r a longer period of time, lower y i e l d s were obtained because part of the carbonate group was removed by the excess base. In addition, a large excess of triethylamine was not required f o r the reaction to go to completion, and only a small excess was used. The reaction of a carbohydrate with acetic anhydride i n the presence of an acid c a t a l y s t , also known as "acetoly-s i s " , has been used f o r cleaving polysaccharides ( 1 0 9 ) , and for removing small aglycons from monosaccharides and disac-charides ( 2 3 , 6 8 , 1 1 0 ) . Typical acid catalysts include acetic acid, s u l f u r i c acid, perchloric acid, and l a t e l y ( 1 1 1 ) , boron t r i f l u o r i d e . The mechanism of t h i s reaction w i l l be d i s -cussed b r i e f l y i n connection with the acetolysis of 6 0 . C r y s t a l l i n e methyl 4,6-0-benzylidene-cc-D-glucopyranoside 2,3-carbonate 6 0 was subjected to several a c e t o l y s i s con-d i t i o n s . It was hoped that l , 4 , 6 - t r i - 0 - a c e t y l - 2 , 3 - 0 - c a r b o -nyl-a-D-glucopyranose 6 1 would be obtained, r e s u l t i n g from the simultaneous removal of the benzylidene and methyl groups and t h e i r concurrent substitution by acetates. Compound 6 l . i f obtained, could be brominated to bromide 6 2 . However, the product iso l a t e d following a c e t o l y s i s was not 6 1 . The pro-Scheme 10 Ac 55 ton magnetic resonance spectrum (Figure 4) of t h i s product indicated : the presence of four acetate groups instead of the expected three. In addition, the spectrum showed two methoxy si n g l e t s , each integrating f o r 1% hydrogens. The spectrum also showed two low f i e l d doublets, at 4.02 and 4.10 respectively having s l i g h t l y d i f f e r e n t coupling constants -'- (3«1H_ and 4,0 H r e s p e c t i v e l y ) , each doublet integrating for % hydrogen. These data strongly suggest that the products is o l a t e d have the structure 6j3 shown i n scheme 10 j two a c y c l i c acetals epimeric at 0-1, r e s u l t i n g from ri n g opening of the pyranose r i n g . The additional acetate signal r e s u l t s from acetylation of the 5-hydroxyl, while the two methoxy signals derive from the two compounds epimeric at C - l . Additional evidence f o r structure 6 j w a s indicated when the material was deacetylated with sodium methoxide, a reac-t i o n which gave glucose only as shown by paper chromatography. Reaction of a hemiacetal of structure 6 j with base would be expected to give the free sugar, while a methyl glucoside or an acetal would be stable i n base. The a c e t o l y s i s conditions t r i e d included the use of s u l f u r i c acid, acetic acid and boron t r i f l u o r i d e as catalysts i n a v a r i e t y of concentrations and reaction times. A l l the conditions, except fo r one, gave one spot by t h i n layer chromatography, and the proton magnetic resonance spectra of the p u r i f i e d materials were i d e n t i c a l . Thus, the same com-ponents were obtained from these reactions. The one excep-t i o n was the reaction of 60 i n 10$ s u l f u r i c acid i n acetic I I I I 1 I I. I I I -r-r - H - > i ,i i i i h i i i i i i i i | i i i 9 . 0 1 0 . 0 57 anhydride heated to 1 0 0 overnight. This removed the methyl groups, but the proton magnetic resonance;.;, spectrum of the p u r i f i e d product gave too many acetates, i n d i c a t i n g that the carbonate group was also removed. This reaction was ac-companied by extensive degradation while the others were not. The attacking species i n the acet o l y s i s reaction i s be-lie v e d to be the acetylium ion CH^C^O ( 1 0 9 ) , which r e s u l t s from cleavage of acetic anhydride i n the presence of acid. This ion can attack the carbohydrate molecule eit h e r at the glucoside oxygen or at the r i n g oxygen. In the case of methyl 4-6-0-benzylidene-cc-D-glucopyranoside 2,3-carbonate 6 0 (Scheme 1 1 ) , attack on the glucoside oxygen r e s u l t s i n the lo s s of the methyl glycoside to give a c y c l i c carbonium ion 64, which on further reaction results i n the formation of the t r i a c e t a t e 6 l . A l t e r n a t i v e l y , the acetylium ion can attack at the r i n g oxygen giving an a c y c l i c , resonance s t a b i l i z e d intermediate 6 5 . Further reaction of t h i s intermediate then gives the acy c l i c compounds 63_. In addition, attack on the interme-diate 6j5 would be expected to give two epimeric compounds i n equal proportion, since attack by acetate from either side of t h i s carbonium ion i s equally probable. This i s i n agree-ment with the proton magnetic resonance spectrum of 6 3 , which indicated the presence of two methoxy signals of equal? i n -t e n s i t y . These two methoxy signals would be expected to be at a s l i g h t l y d i f f e r e n t chemical s h i f t , since t h e i r elec-tronic environments i n the two epimeric compounds are d i f -ferent . 58 Scheme 11 5 9 The r i n g opening of 6b during a c e t o l y s i s i s l i k e l y to be aided by the 2,3 c y c l i c carbonate group, since t h i s group spans two trans hydroxyl functions and i s therefore expec-ted to be r e l a t i v e l y strained. Opening of the pyranose r i n g to the a c y c l i c form r e s u l t s i n the release of t h i s s t r a i n i n the f i v e membered carbonate r i n g . This i s supported by a separate but p a r a l l e l set of experiments conducted i n our laboratory by Bebault and Dutton ( 1 1 2 ) , i n which methyl 4 , 6 -O-benzylidene-d-D-mannopyranoside 2,3-carbonate, the mannose equivalent of 6 0 , was subjected to acet o l y s i s i n 2% s u l f u r i c acid i n acetic anhydride (one of the conditions used for the a c e t o l y s i s of 6 0 ) . This reaction gave the expected t r i -acetate and no a c y c l i c products were obtained. Since the 2,3-carbonate group i n t h i s mannose compound i s c i s and there-fore not as strained, one must i n f e r that the 2,3-trans carbonate group i n the glucose compound 6 0 i s responsible for the r i n g opening observed. B. Syntheses of a-D-glucosides by the 2,3-dibenzyl method The synthesis of disaccharides of the a-D-glucosyl con-f i g u r a t i o n described i n t h i s t h e s is, i s based on the glycoside forming reactions of p_-nitrobenzoyl bromides having a non-p a r t i c i p a t i n g benzyl group at C-2. As noted e a r l i e r , I s h i -kawa and Fletcher reacted these bromides with methanol to give methyl glucosides of the a-D-configuration ( 2 3 ) , but no disaccharide syntheses were reported by these authors. It was therefore necessary to tes t the generality of t h i s 6 0 method i n actual disaccharide synthesis, since glycoside forming reactions with methanol do not necessarily give the same r e s u l t s when larger, l e s s reactive monosaccharides are used as the hydroxylic component. The two bromides used i n the present work were 2,3-di-0-benzyl-4,6-di-0-p_-nitro-benzoyl-g-D-glucopyranosyl bromide £ 1 , which was obtained from 2,3-di-0-benzyl-D-glucose and 2 - 0 - b e n z y l - 3 » 4 , 6 - t r i - 0 -p_-nitrobenzoyl-0-D-glucopyranosyl bromide 8 8 , which was obtained from 2-0-benzyl-D-glucose. 2,3~Di-0-benzyl-4,6-di-O-p_-nitrobenzoyl-0-D-glucopy-ranosyl bromide 7 1 , which can be prepared by a f i v e or six step synthesis s t a r t i n g from methyl 4,6-0-benzylidene-a-D-glucopyranoside, has not appeared i n the l i t e r a t u r e for the purpose of disaccharide synthesis. It was prepared here i n order to test i t s s t e r e o s e l e c t i v i t y for a-D-glucoside syn-thesis, and to compare i t i n t h i s respect with 2-0-benzyl-3,4,6-tri-O-p_-nitrobenzoyl-0-D-glucopyranosyl bromide 88t to be described. Isomaltose was prepared using both bromides. In addition, the disaccharide 6-0-a-D-glucopyranosyl-D-gal-actose, which was prepared by Flowers ( 2 6 ) using the 2-benzyl compound 8 8 , i s described here using 7 1 . Bromide 7 1 was prepared as described i n Scheme 1 2 . Benzylation of methyl 4,6-0-benzylidene-a-D-glucopyranoside 59 with benzyl chloride and potassium hydroxide i n xylene gave c r y s t a l l i n e methyl 2,3-di-0-benzyl-4,6-0-benzylidene-a-D-glucopyranoside 6 6 i n good y i e l d . Hydrolysis of the ben-zyl i d ene group under a c i d i c conditions then gave the cor-6 2 responding methyl 2,3-di-0-benzyl-a-D-gluc'opyranoside 6 7 . 2,3-Di-O-benzyl-D-glucose 69_ was obtained from com-pound 6 2 by eithe r acid hydrolysis or acet o l y s i s as described by Ishikawa and Fletcher ( 2 3 ) . It was found, however, that acid hydrolysis of 6 7 using 0,5 M s u l f u r i c acid under reflux gave a poor y i e l d ( 2 5 $ ) of product 69_. This was presum-ably because 6 £ was degrading i n the acid solution as i t formed, thus lowering the y i e l d . When the methyl glucoside 67 was subjected to ac e t o l y s i s i n a mixture of acetic anhy-dride and acetic acid, the r e s u l t i n g 1 , 4 , 6 - t r i - 0 - a c e t y l - 2 , 3 -di-O-benzyl-a-D-glucopyranoside 6 8 was obtained i n 5 0 $ y i e l d . Deacetylation of 6 8 then afforded 2,3-di-0-benzyl-D-glucose 6 9 . Although the ace t o l y s i s of 67 to the t r i a c e t a t e 6 8 and subsequent deacetylation requires an additional step, the ov e r a l l y i e l d of 6£ was higher (ca 40$) than when the methyl glycoside 6 £ was d i r e c t l y hydrolyzed. p_-Nitrobenzoylation of 2,3-di-0-benzyl-D-glucose gave the corresponding 3»4,6-tri-0-jj-nitrobenzoate 7 0 which on reaction with hydrogen bromide i n dichloromethane afforded the 2,3-di-0-p_-nitrobenzoyl -g-D-glucopyranosyl bromide 7 1 . This bromination reaction was subst a n t i a l l y faster ( l - 2 h ) than the corresponding bromination reaction f o r 2-0-benzyl-l,3,4,6-tetra-0-p_-nitrobenzoyl-a-D-glucopyranose ( 5 - 6 h ) , which w i l l be discussed l a t e r . The i s o l a t e d bromide £ 1 appeared to be unstable and could not be p u r i f i e d w e l l . Attempts to r e c r y s t a l l i z e i t were not completely s a t i s f a c t o r y because some hydrolysis or 6 3 degradation occured, even when dry solvents were used. In addition, traces of hydrogen bromide l e f t over from the reaction could not be removed e n t i r e l y by c o - d i s t i l l a t i o n with dry dichloromethane because again, some hydrolysis would occur. In one run, bromination of the jj-nitrobenzoate 22 with hydrogen bromide i n dichloromethane under the usual conditions (except that many portions of dry dichloromethane were added to the product and removed i n vacuo i n order to remove traces of hydrogen bromide) did not give the expected beta bromide 71, but instead, 2,3-di-0-benzyl-4,6-di-0-p_-nitrobenzoyl-a-D-glueopyranosyl bromide 22 w a s obtained, as indicated by the o p t i c a l r o t a t i o n . The formation of t h i s alpha bromo compound may well be due to anomerization of the beta product i n the presence of traces of hydrogen bromide. This pos-s i b i l i t y i s under further investigation. Bromide 72 was l a t e r used i n a condensation with the isopropylidene com-pound 1 0 (to be discussed). l,2,3,4-Tetra-0-acetyl-g-D-glucopyranose 2it w a s prepared by t r i t y l a t i o n of D-glucose, followed by acetylation and de-t r i t y l a t i o n ( 1 1 9 ) . In order to avoid the formation of excess pentaacetate during acetylation, the t r i t y l a t i o n reaction (which i s carr i e d out by heating glucose and t r i t y l chloride i n pyridine) must be complete. I f any undissolved glucose remained i n the reaction, t h i s was f i l t e r e d out while the mixture was s t i l l hot. Acetylation then gave a good y i e l d of l,2,3,4-tetra-O-acetyl-6-O-trityl-0-D-glucopyranose Tl 64 and p r a c t i c a l l y no pentaacetate was obtained. (a) 6-0-a-D-Glucopyranosyl-D-glucose (isomaltose) This disaccharide i s an important constituent i n plant dextrans, honey and other natural sources ( 6 5 ) . Isomaltose i s also an i n t e g r a l part of such macromolecules as glycogen and amylopectin, i n which i t i s part of the branch points. As mentioned i n the introduction, the disaccharide has been prepared by several d i f f e r e n t chemical methods. ( 1 5 » l 6 , 3 6 , 1 1 7 ) . Reaction of excess bromide 7 1 with 1 , 2 , 3 » 4 - t e t r a - 0 -acetyl-£-D-glucopyranose 7 4 i n absolute nitromethane using mercuric cyanide as condensing agent resulted i n a mixture containing the f u l l y blocked disaccharide 7j> (Scheme - 1 3 ) . Unlike the condensation reactions with the 2-benzyl bromide 8 8 , t h i s reaction was c a r r i e d out at room temperature and the bromide was exhausted within 5 h instead of two days, which r e f l e c t s the higher r e a c t i v i t y of bromide 7 1 compared to the corresponding 2-benzyl compound 8 8 . Because so much of 2 1 was used up i n side reactions, a l a r g e r proportion of the tetraacetate 24 remained unreacted. P u r i f i c a t i o n ( t h i n layer chromatography) of the mixture gave the f u l l y blocked disaccharide 21 a s a syrup. This was deacylated to the 2,3-dibenzyl disaccharide derivative 7 6 which on subsequent hydrogenolysis gave isomaltose 22 a s a syrup i n about 3 5 $ y i e l d from 24. Examination of 22 b y paper chromatography showed a small amount of gentiobiose Scheme 1 3 66 ( t h i s was estimated to be about 5 $ "by paper chromatography and proton magnetic resonance spectroscopy). Acetylation of the isomaltose syrup gave the corres-ponding beta octaacetate £§» which gave melting point, mixed melting point and o p t i c a l r o t a t i o n i d e n t i c a l to beta is o -maltose octaacetate obtained by other methods ( 1 6 ) . (b) 6-0-a-D-Glue opyrano syl-D-galac to se l,2i3»4-Di-0-isopropylidene-a-D-galactopyranose 1 0 was reacted at room temperature with excess 2 , 3 - d i - 0 - b e n z y l - 4 , 6 -di-0-£-nitrobenzoyl-$-D-glucopyranosyl bromide £ 1 i n n i t r o -methane i n the presence of mercuric cyanide. A f t e r 6h, the mixture containing 6 - 0 - ( 2 , 3 - d i - 0 - b e n z y l - 4 , 6 - d i - 0 - j r i - n i t r o b e n -zoyl-a-D-glucopyranosyl)-l,2t3,4-di-0-isopropylidene-a-D-galactose 22 w a s p u r i f i e d by preparative t h i n layer chromato-graphy. The product 22 w a s deacylated, the isopropylidene groups hydrolyzed, and the benzyl group hydrogenolyzed (Scheme 1 3 ) . The r e s u l t i n g 6-0-a-D-glucopyranosyl-D-gal;ac-tose 8 0 was iso l a t e d i n 2 6 $ o v e r a l l y i e l d from 1 0 a f t e r p u r i -f i c a t i o n by paper chromatography. The o p t i c a l r o t a t i o n of compound 8 0 corresponded to the l i t e r a t u r e value ( 2 6 ; 1 1 3 ) • In a separate experiment 2,3-di-0-benzyl-4,6-di-0-p_-nitrobenzoyl-a-D-glucopyranosyl bromide 22 was s i m i l a r l y re-acted with the isopropylidene compound 1 0 . Although the re-action conditions were the same as described for the beta bromide 21» t h i s reaction required 2 - 3 days, a f t e r which some of the isopropylidene compound s t i l l remained unreacted. Ex-67 amination of the product mixture by thin layer chromato-graphy indicated two large disaccharide components, which were isolated by preparative thin layer chromatography. The proton magnetic resonance spectra of the two dis-accharides indicated the presence of benzyl, p_-nitrobenzoate and isopropylidene groups, thus showing that the alpha and beta forms of 6-0-(2,3-di-0-benzyl-4,6-di-0-£-nitrobenzoyl-D-glucopyranosyl)-1,2i3,4-di-0-isopropylidene-a-D-galactose were obtained. The optical rotation indicated that the major product (71$) was the a-linked anomer. However, the 29$ beta disaccharide obtained clearly indicates that the a-bromo compound £2 i s less stereoselective than the cor-responding 0-anomer £1, which gave over 90$ of the a-linked disaccharide. C. Syntheses of a-D-glucosides by the 2-benzyl method The synthesis of a-linked glucosides using 2-0-benzyl--3,4,6-tri-0-£-nitrobenzoyl-p-D-glucopyranosyl bromide 88 i s described in this section. Three disaccharides were pre-pared using this method! (a) isomaltose, which was also pre-pared by the 2,3-dibenzyl method, (b) 6-0-a-D-glucopyranosyl D-mannose and (c) 4-0-a-D-glucopyranosyl-L-rhamnose. The key intermediate in these syntheses was bromide 88, whieh-'Jwras prepared as shown in Scheme 14. 3,4,6-Tri-0-ac etyl-l-deoxy-l-piperidino-g-D-glucopyra-nose 81 was prepared by reaction of 0-D-glucopyranose penta-acetate Z with 3 moles of piperidine (114). Product 81 was Scheme 14 68 glucose 69 i s o l a t e d c r y s t a l l i n e i n about 30$ y i e l d . With a few modifications, 2-0-benzyl-D-glucose 84 was prepared v i a the benzylation of compound 81 with excess ben-z y l bromide and s i l v e r oxide i n benzene as described by Kle-iner (115) . When t h i s procedure was followed, using 5 moles of benzyl bromide per mole of 81 as described i n the above reference, only about 50$ y i e l d of 3 , 4 , 6 - t r i - 0 - a c e t y l - 2 - 0 -benzyl-l-deoxy-l-piperidino-0-D-glucopyranose 82 was obtained. This was p a r t l y due to d i f f i c u l t i e s i n i s o l a t i n g a l l the material i n the presence of the excess benzyl bromide, which could not be removed by steam d i s t i l l a t i o n because the pro-duct decomposes below 80°. The reaction was also sluggish, 4-6 days being required. In addition, a f t e r the isola t e d compound was deacetylated to the corresponding 2 -0-benzyl-l-deoxy-l-piperidino-g-D-glucopyranose 8^, hydrolysis of t h i s compound with Amberlite IR 120 r e s i n at 70° as described i n the l i t e r a t u r e procedure gave incomplete reaction (50-6 0 $ ) . I t was then decided to decrease the molar excess of. benzyl bromide used to 3t 1*5 and f i n a l l y , 0 . 2 molar excess. As the excess bromide decreased, the time required f o r the reaction increased. However, i f a drying agent ( D r i e r i t e ) was added to the medium and i n addition, f r e s h l y prepared s i l v e r oxide was used, the reaction time was su b s t a n t i a l l y l e s s (2-4h) than described i n the l i t e r a t u r e procedure (4 days). With 0 . 2 molar excess benzyl bromide and h a l f the s i l v e r oxide, 90$ y i e l d of c r y s t a l l i n e 3 » 4 , 6 - t r i - 0 - a c e t y l -7 0 2-O-benzyl-l-deoxy-l-piperidino -0-D-glucopyranose 82 was usually obtained. Compound 82 was deacetylated using a c a t a l y t i c amount of sodium methoxide i n methanol to give 2 - 0-benzyl-l-deoxy-l-piperidino-0-D-glucopyranose .82 which was not i s o l a t e d . This compound was immediately hydrolyzed under r e f l u x at pH 3 ( 1 1 6 ) , affording c r y s t a l l i n e 2 -0-benzyl-D-glucose 84 i n 79$ y i e l d from 8 2 . The above f i v e step synthesis of 2-0-benzyl-D-glucose includes one low y i e l d step (conversion of £ a ^ d §1) which cuts down the o v e r a l l y i e l d considerably. In an attempt to obtain t h i s compound by a shorter route and i n higher y i e l d , l , 3 i ^ » 6-tetra - 0-acetyl-a.-D-glucopyranose 1 2 3 (the synthesis of t h i s compound from glucose i s described i n the Appendix) was d i r e c t l y benzylated with benzyl bromide and s i l v e r oxide. It was expected that the r e s u l t i n g 2 - 0-benzyl tetraacetate, on deacetylation, would give 2 -0-benzyl-D-glucose. However, when the benzylation reaction was complete, deacetylation of the product 85_ gave a c r y s t a l l i n e monobenzylated compound 86 which had a melting point ( 1 0 5 - 1 0 7 ° ) considerably lower than 2 -0-benzyl-D-glucose ( 1 7 6 - 1 7 7 ° ) . This suggests; that acetate migration occured during the benzylation reaction. Acetate migration of compound 123 was also encountered dur-ing the synthesis of 3 , ^ , 6 - t r i m e t h y l hexoses described i n the Appendix. jj-Nitrobenzoylation of 84 gave c r y s t a l l i n e 2 - 0-benzyl-l» 3 » ^ . 6-tetra - 0-p_-nitrobenzoyl-a-D-glucopyranose 87_ i n 94$ 7 1 y i e l d . Bromination of t h i s compound with hydrogen bromide i n dichloromethane then afforded c r y s t a l l i n e 2-0-benzyl-3»4,6-tri-O-p_-nitrobenzoyl-0-D-glucopyranosyl bromide 88 i n 2 5 $ y i e l d . This low y i e l d of 88 was i n part due to some degra-dation material present i n the reaction. In addition, some alpha anomer usually c r y s t a l l i z e d from the mixture. Although 88 gave melting point and o p t i c a l r o t a t i o n i d e n t i c a l to,the l i t e r a t u r e values, when t h i s compound was spotted on a t h i n layer chromatography plate two undefined spots usually re-sulted. Since t h i s did not change subs t a n t i a l l y on further p u r i f i c a t i o n , i t was assumed that the bromide was reacting on the plate. In a separate experiment, the jj-nitrobenzoate 8£ was brominated using hydrogen bromide i n g l a c i a l acetic acid i n Order to see i f any beta bromide could be obtained t h i s way. However, the product obtained had a high o p t i c a l rotation, suggesting that 2-0-benzyl-3,4,6-tri-0-£-nitrobenzoyl-a-D- ; glucopyranosyl bromide 8£ was obtained, not the beta anomer. Compound 8£ was not used f o r disaccharide synthesis. (a) 6-0-a-D-Glucopyranosyl-D-glucose (isomaltose) Reaction of 2-0-benzyl-3»4,6-tri-0-p_-nitrobenzoyl-$-D-glucopyranosyl bromide 88 with 1,2,3,4-tetra-O-acetyl-g-D-glucopyranose 2ii (Scheme 1 5 ) i n absolute nitromethane us-ing mercuric cyanide as acid acceptor at room temperature gave a rather slow reaction, a f t e r two days most 24 remaining unreacted. The use of a c e t o n i t r i l e as solvent did not change 7 2 Scheme 1 5 glucose (or mannose) 3 steps 73 t h i s s i t u a t i o n appreciably. However, when the temperature was increased to 4 0 - 5 0 ° , a substantial reaction did occur, although not a l l the tetraacetate reacted. A f t e r 2-3 days, the r e s u l t i n g l , 2 , 3 , 4 - t e t r a - 0 - a c e t y l - 6 - 0 - ( 2 - 0 - b e n z y l - 3 , 4 , 6 -tri-O-£-nitrobenzoyl-a-D-glucopyranosyl)-0-D-glucopyranose 9 0 was d i f f i c u l t to i s o l a t e from the mixture because of the s i m i l a r chromatographic mobility of some of the other compo-nents. However, a small portion was obtained by c a r e f u l p u r i -f i c a t i o n by preparative t h i n layer chromatography. The proton magnetic resonance spectrum obtained ascertained that i t was i n fact the disaccharide £ 0 , except that the nature of the anomeric linkage i n t h i s derivative was unclear. The bulk of the condensation mixture containing £ 0 was deacylated (sodium methoxide) to give a mixture of 6 - 0 - ( 2 - 0 -benzyl-a-D-glucopyranosyl)-D-glucose 9 1 , 2-0-benzyl-D-glucose, and glucose. The methyl p_-nitrobenzoate by-product was removed by chloroform extraction and the r e s u l t i n g mix-ture of sugars was conveniently p u r i f i e d by "short column chromatography" on s i l i c a gel with butanone-water azeotrope as solvent. This highly e f f i c i e n t chromatographic method, f i r s t described by Hunt and Rigby ( 1 2 0 ) , has been used rou-t i n e l y i n our laboratory f o r the p u r i f i c a t i o n of complex mix-tures. The method i s f a s t and separations comparable to those r e s u l t i n g from t h i n layer chromatography can be ob-tained. A portion of the 2-0-benzyl compound £ 1 was acetylated to give 1 , 2 , 3 , 4 - t e t r a - 0 - a c e t y l - 6 - 0 - ( 2 - 0 - b e n z y l - 3 , 4 , 6 - t r i - O -74 acetyl-a-D-glucopyranosyl)-D-glucopyranose £ 2 , the structure of which was confirmed by proton magnetic resonance spectro-scopy except f o r the anomeric linkage. C a t a l y t i c hydrogenation of the bulk of £ 1 using p a l l a -dium on carbon as cat a l y s t gave the free disaccharide 6-0-a-D-glucopyranosyl-D-glucose 22 (isomaltose) i n 48$ o v e r a l l y i e l d from 24• The proton magnetic resonance spectrum of 22 (Figure 5 ) showed a 1-hydrogen doublet at 5.02 with a coupling constant of 3*5 H , c l e a r l y indicating that the z a-D-1inked disaccharide was obtained. In addition, the anomeric hydrogens at the reducing glucose residue showed the r a t i o of alpha to beta anomer of about 2. This mixture of the two anomeric forms explains why isomaltose r a r e l y cry-s t a l l i z e s as the free sugar. The'-beta link e d disaccharide 6-0-e-D-glucopyranosyl-D-glucose (gentiobiose) could not be detected by proton mag-netic resonance spectroscopy, which suggests that the concen-t r a t i o n of t h i s compound was l e s s than f i v e percent. However, paper chromatographic analysis of the mixture indicated a f a i n t spot corresponding to gentiobiose. This was estimated to be l e s s than f i v e percent by v i s u a l comparison with a standard. Acetylation of the syrup 22 gave 3-isomaltose octaacetate, which gave melting point and mixed melting point as the product previously obtained. 7 6 (b) 6-0-a-D-Glue opyrano syl-D-manno se l,2,3,4-Tetra-0-acetyl-#-D-mannopyranose £ 4 was prepared e s s e n t i a l l y as described f o r the glucose analog. T r i t y l a -t i o n and acet y l a t i o n ( 1 2 1 ) of D-mannose, gave 1 , 2 , 3 , 4 - t e t r a -O-acetyl-6-O-trityl-0-D-mannopyranose £3_. Hydrolysis of the t r i t y l group afforded £ty. Condensation of £ 4 with 2 - 0 -benzyl-3,4,6-tri-0-£-nitrobenzoyl-g-D-glucopyranosyl bromide 8 8 under the conditions used f o r the synthesis of isomaltose (Scheme 1 5 ) gave the f u l l y blocked product l , 2 , 3 , 4 - t e t r a - 0 -ac etyl-6-0-(2-0-benzyl-3,4,6-tri-0-£-nitrobenzoyl-a-D-glueo-pyranosyl)-3-D-mannopyranose £5_. Even when a 0.8 molar ex-cess of bromide was used, not a l l the tetraacetate £ 4 reac-ted. This u n r e a c t i v i t y of £ 4 at the end of the reaction was also observed f o r the isomaltose condensation. P u r i f i c a t i o n of the condensation mixture was again car-r i e d out a f t e r removal of the acyl functions, except that column chromatography was c a r r i e d out using chloroform-methanol (3»l) as solvent, which was found to give as good a separation as butanone-water azeotrope, but was much fa s t e r . Chromatographically pure 6-0-(2-0-benzyl-a-D-gluco-pyranosyl)-D-mannose £6 was obtained i n 4 4 $ o v e r a l l y i e l d from £ 4 . A small portion of £6 was converted to the correspon-ding l , 2 , 3 , 4 - t e t r a - 0 - a c e t y l - 6 - 0 - ( 2 - 0 - b e n z y l - 3 , 4 , 6 - t r i - 0 -acetyl-a-D-glucopyranosyl)-D-mannopyranose ££» the structure of which was confirmed by proton magnetic resonance spectro-scopy. The r a t i o of a to 0 anomer at the reducing mannose 77 residue i n 9_Z w a s about 3. The nature of the 1-6 g l y c o s i d i c linkage (a or 3) was more conveniently determined i n the free sugar. The 2-0-benzyl intermediate £ 6 was hydrogenated using 1 0 $ palladium on carbon as c a t a l y s t . However, the proton magnetic resonance spectrum of the free sugar thus obtained showed that the integration f o r the combined alpha and beta forms of the reducing mannose' residue was l e s s than one hydro-gen. This e f f e c t , which was not observed i n the correspon-ding reaction with the isomaltose derivative, could be due to a p a r t i a l reduction of the disaccharide to the a l d i t o l during hydrogenation. When 5 $ palladium on carbon was used, the effe c t was minimized and 6-0-a-D-glucopyranosyl-D-mannose £ 8 was obtained as a syrup. The a-D-glucosidic linkage was es-tablished by proton magnetic resonance spectrum, i n which the 1-hydrogen doublet at 5 . 0 5 corresponding to the non-reducing glucose unit had a coupling constant of 3.2 H (Fig-ure 6 ) . No beta l i n k e d disaccharide was detected by proton mag-netic resonance, but paper chromatography did show a trace amount of t h i s component. It i s of interest to note that the small amount of beta linked disaccharide obtained i n both the isomaltose and 6-0-a-D-glucopyranosyl-D-mannose synthesis could not be c l e a r l y seen by chromatography of the blocked or p a r t i a l l y blocked sugars. Only when the free disaccharide was obtained was t h i s small amount of beta anomer observed. No s a t i s f a c t o r y c r y s t a l l i n e derivative of £ 8 has yet 78 79 been found. Acetylation gave a syrupy octaacetate £ 2 which was shown by proton magnetic resonance spectroscopy to con-t a i n a mixture of alpha and beta forms (at the mannose reduc-ing end) i n a r a t i o of about 3 . This octaacetate could not be c r y s t a l l i z e d . Takiura and co-workers (117) have recently described the synthesis of isomaltose using the alpha anomer of 88, namely 2-0-benzyl-3»4,6-tri-0-p_-nitrobenzoyl-a-D-glucopyranosyl bro-mide 8£. Their synthesis, l i k e the one reported i n t h i s thesis, was conducted using mercuric cyanide as condensing agent, nitromethane as solvent, and tetraacetate a s agly-con, but the reaction was c a r r i e d out at room temperature for a longer period (5 days). The o v e r a l l y i e l d of isomal-tose obtained (18$) was lower than that obtained i n our lab-oratory (48$), which may r e f l e c t the difference i n reac-t i v i t y between the two bromides. It i s i n t e r e s t i n g to note that the r a t i o of alpha to beta disaccharides (isomaltose to gentiobiose) obtained by these workers was 84il6 while the synthesis described i n t h i s work, i n which the beta bromide was used, gave a r a t i o of at l e a s t 9 5 » 5 » i n d i c a t i n g that the beta bromide i s s i g n i -f i c a n t l y more stereoselective than the alpha compound. In t h e i r study on the methanolysis of D-glueopyranosyl bromides having a non-participating benzyl group at C-2, Ishikawa and Fletcher found that i n the presence of added bromide ion, the rate of methanolysis and product composi-80 t i o n of the beta bromide 88 and the corresponding alpha anomer 89 were the same. Under these conditions the h a l f l i f e of both bromides was l4h while the r a t i o of alpha to beta methyl glycoside obtained was about 95*5* "the same as obtained i n our isomaltose synthesis. In the absence of excess bromide ion, the alpha bromide had a h a l f l i f e of 18 days and gave an a/g r a t i o of 91«9» while the beta bromide had a h a l f l i f e of 21 minutes with the a/g r a t i o of methyl glucosides being 94i6. In order to explain the predominant formation of the alpha glycoside from both bromides, Ishikawa and Fletcher proposed a mechanism i n which an equilibrium between the alpha and beta bromides exists 1 a-glycoside As the glucosyl bromide reacts, t h i s equilibrium condition i s aided by the released hydrogen bromide. The alpha bromide 81 reacts with methanol to give the beta g lycos ide by Walden i n -vers ion while the beta bromide w i l l give the beta g lycos ide. Since the beta bromide i s much more reac t i ve , the alpha g l y -coside w i l l be the predominant product regardless of which bromide i s the s t a r t i n g mater i a l . When the alpha bromide i s the s t a r t i n g mater ia l however, t h i s compound reacts f i r s t to give the beta g lycos ide (thus accounting fo r the higher proport ion of t h i s g lycos ide obtained) and as the hydrogen bromide i n so lu t ion increases, equ i l ibr ium to the more reac-t i v e beta bromide i s estab l i shed and alpha g lycos ide formation predominates. Under condensation condit ions however, the mechanism must include an add i t i ona l f ac to r , the mercuric cyanide used as ac id acceptor. The in f luence of the cyanide i s c l e a r l y ind icated by the f a c t that the ha l f l i f e of the alpha bromide was 18 days when reacted with methanol, while t h i s same bro-mide reacted f o r l e s s time (5 days) with a more complex, l e s s react ive carbohydrate a lcoho l under condensation condi t ions The f i r s t step i n the condensation reac t ion i s probably the reac t ion of the g lycosy l ha l ide with mercuric cyanide to give a c l o se l y associated ion p a i n (117). 82 a beta bromide would give a beta ion p a i r while an alpha bro-mide would give an alpha ion p a i r . Since the anomeric e f f e c t (91) makes the beta bromide more reactive than the alpha one, the corresponding beta ion p a i r should be easier to form. Reaction with Walden inversion, of these ion pai r s with the carbohydrate hydroxylic component then r e s u l t s i n the f o r -mation of the a-glycoside from the beta ion p a i r and the 0-glycoside from the alpha ion pa i r . To account f o r the isomeric mixtures obtained, an ano-merization mechanism s i m i l a r to the one proposed by I s h i -kawa and Fletcher can be assumed, except that now mercuric cyanide i s involved. Thus, i f an equilibrium between the alpha and beta ion pairs e x i s t s , the beta ion p a i r w i l l react p r e f e r e n t i a l l y to give alpha glycoside. When the beta bromide i s used, as i n our synthesis of isomaltose, the percent product composition of a-disaccharide i n the mixture should be higher because the more reactive beta ion p a i r i s immediately formed without having to go through an anomerization step. When the alpha bromide i s used, the alpha ion p a i r forms f i r s t , a portion of i t reacts to give the beta glycoside (although at a much shower rate) but as ano-merization proceeds, the highly reactive beta ion p a i r gives the alpha ( l r 2 - c i s ) glycoside. Because of the i n i t i a l alpha ion p a i r formation, the proportion of the unwanted beta l i n k -ed disaccharide i s higher when the a-bromide i s used. A d i f f e r e n t mechanism has been proposed by Flowers (118) to account f o r the p r e f e r e n t i a l formation of the 1,2-8 3 c i s glycoside. This mechanism involves long range p a r t i c i pation hy the ester groups at other positions i n the mole-cule, such as 3,4 or 6. This was proposed f o r a-L-fucopy-ranosyl "bromides such asi i n which the a x i a l acetate group at C-4 can p a r t i c i p a t e on C - l , thus f o r c i n g the alcohol to attack from the opposite d i r e c t i o n g i v i n g the 1 , 2 - c i s glycoside. In the case of the D-glucopyranosyl "bromides, i n which there are no a x i a l groups, p a r t i c i p a t i o n from the equatorial esters i s l e s s e f f e c t i v e . Therefore, i f t h i s mechanism i s operative, the main e f f e c t of the p a r t i c i p a t i n g groups would have to be from the C-6 ester. That the C-6 ester does have an influence on the outcome of these reactions has been discussed by Frechet and Schuerch ( 2 ? ) . (c) 4-0-a-D-Glucopyranosyl-L-rhamnopyranose Polysaccharides containing L-rhamnose are important constituents i n plants and bacteria ( 7 5 . 1 2 2 , 1 2 3 ) . As part of a continuing i n v e s t i g a t i o n of the structure of K l e b s i e l l a benzyl 84 capsular polysaccharides i n progress i n our laboratory (66, 124), i t i s of intere s t to prepare some of the disaccharide units which may be found i n these bacteria. The synthesis of 4-0-a-D-glucopyranosyl-L-rhamnopyranose 114 and the corres-ponding 0 - l i n k e d analog described by Bebault and Dutton ( 1 2 5 ) constitute the f i r s t p a i r of disaccharides which were pre-pared as model compounds f o r subsequent s t r u c t u r a l and im-munological studies related to b a c t e r i a l polysaccharides, es-p e c i a l l y of the genus K l e b s i e l l a . In addition, the synthesis of 114 constitutes an additional example of the use of 2-0-benzyl-3»4,6-tri-O-jj-nitrobenzoyl-0-D-glucopyranosyl bromide as a general method f o r the synthesis of disaccharides of the a-D-glucosyl configuration. It i s rather surprising to note that although rhamnose containing polysaccharides are widely d i s t r i b u t e d i n nature and many investigations pertaining to these systems are now i n progress i n several laboratories, i t i s only r e l a t i v e l y recently that the f i r s t synthesis of a disaccharide contain-ing L-rhamnose as an aglycon has been reported"(126). The synthesis of 4-0-a-D-glucopyranosyl-L-rhamnose described here w i l l therefore represent the f i r s t a-D-glucosyl-L-rhamnose to be synthesized. The rhamnose aglycon used i n t h i s synthesis was methyl 2»3-0-isopropylidene-a-L-rhamnopyranoside 101, which was pre-pared by the method of Levene and Muskat ( 1 2 7 ) , except that the product was is o l a t e d v i a the c r y s t a l l i n e 4-acetate ( 1 2 5 , 128). 8 5 L-Rharanose monohydrate (Scheme 1 6 ) was refluxed i n metha-no l i c hydrogen chloride and methyl a-L-rhamnopyranoside 1 0 0 was obtained i n about 6 0 $ y i e l d . This compound was subse-quently converted to the corresponding methyl 2,3-0-isopro-pylidene-a-L-rhamnopyranoside 1 0 1 by treatment with acetone i n the presence of cupric s u l f a t e and a c a t a l y t i c amount of s u l f u r i c a c i d . The r e s u l t i n g compound 1 0 1 could be d i s t i l l e d when an e f f i c i e n t d i s t i l l a t i o n system was used, but often the product was contaminated with some methyl rhamnoside (maybe 5 $ ) . In order to prevent the excessive formation of unwanted disaccharides from condensations involving methyl rhamnoside, the mixture 1 0 1 was acetylated and the corresponding methyl 4-0-acetyl-2,3-0-isopropylidene-a-L-rhamnopyranoside 1 0 2 was obtained as a c r y s t a l l i n e compound. The acetate 1 0 2 was then deacetylated (sodium methoxide) and methyl 2 , 3 - 0 - i s o p r o p y l i -dene-a-L-rhamnopyranoside 1 0 1 was obtained as a syrup. Only a small amount of methyl rhamnoside (maybe 1%) contaminant was usually present when 1 0 1 was obtained i n t h i s manner. j This contaminant was obtained because of deacetalation which occured when the sodium methoxide solution of 1 0 1 was neu-t r a l i z e d with an a c i d i c r e s i n (Amberlite IR 1 2 0 H +). Reaction of methyl 2,3-0-isopropylidene-a-L-rhamnopy-ranoside 1 0 1 with excess 2-0-benzyl-3,4,6-tri-0-|>-nitroben-zoyl-0-D-glucopyranosyl bromide 8 8 i n nitromethane using mercuric cyanide as condensing agent gave methyl 4 - 0 - ( 2 - 0 -benzyl- 3,4,6-tri-0-£-nitrobenzoyl-a-D-glueopyranosyl)-2,3-0-isopropylidene-a-L-rhamnopyranoside 1 0 3 i n high y i e l d . Un-Scheme 16 L-rhamnose HCl/CH3OH H2/Pd see over 87 88 l i k e the condensations of 88 previously described, t h i s reac-t i o n required only 5 h at 40° f o r completion, which may well r e f l e c t the higher n u c l e o p h i l i c i t y of 101 compared to the 1,2,3»4-tetraacetates of glucose and mannose. The main product 103 was iso l a t e d by short column chro-matography on s i l i c a gel using benzene-ether (9«l)as solvent and the syrup 103 was iso l a t e d i n 80$ y i e l d . Except f o r the anomeric linkage which was unclear, the proton magnetic re-sonance spectrum of 103 confirmed that i t was a disaccharide, indicated by the presence of benzyl, p_-nitrobenzoate, isopropylidene, methoxy, and methyl groups. In addition to 103t the column separation gave a major by-product 104, of higher mobility than 103. Compound 104 was again p u r i f i e d (preparative t h i n layer chromatography) and the proton magnetic resonance spectrum obtained indicated that i t was a monosaccharide tri-0-p_-nitrobenzoate. Deacylation of 104 with sodium methoxide gave a compound which moved fas t e r than 2-0-benzyl-D-glucose on t h i n layer chromatography. A l -though no additional data were obtained f o r t h i s by-product, the indications are that 104 i s 2-0-benzyl-3,4,6-tri-0-p_-nitr0-benzoyl-D-glucopyranosyl cyanide, which r e s u l t s from reaction of the glucosyl bromide with mercuric cyanide. The reaction of glycosyl bromides with mercuric cyanide has been reported i n the l i t e r a t u r e (129, 130). The f u l l y blocked disaccharide 103 was deacylated i n two ways, by re f l u x i n a mixture of potassium hydroxide, ethanol and water, and with a d i l u t e solution of sodium methoxide i n 8 9 methanol. Deacylation with potassium hydroxide was suitable f o r l a r g e r amounts of material because the product could be extracted with ether from the insoluble potassium s a l t s . The conditions used however, were somewhat vigorous. The milder sodium methoxide method was suitable f o r smaller quantities because p u r i f i c a t i o n could be ca r r i e d out by preparative t h i n layer chromatography. The r e s u l t i n g me-t h y l 4-0(2-0-benzyl-a-D-glucopyranosyl)-2,3-0-isopropylidene-a-L-rhamnopyranoside 1 0 5 obtained by deacylation with sodium methoxide c r y s t a l l i z e d on standing. These c r y s t a l s were-sub-sequently used f o r seeding the potassium hydroxide prepara-tions. The proton magnetic resonance spectrum of 1 0 5 . unlike the preceeding compound 1 0 3 , showed the anomeric hydrogen f o r the non-reducing glucose u n i t . The 2 value was 3«7 H z» i n d i c a t i n g ( 9 5 . 9 6 ) the a-D-glucosyl linkage. Although a small amount of a second disaccharide com-ponent (the beta anomer of 1 0 5 ) was detected by t h i n layer chromatography of the syrupy mixture, t h i s was not easy to i s o l a t e at t h i s point, but was more conveniently i s o l a t e d following hydrogenolysis. The next step i n the synthesis consisted i n the removal of the benzyl group by hydrogenolysis. In one t r i a l run, the isopropylidene group i n 1 0 5 was hydrolyzed f i r s t to give methyl ,^-0-(2^0-r,benzyl-a-D-glucopyranosyl)-a-L-rhamnopyrano-side 1 0 6 but when t h i s compound was subjected to hydrogenolysis, about 1 0 $ of the methyl glycoside was apparently removed during 9 0 t h i s reaction. At t h i s point however, not enough material was available and t h i s was not repeated. Hydrogenolysis of the c r y s t a l l i n e intermediate 1 0 5 gave methyl 4-0-a-D-glueopyranosyl-2,3-0-isopropylidene-a-L-rham-nopyranoside 1 0 7 as a syrup i n almost quantitative y i e l d . The hydrogenation was also conducted on a syrupy sample of 1 0 5 (containing some beta disaccharide). This gave the ex-pected 1 0 7 and a second component l a t e r i d e n t i f i e d as me-t h y l 4-0-0-D-glucopyranosyl-2,3-0-isopropylidene-a-L-rham-nopyranoside 1 0 8 . Compounds 1 0 7 and 108 were separated by preparative t h i n l a y e r chromatography and 108 was i s o l a t e d as 3 . 3 $ of the mixture. The proton magnetic resonance spectra of 1 0 7 and 1 0 8 (the l a t t e r using Fourier transform) did not di s t i n g u i s h between these two compounds. It was then decided to make the t r i -m e t h y l s i l y l ethers and i d e n t i f y them by proton magnetic re-sonance, a technique which has been used i n the laboratory for the i d e n t i f i c a t i o n of the number of free hydroxyl groups i n sugars ( 1 3 1 ) . Compounds 1 0 7 and 108 were thus converted to the corresponding t r i m e t h y l s i l y l ethers 1 0 9 and 1 1 0 . The proton magnetic resonance spectrum of 1 0 9 c l e a r l y indicated the a-D-glucosyl linkage, the non-reducing anomeric hydrogen having a 2 value of 4.0 H 2. S i m i l a r l y , the coupling con-stant f o r the non-reducing anomeric hydrogen i n 1 1 0 was 7.3 H , which indicates a 0-D-linkage. The spectrum of 1 1 0 was also compared and found to be i d e n t i c a l to the trimethyl-s i l y l derivative of an authentic sample of methyl 2 , 3 - 0 - i s o -p r o p y l i d e n e - 4 - 0 - ( 2 , 3 , 4 , 6 - 1 e t r a - 0 - t r i m e t h y l s ilyl-g-D-gluc o-pyranosyl)-a-L-rhamnopyranoside 1 1 0 which was obtained by deacetylation and subsequent t r i m e t h y l s i l y l a t i o n of a cry-s t a l l i n e sample of methyl 2 , 3 - 0 - i s o p r o p y l i d e n e - 4 - 0 - ( 2 , 3 , 4 , 6 -tetra-O-acetyl-0-D-glucopyranosyl)-a-L-rhamnopyranoside pre-v i o u s l y prepared i n the laboratory ( 1 2 5 ) . The spectra of 1 0 9 and 1 1 0 are shown i n Appendix 1 , The isopropylidene group i n 1 0 7 was removed by hydrolysis i n a 1 0 $ s o l u t i o n of t r i f l u o r o a c e t i c acid i n chloroform containing a small amount of water, and the r e s u l t i n g methyl 4-0-a-D-glucopyranosyl-a-L-rhamnopyranoside 1 1 1 was obtained as a syrup i n high y i e l d . The proton magnetic resonance spectrum of 1 1 1 taken at 6 0 ° (Figure 7 ) c l e a r l y indicated the H-l signals corresponding to the glucose (at 4 , 9 7 , »3 H ) and rhamnose (at 5»35» J i o^l.? H ) units, again showing that the a-linked glucoside was obtained. Compound 1 1 1 was then acetylated and the corresponding methyl 2 , 3 - d i - 0 - a c e t y l -4-0-(2,3,^»6-tetra-0-acetyl-a-D-glucopyranosyl)-L-rhamnopy-ranoside 1 1 2 was obtained c r y s t a l l i n e . The next step i n the synthesis consisted i n the removal of the methyl glycoside i n 1 1 2 without cleaving the disaccha-r i d e . This was c a r r i e d out by a c e t o l y s i s i n 2 $ s u l f u r i c acid i n acetic anhydride, conditions used previously during the attempted stereoselective synthesis of 3-0-0-D-glucopyranosyl D-mannose, and also i n the unusual a c e t o l y s i s of the carbo-nate 6 0 . The r e s u l t i n g syrup contained 1 , 2 , 3 - t r i - 0 - a c e t y l -4 - 0 - ( 2 , 3 , 4 , 6 - t e t r a - 0 - a c etyl-a-D-glucopyranosyl)-L-rhamnopy-Figure 7. P.M.R. Spectrum (D^O) of compound 111 9 3 ranose 1 1 3 was then isol a t e d i n 9 3 $ y i e l d a f t e r p u r i f i c a t i o n "by preparative t h i n layer chromatography. Although the chromatographic mobility of 1 1 3 was i d e n t i -c a l to 1 1 2 i n several solvents, the proton magnetic resonance spectrum showed no methoxy signal and seven acetates. The o p t i c a l r o t a t i o n of 1 1 3 (+55° i n chloroform) was somewhat lower than that of the hexaacetate 1 1 2 (+62° i n chloroform). Deacetylation of 1 1 3 f i n a l l y gave 4-0-a-D-glucopyranosyl-L-rhamnose 114 as a syrup (40$ o v e r a l l y i e l d from 1 0 1 ) which on paper chromatography showed one component only. The proton magnetic resonance spectrum showed the H-l glucosyl signal at 4.94 having J 1 2 value of 3.8 H z > which indicates (95.96) the a-D-glucosyl configuration. The a-D-glucosyl linkage i n 114 was once more demonstra-ted by enzymatic hydrolysis with maltase, which gave more than 9 0 $ cleavage i n 30 minutes. On the other hand, reaction of 114 with beta glucosidase gave only trace cleavage i n l 6 h . The disaccharide 114 was reduced to the a l d i t o l 1 1 5 with sodium borohydride i n water. The proton magnetic resonance spectrum of the r e s u l t i n g syrup 1 1 5 indicated a low f i e l d dou-b l e t at 4.?6 ( J ^ ^ " ^'^ H z ^ ' showing the a-D-glucosyl linkage. Compound 1 1 5 was subsequently converted to the a l d i t o l acetate 1 1 6 and per ( t r i m e t h y l s i l y l ) a l d i t o l f o r eventual; gas chro-matographic analysis. Methylation- Although the a-D-glucosyl linkage of the disaccharide was well .demonstrated by the proton magnetic 94 resonance spectra of 1 0 5 . 1 0 9 . 1 1 1 . 114 and 115. and by enzy-matic hydrolysis of the disaccharide, these techniques could not establish the 1-4 linkage. This was ascertained by methy-l a t i o n and periodate oxidation analysis of the disaccharide methyl glycoside 1 1 1 . Compound 1 1 1 (instead of the free d i -saccharide 114) i s more suitable f o r these analyses because i t i s more stable under methylation conditions and the perio-date oxidation can only give one possible r e s u l t . When the free sugars are used (as found with the 4-0-methyl disaccharide 3 9 ) extensive degradation may occur during the methylation and t h i s i s l i k e l y to be enhanced when a reducing 6-deoxy unit (L-rhamnose) i s involved. Methyl 4-0-a-D-glucopyranosyl-a-L-rhamnopyranoside 1 1 1 was methylated (Scheme 1 7 ) by the Hakomori method e s s e n t i a l l y as previously described f o r 22* The r e s u l t i n g f u l l y methyla-ted disaccharide 1 1 7 was p u r i f i e d by preparative t h i n layer chromatography and the proton magnetic resonance spectrum ob-tained (Figure 8 ) showed seven methyl groups and the two ano-meric hydrogens, which were c l e a r l y v i s i b l e i n t h i s spectrum. Figure 9 i l l u s t r a t e s the anomeric region of 1 1 1 , 1 1 5 and 1 1 7 . as compared to the 0-linked analogs prepared i n t h i s labora-tory by Bebault and Dutton ( 1 2 5 ) . In a l l three cases, the chemical s h i f t s of the a-anomers are downfield of -the 0-anomers, while the coupling constants of the a-anomers (ca 3*8 H ) are smaller than those of the corresponding g-anomers (ca 7.6 H ). z A portion of 1 1 7 was subjected to methanolysis and the r e s u l t i n g mixture containing methyl 2,3-di-0-methyl-L-rhamno-Scheme 17 95 NaH/DMSO CH I -OCH OCH3 CH 0 OCH3 46 . 118 NaBH /wat Ac o0/pyr g . l . c . g . l . c . g . l . c . m/s 96 a - l i n k e d j5- linked T 4 . 8 8 7.5 — i 1 — 5.12 5 . 2 6 3.7 G l c — R h a OMe-7.7 r —i 1 r - 4 . 9 5 5 . 2 5 5 . 3 7 3.8 K G l c — R h a — o l 7 2 4 . 7 0 — i 5 . 3 7 Figure 9. P.M.R. spectra of 1 1 1 , 1 1 £ and 1 1 Z a n d 3-linked analogs 98 pyranosides 118 (a and a n a methyl 2,3,4,6-tetra-O-methyl-D-glucopyranosides 46 (a and 0 ) was converted to the trimethyl-s i l y l d erivatives, affording methyl 2,3-di-0-methyl-4-G-tri-m e t h y l s i l y l - ! - rhamnopyranosides 119 (a and 0 ) and unchanged 46. Fragments 46 and 119 were analyzed hy g a s - l i q u i d chroma-tography and the retention times were i d e n t i c a l to those of authentic standards. A second portion of 117 was hydrolyzed and 2,3,4,6-tetra-O-methyl-D-glucose 48 and 2,3-di-0-methyl-L-rhamnose 120 were obtained and were i d e n t i f i e d by paper chromatography. The mix-ture of compounds 48 and 120 was then acetylated and the r e s u l -t i n g l-0-acetyl-2,3,4,6-tetra-O-methyl-D-glucose (a and 0 ) and 1.4- di-0-acetyl-2,3-di-0-methyl-L-rhamnose (a and 0 ) were i d e n t i f i e d by gas - l i q u i d chromatography as compared to authen-t i c standards. A portion of the hydrolyzate from 117 above was reduced with sodium borohydride i n water and then acetylated. The r e s u l t i n g l,4,5-tri-0-acetyl-2,3-di-0-methyl-L-rhamnitol and 1.5- di-0-acetyl-2,3,4,6-tetra-0-methyl-D-glucitol were again i d e n t i f i e d by gas-liquid chromatography. Samples were c o l -l e c t e d and the mass spectra obtained were consistent with these two structures. Periodate oxidation- The methyl glycoside 111 was oxi-dized (Scheme 18) with sodium metaperiodate and the uptake ( 3 moles) was monitored spectrophotometrically as described f o r The polyaldehyde obtained was reduced with sodium boro-Scheme 18 1 0 0 hydride i n water to give a polyalcohol, which was subjected to methanolysis. The r e s u l t i n g 1-deoxy-D-erythritol 1 2 1 and g l y c e r o l 1 2 2 were i d e n t i f i e d by paper chromatography by compar-ison with standards. Acetylation of these fragments 1 2 1 and •: 1 2 2 gave the corresponding peracetates which were i d e n t i f i e d by ga s - l i q u i d chromatography and mass spectrometry. Standards of 2,3,4,6-tetra-O-methyl-D-glucose and methyl 2,3,4,6-tetra-O-methyl-D-glucopyranoside (a and 0 ) ( 1 3 2 ) , and 2,3-di-O-methyl-L-rhamnose and 1-deoxy-D-erythritol ( 1 2 5 ) were available i n the laboratory from previous studies. The above methylation and periodate oxidation analysis c l e a r l y prove that the 1-4 linkage was obtained, since any other linkage would have given d i f f e r e n t r e s u l t s . As a point of interest i t should be mentioned that the gas- l i q u i d chromatographic analysis of the fragments from the periodate oxidation gave the expected peaks corresponding to the acetates of 1 2 1 and 1 2 2 . However, and as previously noted i n connection with the periodate oxidation of J2» other minor unknown components were also detected. This could be impor-tant when unknown disaccharides (or oligosaccharides) are analyzed by periodate oxidation, since on some occasions these by-products may "be present i n f a i r l y high proportion and could be confused with primary fragments. This i s one of the reasons why known standards should be used whenever possible, as an aid f o r i d e n t i f i c a t i o n during analysis by gas chromatography. In addition, retention times recorded i n the l i t e r a t u r e depend on l o c a l conditions (column, equipment, etc.) and may not be re-101 producible, giving r i s e to uncertainties. The synthesis of some methylated sugars which are to be used as standards f o r polysaccharide analysis i s described i n the Appendix. 1 0 2 CONCLUSIONS When t h i s research started, the synthesis of a-linked disaccharides using g-nitrobenzoyl-D-glucopyranosyl bromides having a non-participating group at C-2 had not been reported. The present work demonstrates that disaccharides of the a-D-glucosyl configuration can be obtained i n high stereoselec-t i v i t y , and precedes some of the groundwork necessary f o r sub-sequent studies i n the synthesis of oligosaccharides con-t a i n i n g a-linked glucose residues. In addition, the synthesis of four a-linked disaccharides suggests that the method tested i s a general one, and therefore can be used f o r the synthesis of other disaccharides containing the glucosyl u n i t . The 2-benzyl beta bromide 8 8 appears to be superior to the 2,3 dibenzyl beta bromide 7J. because the l a t t e r i s more un-stable, and gives more side products. In addition, 2 i c a n ano-merize during work up to the alpha anomer, which i s much l e s s stereoselective i n i t s glycoside forming reactions. As f a r as the s t e r e o s e l e c t i v i t y goes, the 2-benzyl method i s probably better than other procedures available f o r the synthesis of a-D-glucosides. The main disadvantage however, i s the fact that the method i s rather long and the o v e r a l l y i e l d (from glucose) of disaccharide i s low because there are two low y i e l d steps i n the preparation of the 2-benzyl beta bromide 8 8 . As a r e s u l t of t h i s , only small amounts of d i -saccharide were available to obtain c r y s t a l l i n e derivatives and to conduct proof of structure studies, operations which 1 0 3 are considerably more d i f f i c u l t to perform when only small amounts of material are a v a i l a b l e . Although i t i s possible to use the 2-benzyl method to syn-thesize higher oligosaccharides ( f o r example, the 2-0-benzyl^ disaccharides heptaacetates £2 and £7, could be debenzylated giving the corresponding 2-hydroxy compounds, which can then condense with glycosyl halides) i n low y i e l d , t h i s i s l i k e l y to be impractical f o r making oligosaccharides having more than 3 or 4 sugar units . In t h i s respect the method has to be improved or new methods w i l l have to be found f o r the synthe-s i s of higher oligosaccharides. It was noted i n the introduction that disaccharides are being used i n other laboratories f o r b i o l o g i c a l investigations. Recently, i n immunological studies involving b a c t e r i a l poly-saccharides of the genus Sh i g e l l a , i t was believed that the disaccharide 4-0-a-D-glucopyranosyl-L-rhamnose 114 was an antigenic determinant i n t h i s genus (2). This was based on i n h i b i t i o n studies i n which a disaccharide believed to be 114 was used. Later however, ( 1 3 3 ) the authors discovered that the disaccharide used i n t h i s investigation was not 114, but a d i f f e r e n t one, thus i n v a l i d a t i n g t h e i r f i r s t r e s u l t s . This points to the fact that i n studies l i k e t h i s and others now taking place i n many laboratories, i t w i l l often be a necessity to have disaccharides and higher oligosaccha-* rides of unambiguous structure. It i s i n t h i s area that syn-t h e t i c routes, even i f long and tedious, may be of much use i n providing some of these compounds. 104 EXPERIMENTAL General conditions Thin l a y e r chromatography ( t . l . c . ) was ca r r i e d out using 10cm x 20cm glass plates coated with s i l i c a gel G (EM reagents). Plates were developed hy spraying with 35$ ethanolic s u l f u r i c acid and heating to about 1 5 0 ° f o r 3-5 min. Preparative t h i n layer chromatography (13*0 was conducted using 20cm x 20cm plates coated with a 0.75mm layer of s i l i c a gel G. Paper chromatography was carr i e d out on Whatman # 1 paper, v i s u a l i z a t i o n was accomplished by using s i l v e r n i t r a t e i n ace-tone (135) f o r non-reducing sugars, p_-anisidine spray (136) i n t r i c h l o r o a c e t i c acid f o r reducing sugars, or periodate-benzidine (137) f o r non-reducing sugars. R indicates with respect to 2 , 3 , o 4,6-tetra-O-methyl-D-glucose, R f with respect to the solvent front. The following solvent systems were usedi A ethyl ether-toluene ( 2 i l ) B butanone-water azeotrope C ethyl acetate-pyridine-water (8 I 2 I 2 ) D ethyl acetate-acetic acid-formic acid-water (18 I 3 I 1 I 4 ) E 1-butanol-acetic acid-water ( 2 i l t l ) F benzene-ether ( 4 i l ) G 1-butanol-ethanol-water ( 4 i l » 5 ) Short column chromatographic separations (120) were c a r r i e d out using s i l i c a gel H (without CaSO^) at pressures not exceeding 20 p . s . i . (N 2). Gas-liquid chromatography (g.l.c.) was conducted on a 1 0 5 F and M 7 2 0 instrument using the following columns» (a) 8 f t x i i n 2 0 $ SF 9 6 on 8 0 - 1 0 0 mesh Diatoport S. (b) 4 f t x i i n 5 $ butanediol succinate on 8 0 - 1 0 0 mesh Diatoport S. (c) 2 f t x i i n 2 0 $ SE 30 (F and M d i v i s i o n , Hewlett-Packard, Avondale, Pennsylvania), (d) 6 f t x i i n OS 1 3 8 on 1 0 0 - 1 2 0 mesh Gas Chrom Q Proton magnetic resonance (p.m.r.) spectra were recorded on Varian XL-100, HA-100, or T-60 (when noted) instruments using inte r n a l tetramethylsilane (TMS) as int e r n a l standard f o r orga-nic solutions, or either i n t e r n a l sodium 2,2-dimethyl-2-silapen-tane-5-sulfonate (DSS) or external TMS f o r aqueous solutions. Fourier transform equipment coupled to the XL-100 instrument was used f o r small samples. A l l chemical s h i f t s are expressed i n t values. Mass spectra were determined on an A t l a s AEI MS9 spectro-meter. U l t r a v i o l e t spectra were recorded using a Varian Cary-15 instrument. Infrared spectra were measured on a Perkin-Elmer model 4 5 7 spectrophotometer. Optical rotations were measured at 2 3 - 1 ° on a Rudolph model 2 1 9 polarimeter and l a t e r on a Perkin-Elmer model 141 polarimeter. Melting points are uncorrected. Solvents were removed below 50° unless otherwise stated. Model syntheses of g-D-glucosides 106 Preparation of 6-0-0-D-glueopyranosyl-D-galactose 0-D-Glucopyranose pentaacetate 7 A suspension of 25g of anhydrous sodium acetate i n 350ml of acetic anhydride i n a 1 - l i t e r , round bottomed flask was h heated over a flame to the b o i l i n g point in a e f f i c i e n t fume-hood. About 2g of anhydrous D-glucose (from a 50g supply) was added and the flask was heated without shaking at the point nearest the sugar l y i n g at the bottom of the f l a s k . I n i t i a -t i o n of the reaction was indicated by continued b o i l i n g a f t e r removal of the flame. A f t e r extinguishing the flame, the rest of the sugar was then added at such a rate as to maintain the b o i l i n g temperature of the mixture (about 20 minutes). The mixture was shaken occasionally to prevent accumulation of s o l i d sugar at the bottom of the f l a s k . I f too much sugar i s added at once,&a(too vigorous reaction follows and the contents of the flask may s p i l l over. Therefore, i f the reaction stops, i t should be started again by heating before much more sugar i s added. The flame should be immediately extinguished. When the addition was complete and b o i l i n g stopped, the mixture was heated to the b o i l i n g point, cooled, and poured with s t i r r i n g onto cracked i ce (1 l i t e r ) . A f t e r standing f o r 3h with occa-sional s t i r r i n g the c r y s t a l s were f i l t e r e d , washed with cold water, and dried (vacuum oven at 60°), 77g (70$). Dissolution i n ethanol (400ml), f i l t r a t i o n while hot with decolorizing 107 carbon, and cooling to room temperature, then to 10" f o r 2h, gave the pure product which was dried at 60° (vacuum oven, 15mm), m.p. 130-132°* [ a ] D +4° (c 3. chloroform); ( l i t . (138) m.p. 1 3 2 ° , [ a ] D +4 (chloroform)). 0-D-G:lucose pentaacetate was prepared i n smaller quanti-t i e s (5g glucose) by heating the mixture on a steam bath with occasional shaking u n t i l the sugar i s dissolved ( l - 3 h ) . The reaction was conveniently followed by t . l . c . using solvent A. Workup as above gave about 90$ y i e l d . a-D-Glucopyranose pentaacetate 8 To a mixture of acetic anhydride (70ml) and perchloric acid (60$, 0.5nil) was showly added (about 30 min) anhydrous a-D-glucose (lOg) with s t i r r i n g while keeping the int e r n a l tem-perature between 30 and 40°. The s t i r r i n g was continued f o r 30 min, and the mixture was poured into 300ml of ice water. After 2h, the c r y s t a l s were f i l t e r e d , washed with cold water, and dried at 50° under vacuum. Y i e l d 17g (79$). Recrystal-l i z a t i o n from hot ethanol gave the pure product, m.p. 1 1 1 - 1 1 2 ° } [ a ] D 4-100° (c 2, chloroform); ( l i t . (138) m.p. 1 1 2 - 1 1 3 ° , [ a ] D '+102° (chloroform)). Tetra-O-acetyl-a-D-glucopyranosyl bromide 9_ This compound was prepared by bromination of the penta-acetates (a), and by the procedures (b and c) of Lemieux (62) and Dale (63). (a) . D-Glucose pentaacetate (25g» alpha or beta) was dissolved 108 i n cold 2>2% hydrogen bromide i n acetic anhydride (75ml) and the mixture was allowed to stand at room temperature f o r 2h under anhydrous conditions. Chloroform (100ml) was added and the mixture was shaken with cold water (50ml) i n a separatory funnel. The chloroform layer was withdrawn and the water layer was washed with chloroform (2x 20ml). The combined chloroform extracts were washed with cold water, cold saturated sodium b i -carbonate and cold water. A l l aqueous washings were done at a reasonable speed i n order to avoid hydrolysis of the bromide. Drying (CaCl2)» f i l t r a t i o n and evaporation below 35° gave the product. R e c r y s t a l l i z a t i o n from anhydrous d i e t h y l ether gave pure £ (ca. 22g), [ a ] D +199°(c 2, chloroform) m.p. 89-90°; ( l i t . (62) m.p. 88-89°, [ a ] D +198° (c 2, chloroform)). Compound £ can be stored i n a vacuum desicator over phosphorus pentoxide at room temperature f o r several months without much degradation. S i m i l a r l y , i t can be stored at -10° i n a well stoppered f l a s k . However, bromide contaminated with hydrogen bromide may decom-pose within a few days. (b) Acetic anhydride (400ml) was placed i n a 1 - l i t e r 3-necked f l a s k equipped with an overhead s t i r r e r and thermometer. I t was cooled i n an ice-water bath and perchloric acid (60-70$, 2.4ml) was added dropwise. The solution was warmed to room temperature and anhydrous D-glucose (lOOg) was slowly added (about 30 min) with s t i r r i n g while keeping the in t e r n a l tem-perature between 30 and 40°, The reaction mixture was cool-ed to 20° and red phosphorus (30g) was added. Bromine (58ml) was then added while keeping the temperature below 20°, f o l -109 lowed by water (36ml) , which was added dropwise (about 30 min) at the same temperature. The reaction mixture was kept 2h at room temperature. Chloroform (300ml) was added and the mixture was f i l t e r e d through a f i l t e r bed of fi n e glass wool. The f l a s k and the f i l t e r were washed with chloroform (about 75ml) . The f i l t r a t e was shaken with cold water (800ml, about 5°) i n a large separatory funnel, the chloroform drawn o f f , and the water ex-tracted with chloroform (2x 5 0 m l ) . The combined chloroform extracts were washed with cold water. (2x 200ml), saturated cold sodium hydrogen carbonate ( 3x 300ml, at f i r s t i n a large beaker, then i n the separatory funnel), and cold water (2x200ml). A l l aqueous washings were done at a reasonable speed i n order to avoid hydrolysis of the bromide. Drying (CaClg) f i l t r a t i o n and evaporation below 35° gave a c r y s t a l l i n e mass. R e c r y s t a l l i z a -t i o n from anhydrous d i e t h y l ether gave the pure product, 170g, m.p. 88-89°. The bromide was stored as indicated under (a) above. (c) Anhydrous D-glucose (30g) was placed i n a 500ml 2-necked fl a s k equipped with a thermometer. A saturated solution of hy-drogen bromide i n acetic anhydride (li|0ml) was then added. The f l a s k was f i t t e d with a calcium sulfate drying tube and gently s t i r r e d (magnetic s t i r r e r ) . Within a few minutes, the reaction became exothermic with the rapid los s of hydrogen bromide gas. S t i r r i n g was stopped, and the reaction mixture was cooled i n an ice-water bath i n order to keep the in t e r n a l temperature below 3 5 ° . S t i r r i n g was continued i f a l l the sugar 110 did not dissolve. The reaction mixture was allowed to stand at room temperature f o r 40 min. Chloroform (300ml) was added and the solution was poured onto ice i n a large beaker. The chloroform layer was placed i n a separatory funnel and washed with cold water (2x 100ml), cold 50$ sodium hydrogen carbonate (about 3x 200ml, at f i r s t i n a large beaker) and cold water (2;x 100ml). Drying (CaClg), f i l t r a t i o n and evaporation gave a syrup. The syrup c r y s t a l l i z e d a f t e r adding portions (2x 50 ml) of anhydrous d i e t h y l ether and evaporating at about 3 5 ° . R e c r y s t a l l i z a t i o n from anhydrous d i e t h y l ether gave the pure product (about 40g) which was stored i n a vacuum over phos-phorus pentoxide. 1,2 i3 ,4-Di-0-isopropylidene-a-D-galactopyranose 10 Freshly fused zinc chloride (21,6g) was ra p i d l y weighed into a 500ml Erlenmeyer f l a s k containing a magnetic s t i r r i n g bar. Acetone (225ml, pre-dried with D r i e r i t e , f i l t e r e d and d i s t i l l e d ) was ra p i d l y added and the mixture was s t i r r e d under anhydrous conditions (CaSO^ drying tube, or just stoppered) u n t i l most of the chloride dissolved. Concentrated s u l f u r i c acid (0.72ml) was rapidl y added dropwise from a pipet, with-out the acid touching the neck of the f l a s k . F i n e l y powdered anhydrous D-galactose (I8g) was quickly added and the reaction mixture was s t i r r e d at room temperature f o r 4h under anhydrous conditions. A suspension of anhydrous sodium carbonate (36g) i n water (63ml) was added as rapidl y as possible and s t i r r e d (at f i r s t cautiously, then rapidly) u n t i l the supernatant was I l l free from zinc ions. The suspension was f i l t e r e d and the s a l t s were washed by suspending i n acetone ( 3 x 50ml) and f i l -t e r i n g . The combined f i l t r a t e and washings were evaporated under reduced pressure u n t i l the acetone was removed. The pro-duct then separated as an o i l y upper layer. The mixture was extracted with ether ( 3 x 50ml), and the combined extracts dr-ied (Na 2S0^). F i l t r a t i o n and evaporation gave a syrup (23g» 89$). D i s t i l l a t i o n at 140°, 0.2mm gave the pure product, [ a ] D -55° (c 3, chloroform)} ( l i t . (64) [ a ] D -55° (c 3.6, chloro-form) ). 1,2 i3,4-Di-0-isopropylidene- 6-0- (tetra-0-ac etyl-g-D-gluco-pyranosyl)-a-D-galactopyranose 11 1,2«314-Di-0-isopropylidene-a-D-galactopyranose (10, 2.0g), f r e s h l y prepared (p.122) s i l v e r oxide (2.5g) and Drie-r i t e (3»0g, preheated to 200° i n vacuo f o r 2h) were s t i r r e d under anhydrous conditions i n the dark f o r 2h e Tetra-0-acetyl-a-D-glucopyranosyl bromide (£, 2.2g) was then added and the reaction monitored by t h i n layer chromatography using solvent A. values were as follows* bromide £, 0.58} isopropylidene compound 10, 0.41; disaccharide j5, 0.51. The reaction mix-ture was s t i r r e d f o r 5b and f i l t e r e d . The f i l t r a t e was eva-porated down to a syrup which was dissolved i n 95$ ethanol (12ml) and poured onto i c e water (30ml). The r e s u l t i n g paste was t r i t u r a t e d with a glass rod and the hardened paste obtain-ed was dissolved i n methanol (40ml) and allowed to stand. Cry-s t a l l i z a t i o n occurred a f t e r standing for one day, y i e l d 2.0g, 112 m.p. 1 3 7 - 1 3 9 ° } ( l i t . (61) m.p. 141°). 6-0-0 -D-flue opyrano sy1»D-galac to s e 13 Compound 11 (500mg) was dissolved i n dry methanol (15ml) and sodium methoxide i n methanol (0.1M, 5ml) was added. A f t e r standing at room temperature f o r 45 min, the mixture was neu-t r a l i z e d with Amherlite IR 120 H + r e s i n and concentrated. The re s u l t i n g l,2i3 , 4-di-O-isopropylidene - 6-O-0-D-glucopyranosyl-D-galactose 12 was obtained as a syrup of R f 0.27 ( t . l . c . s o l -vent B). Hydrolysis of the isopropylidene groups i n 12 (0.05M s u l f u r i c acid, 6 0 ° , ca 50 min) gave the free disaccharide 12 as a syrup of R g l u c o s e 0;23 (paper, solvent C)j [ a ] D +10° (c 0.5, water); p.m.r. (DgO, external TMS), 4.28 (H-l doublet, 2 -3.0 H , a-form of reducing galactose u n i t ) , 4.96 and 5.03 Zi (H-l doublets, J-^ 2 "7,5 and 7.7 H z # 0-forms of the galactose and glucose units', ( l i t . (26) [ a ] D +19° (c 1, water)). Attempted stereoselective synthesis of 3-0-0-D-glucopyranosyl-D-mannose 21 Methyl 4,6-0-benzylidene-a-D-mannopyranoside 14 Finely powdered methyl a-D-mannopyranoside (lOg) was d i s -solved as ra p i d l y as possible i n 98-100$ formic acid (50ml). Benzaldehyde(50ml f r e s h l y d i s t i l l e d , technical grade may be used with a s l i g h t l y lowered y i e l d of product) was added at once and the solution was allowed to stand at room temperature f o r 5 min (longer time r e s u l t s i n the formation of a large amount of dibenzylidene compound). The reaction mixture was 113 poured as ra p i d l y as possible, and with good s t i r r i n g , into a previously prepared solution of anhydrous potassium carbonate (137g) i n water (200ml). Excess benzaldehyde was removed by immediate steam d i s t i l l a t i o n of the solution. Extraction of the r e s u l t i n g aqueous mixture with chloroform (5 x 200ml) and subsequent evaporation gave 14 (4.8g), contaminated with the d i -benzylidene compound. R e c r y s t a l l i z a t i o n from benzene gave pure product, m.p. 143-145°t ( l i t . (139,140) m.p. 143 -145° , 147-148°). Condensations of 14 with tetra-O-acetyl-a-D-glucopyranosyl bromide £ ( i ) Methyl 4,6-0-benzylidene-a-D-mannopyranoside 14 (500mg), s i l v e r oxide (2g, prepared as on p.122 ), and D r i e r i t e (2g, f i n e l y ground and preheated to 150° f o r 2h under vacuum), were s t i r r e d i n absolute chloroform f o r 45 min. Tetra - 0 -acetyl-a-D-glueopyranosyl bromide £ (2.5g) was then added at once and the reaction mixture was s t i r r e d at room tempera-ture i n the dark f o r lOh. ( i i ) As ( i ) , except that the bromide was added i n ten por-ti o n s during a period of 5h.. ( i i i ) As ( i ) , except that the bromide was added i n ten por-tions during a period of 3h and s i l v e r perchlorate c r y s t a l s (50mg) were added to the reactions mixture. (iv) As ( i ) , but the bromide was added over a period of 3h 114 from chloroform (10ml). In addition, iodine c r y s t a l s (lOOmg) werepadded. (v) As ( i ) , but the bromide was added as i n ( i v ) . Iodine (lOOmg) and s i l v e r perchloroate (50mg) were added. (vi) Compound 14 (300mg), s i l v e r carbonate (1.5g) and D r i e r i t e (2g) were s t i r r e d i n chloroform (50ml). Iodine (50mg) and bromide £ (2g) were then added and the reaction was s t i r r e d i n the dark f o r l6h. V ( v i i ) As (v), using dry benzene as solvent. ( v i i i ) As (v), using carbon tetr a c h l o r i d e as solvent. (ix) As ( i i ) , but the reaction mixture was cooled at 0 ° for 5h, then allowed to warm up to room temperature overnight. (x) As ( i i ) , but the reaction was carri e d out i n carbon te-t r a c h l o r i d e at 0 0 ° f o r 5b, then allowed to warm up to room temperature overnight. (xi) As ( i i ) , but i n dry benzene as solvent, cooled to 0° f o r 5b, then allowed to warm up to room temperature overnight. ( x i i ) The bromide was added as i n ( i i ) , iodine and s i l v e r per-chlorate were added as i n (v). The reaction was conducted i n chloroform and was cooled to 0 ° f o r 5 h and then allowed to warm up to room temperature overnight. Workup« the reaction mixture was f i l t e r e d and the f i l t r a t e was 115 washed with water i n a separatory funnel. Drying and evapora-t i o n gave syrups. The reactions were followed by t . l . c . using chloroform-methanol (9«1)» which gave t y p i c a l R f values as followsi 14, 0.44; bromidef£, 0.79; hydrolysis of bromide £, 0,59? the products were not c l e a r l y resolved. More conveniently, the syrups were deacetylated (0.1M sodium methoxide) and care-f u l l y neutralized (Amberlite IR 120 H + resin) i n order to pre-vent hydrolysis of the benzylidene group. T . l . c . of the deace-t y l a t e d mixtures i n solvent B improved the r e s o l u t i o n between products and by-products. Typical R^ values werei unreacted acetal 14, 0.66$ main products, 0.30; other products, 0.49, O.85. In addition, glucose (from excess bromide) and methyl mannoside (from deacetalation) were present. In order to mini-mize deacetalation, the products from the sodium methoxide de-acetyl a t i o n were kept s l i g h t l y basic ( i . e . by treatment with a def i c i e n t amount of Amberlite IR 120 H + r e s i n , preferably while cold)• ( x i i i ) Methyl 4,6-0-benzylidene-a-D-mannopyranoside (300mg) and mercuric cyanide (500mg) were s t i r r e d i n absolute aceto-n i t r i l e . Excess tetra-O-acetyl-a-D-glucopyranosyl bromide (1.3g) was then added over a period of 5h and the mixture was s t i r r e d at room temperature overnight. Evaporation of the solvent gave a syrup which was dissolved i n chloroform (25ml) and washed with water (3 x 10ml), sodium hydrogen carbonate (2 x 10ml) and water (2 x 10ml). Drying and evaporation gave a syrup which was deacetylated by fres h l y prepared sodium 1 1 6 methoxide i n methanol ( 0 . 1 M , 3 0 m l ) . P a r t i a l n e u t r a l i z a t i o n at 5 ° (Amberlite IR 1 2 0 H + resin) gave a s l i g h t l y basic solu-t i o n ( to prevent deacetalation) which was evaporated to a syrup. (xiv) As ( x i i i ) , but i n nitromethane. (xv) As ( x i i i ) , using mercuric cyanide ( 1 5 0 m g ) and mercuric bromide ( 2 1 5 m g ) . The workup included washing the chloroform solution with water, 1M potassium bromide and water. T . l . c . of the deacetylated reaction mixtures i n solvent B were s i m i l a r to those already described, but an addit i o n a l spot (R f 0 . 3 1 ) moving just ahead of the product (R f 0 . 3 0 ) spot was detected. The products of R f 0 . 3 0 were separated by preparative t h i n layer chromatography or by short column chromatography ( 1 2 0 ) using solvent B. Acetylation then gave syrups which on p.m.r. spectroscopy showed the presence of methoxy, acetates, and benzylidene functions, i n d i c a t i n g the presence of disa-ccharides. Acetolysis The condensations mixtures ( i ) to (xv) ( 3 0 0 m g each before deacetylation) were subjected to acet o l y s i s i n 2% (v/v) s u l -f u r i c acid i n acetic anhydride ( 2 0 m l ) f o r 4 h at room, tempera-ture. Each mixture was poured into saturated sodium hydrogen carbonate and s t i r r e d u n t i l the evolution of gas ceased. The 1 1 7 organic layer was dried (MgSO^), f i l t e r e d and the solvent was removed under reduced pressure. The r e s u l t i n g syrup was deace-t y l a t e d i n 0.1M sodium methoxide i n anhydrous methanol ( 1 5 m l ) and the solution was neutralized (Amberlite IR 1 2 0 H + r e s i n ) . The mixture of free sugars thus obtained was analyzed by paper chromatography. A portion ( 2 5 m g ) of the deacetylated condensation mix-ture was p u r i f i e d by t . l . c . and was s i m i l a r l y acetolyzed and deacetylated to the free sugars. The mixture of disaccharides obtained was si m i l a r , i n d i c a t i n g that a c e t o l y s i s could be done on either the f u l l y blocked or the p a r t i a l l y blocked sugars. Summary of r e s u l t s i 1 . Condensations using s i l v e r oxide were better than those using s i l v e r carbonate, which gave l e s s product. 2. Condensations using s i l v e r oxide were s l i g h t l y better than those using mercuric cyanide because the l a t t e r ones gave, on deacetylation, an impurity of si m i l a r mobility to the disaccharide products. However, the amount of product was s i m i l a r i n both conditions. 3. Mercuric cyanide condensations i n a c e t o n i t r i l e and i n nitromethane gave s i m i l a r r e s u l t s . 4. S i l v e r oxide condensations containing s i l v e r perchlorate gave better y i e l d s than those without added perchlorate. Iodine gave s l i g h t l y better y i e l d s when used together with s i l v e r perchlorate. 118 5. Bromide added slowly over a period of 5 h t or i n portions during the same period, gave l e s s degradation products than i f added at once ( s i l v e r oxide condensations). 6. S i l v e r oxide condensations i n chloroform were somewhat u cleaner than the corresponding ones i n benzene or carbon tetrachloride, due to l e s s degradation of the bromide. 7. The above condensation conditions gave a mixture of two disaccharides and slower moving components, as shown by paper chromatography. The two disaccharides were pre-sent i n a r a t i o of about 3«2 i n the better condensation mixtures. 8. The separation by t . l . c . of the condensation mixtures ; before deacetylation was attempted using many solvents, but t h i s was not successful. Although deacetylation se-parated the disaccharides from the by-products, p u r i f i -c ation was d i f f i c u l t because the l a b i l e benzylidene group would f a l l o f f during column or t . l . c . separation. This was much improved by working with a s l i g h t l y basic mixture. ^* -glucose v a l u e s o f " t h e f-ree sugars were as follows (paper chromatography)* Solvent Solvent Solvent C D E Disaccharide 0.41 0.42 0.50 (major) Disaccharide 0.27 0.32 0.37 (minor) Glucose 1.00 1.00 1.00 Mannose I.36 1.20 Others 0.09; 0.11 0.05; 0.15 0.08; 0.16 119 Although t h i s method would be a convenient one f o r the synthesis of a few milligrams of the disaccharides, i t does not seem to be a good preparative method. Because of the seemingly unselective reactions at positions 2 and 3 of the mannose moeity, and the d i f f i c u l t y i n separating the two disaccharides (except i n small quantities by paper chromato-graphy) , the synthesis was not pursued further. The nature of the linkages (1-2 or 1-3) of the two disaccharides was not investigated. Synthesis of 6-0-(4-O-methyl-0-D-gluc opyrano syl)-D-galac to se D-Glucose d i e t h y l t h i o a c e t a l 23 Anhydrous D-glucose (60g) and concentrated hydrochloric acid (60ml) were shaken at room temperature f o r about 5 min i n an e f f i c i e n t fumehood. The mixture was cooled to 0° and technical grade ethanethiol (60ml) was added and s t i r r e d Vig-orously u n t i l c r y s t a l l i z a t i o n occurred(l-2h). When any warm-ing was detected, the reaction mixture was cooled i n ice water. Methanol (400ml) was added and the mixture was neutralized with lead carbonate and f i l t e r e d . The lead s a l t s , which con-tained some of the product, were washed with hot methanol (ca 100ml). The product obtained on cooling the methanol solution was f i l t e r e d , washed with cold methanol (ca 20ml) and r e c r y s t a l l i z e d from hot methanol (ca 400ml). The product was a i r dried f o r l h , then overnight at 4 5 - 5 0 ° i n a vacuum 120 oven at 15mm; y i e l d , 60g, m.p. 124 -125° ; ( l i t . (77) m.p. 119-120°). Di-O-isopropylidene-D-glucose d i e t h y l thioacetals (mixture)124 D-Glucose d i e t h y l t h i o a c e t a l (40g) was shaken i n dry ace-tone (400ml) but did not dissolve completely. Concentrated s u l f u r i c acid (9»8g, 5.3ml) was added dropwise with s t i r r i n g . A f t e r s t i r r i n g f o r 45 min a l l the sugar had dissolved and the mixture was allowed to stand f o r 48h. At t h i s time, t . l . c . (solvent A) indicated the presence of a major product (R f 0.57), a minor product (R f 0.70, and a small amount of s t a r t i n g mat-e r i a l (R f 0.0). In addition, small quantities of slower (R f 0.09, 0.15) moving components were present. The mixture was neutralized with 10$ barium carbonate i n methanol. Since f i l -t r a t i o n of the barium s a l t s was d i f f i c u l t , they were separated by centrifugation and the supernatant was c o l l e c t e d . The ace-tone was removed and the remaining.syrup was dried at room temperature f o r 4h at 0.5mm, y i e l d 45g. 2,3*£?6-Di-0-isopropylidene-D-glucose dimethyl acetals (mixture) 25. Syrup 24, containing 2 , 3 « 5 ,6-di-0-isopropylidene-D-glucose di e t h y l t h i o a c e t a l and other isopropylidene compounds was d i s -solved i n dry methanol (500ml). Cadmium carbonate (55g) and mercuric chloride (150g) were added to the solu t i o n while s t i r r i n g . The mixture was s t i r r e d f o r about 2h under ref l u x , then i t was cooled, f i l t e r e d , and the f i l t r a t e was shaken with 1 2 1 water and chloroform i n a separatory funnel. The chloroform layer was separated, washed with water u n t i l free from chloride ion (ca 5 l i t e r ) , and dried (sodium s u l f a t e ) . Evaporation of the solvent gave a syrup 2j> ( 2 2 g ) . 2,3«5,6-Di-0-isopropylidene-4-2-nitrobenzoyl-D-glucose dimethyl acetal 2 6 The mixture of isopropylidene compounds obtained above ( 2 2 g ) was dissolved i n dry pyridine ( 1 3 0 m l ) and f r e s h l y d i s -t i l l e d E-nitrobenzoyl chloride ( 2 2 g ) was added with s t i r r i n g while keeping the temperature below 2 5 ° . The mixture was s t i r r e d overnight and poured slowly into ice water. This was extracted with petroleum ether (b.p. 6 5 - 1 1 0 ° , 3 5 0 m l x 1 0 ) . The petroleum ether layer was washed with 5 $ sodium hydrogen car-bonate ( 2 0 0 m l x 2 ) and water ( 2 0 0 m l x 2 ) , then dried (sodium sulfate).Evaporation gave a c r y s t a l l i n e mass which was recrysta-l l i z e d from absolute ethanol ( 1 2 5 m l ) j y i e l d 1 7 . 5 g t m.p. 1 0 3 -105°j ( l i t . ( 7 9 ) m.p. 1 0 6 - 1 0 7 ° ) . 2,3»5 i6-Di-O-isopropylidene-D-glucose dimethyl acetal 2j5 The 2-nitrobenzoate 26 ( 1 7 . 3 g ) was placed i n methanol ( 1 0 0 m l ) and f r e s h l y prepared sodium methoxide solution ( l g sod-ium metal i n 1 0 0 m l methanol) was added and the mixture was allowed to stand at room temperature f o r 30 min. Thin layer chromatography (solvent A) showed that deacylation was com-ple t e . The solution was poured into 4 l i t e r of 0.15M sodium 122 hydroxide and warmed u n t i l a small amount of s o l i d material dissolved and the cloudiness of the solution disappeared. The solution was allowed to stand at room temperature f o r 3 h and then i t was divided into two equal portions i n order to f a c i l i -tate the workup. Each portion was extracted with chloroform ( 5 x 250ml) and the combined chloroform extracts (1250ml) were washed with 5 $ sodium hydrogen carbonate (2 x 200ml) and water (2 x 400ml). Drying (sodium sulfate) and evaporation gave a syrup (11.3g) of R f 0.41 (solvent A); p.m.r. (CDCl^)1 6 . 6 5 , 6.62 (3H s i n g l e t s , OCH^), 8.62, 8.67, 8.72 (6H, 3H and 3H sing-l e t s respectively, isopropylidene CH^). 2,3»5»6-Di-0-isopropylidene-4-0-methyl-D-glucose dimethyl acetal 27 (a) Purdie methylation S i l v e r oxide- S i l v e r n i t r a t e (15.0g) was dissolved i n hot water ( 8 5 ° , 150ml). Sodium hydroxide (3 . ^5g) i n hot water ( 8 5 ° , 150ml) was then added i n a fin e stream while s t i r r i n g (141). The mixture was f i l t e r e d by suction on a sintered glass funnel and s t i r r e d i n the funnel with several portions of hot water (10 x 150ml) and absolute ethanol ( 3 x 25ml). The s o l i d was a i r dried f o r 30 min, then dried to constant weight i n a vac-uum desiccator over phosphorus pentoxide i n the dark. I t was then f i n e l y ground and passed through a 60-100 mesh sieve and stored i n the dark over phosphorus pentoxide. S i l v e r oxide prepared i n t h i s manner was found to be more active than the product obtained commercially. 123 The acetal 25_ (lOg), f r e s h l y prepared dry s i l v e r oxide (15g, added i n f i v e portions during 6h) and methyl iodide (30 ml) were heated under ref l u x while s t i r r i n g on a water bath at about 50° (80). The reaction mixture was refluxed f o r I8h, af-t e r which t . l . c . (solvent A) showed about 50$ reaction. The st a r t i n g material had an R f of 0.41, the product O.65. The mixture was f i l t e r e d and washed with chloroform, and the s i l v e r s a l t s were washed overnight with 200ml of chloroform i n a Sohxlet extractor i n order to recover any bound carbohydrate. The o r i g i n a l chloroform f i l t r a t e and the chloroform from the extraction were combined and evaporated to a syrup. This was dried by adding 50ml portions of dry acetone and evaporating, thus removing any residual water azeotropically. The syrup was methylated twice more as described above, except that each time the mixture was refluxed f o r two days. About 80$ of the acetal was methylated at the end of the t h i r d methylation. A fourth methylation, using 30g s i l v e r oxide and 60ml methyl iodide, gave over 90$ product as shown by t . l . c . (b) Hakomori methylation Methylsulfinyl anion- Sodium hydride i n mineral o i l (3g» 50$) was placed i n a 100ml 3-necked fl a s k containing a magnetic s t i r r i n g bar and was washed with dry petroleum ether (b.p. 3 0 - 6 0 ° , 3 x 50ml) by decantation (about 5ml petroleum ether i s l e f t i n the f l a s k i n order to prevent the hydride from going dry). The flask was f i t t e d with a thermometer, a condenser with a drying tube, and a rubber serum cap through which a hypodermic meedle (connected to a nitrogen suppy) was 124 inserted. Nitrogen was passed continuously through the needle i n order to remove the excess petroleum ether. Methyl s u l f -oxide (45ml , d i s t i l l e d at 15mm from calcium hydride and stored over dried Linde 4A molecular sieve),, was then added using a hypodermic syringe. With a f i n e stream of nitrogen, the mixture was heated at 50-55° with s t i r r i n g u n t i l the evolution of gas ceased and the solution became green or dark (l-2h) (82). The concentration of anion was determined by t i t r a t i o n (142) under anhydrous conditions of 1.0ml aliquots with formanilide (300mg/ml methyl sulfoxide) using triphenylmethane as indicator. The concentration of the anion thus obtained was normally about 1.4M. I t was usually stored f o r a few weeks at about 5° i n a desiccator over D r i e r i t e . 2 ,3«5»6-Di-0-isopropylidene-D-glucose dimethyl acetal 2J (lOg) was dissolved i n dry methyl sulfoxide (100ml). A solu-t i o n of methyl s u l f i n y l anion (0.6M, 120ml) was added (81) and the solu t i o n was l e f t 45 min at room temperature. The mixture was cooled i n kn ice water bath and excess methyl iodide (55ml) was added slowly while keeping the temperature below 25°. As soon as warming was detected the reaction mixture was cooled again. Thin layer chromatography i n solvent A showed the re-action to be complete i n 30 min. Water (200ml) was added to the mixture and t h i s was extracted with petroleum ether (b.p. 65-110°, 12 x 100ml). The combined solvent extracts were washed with a small amount of water (25ml), dried(calcium s u l f a t e ) , f i l t e r e d , and concentrated to a syrup 2J_ (9.6g). 125 4-0-Methyl-D-glucose 28 2,3i5,6-Di-O-isopropylidene-4-0-methyl--D-glucose dimethyl acetal 2£ (9.6g) in .025M sulfuric acid (100ml) was heated for 4h at 60° with sti r r i n g . The solution was cooled, neutral-ized with barium carbonate, f i l t e r e d and concentrated to a syrup. Residual water was removed azeotropically by adding portions of dry benzene (4 x 30ml) and evaporating. Traces of solvent were removed on the vacuum pump at about 0.5 torr. The dried syrup (5.7g) gave [ a ] n +49° indicating the presence of impurities. No carbohydrate impurities were detected by paper chromatography, but a small amount of "degradation pro-ducts^' having Rf 0.0 was detected. A pure sample of 4-0-methyl-D-glucose obtained by sodium methoxide deacetylation of the crystalline tetraacetate had [of) n +60.0° (c 2 .8, water)} ( l i t . (84) [ c t ] D + 6 1 . 1 ° (c 2, water))} R g l u c o s e 2.65 (solvent C). 4-0-Methyl-D-arabino-hexose phenylosazone 29 4-0-Methyl-D-glucose (lOOmg), glacial acetic acid (0.13ml) and water (0.2ml) were heated on a steam bath to about 80°. Freshly d i s t i l l e d phenylhydrazine (0.26ml) was then added and the mixture was heated for 40 min. The reaction was cooled and the crystals were fi l t e r e d , washed with 10$ acetic acid (1ml) and water (1ml). Recrystallization from hot ethanol (0.5ml) and drying(vacuum desiccator over phosphorus pentoxide) gave pure 2£, m.p. 158-160 0 . ( l i t . (7:9,84) m.p. 158-160 0 ). 1»2,3»6-Tetra-0-acetyl-4-0-methyl-3-D-glueopyranose JO V 126 Crude 4-0-methyl-D-glucose ( 4 . 5 g ) , d i s t i l l e d acetic anhy-dride (32ml) and anhydrous sodium acetate (2.25g) were heated on a steam bath f o r 2h, Chloroform (30ml) was added to the cooled mixture which was then washed with water. Removal of the chloroform gave a syrup s t i l l containing a small amount of acetic anhydride which was removed (as ethyl acetate) by adding portions (10 x 20ml) of absolute ethanol and concen-t r a t i n g . C r y s t a l l i z a t i o n then occurred. The c r y s t a l s were washed with l -2ml coldethanol and dried; y i e l d 3»5gf m.p. 98-1 0 0 ° . R e c r y s t a l l i z a t i o n from cyclohexane gave c r y s t a l s of m.p. 102-103°? [ a ^ -8 . 1 ° (c 2.3, chloroform); ( l i t . (84) m.p. 98-9 9 ° , [aOo 2 , 0 » chloroform)). The p.m.r. spectrum of 30 showed the anomeric doublet centered at 4.40, 2 ^ 8.0 H z, thus i n d i c a t i n g the beta anomer (83). Other signals included 6.63 (0CH 3), 7.96, 7.96, 8.01, 8.05, (OAc's). Anal. Calcd. f o r ^i^2Z010x C ' ^9.70; H » 6.12. Found 1 C, 49.62; H, 6.05. I s o l a t i o n of 4-0-methyl-D-glucose 28 from mesquite gum (i ) P a r t i a l hydrolysis of mesquite gum Mesquite gum (200g) was l e f t standing i n water with oc-casional s t i r r i n g u n t i l dissolved (ca 2 days). The solution was f i l t e r e d i n order to remove insoluble material. Concen-trated s u l f u r i c acid (4.5ml) was added dropwise, bringing the f i n a l acid concentration to about .08M (87). The solution * Sourcei Martin Drug Co., Tucson, Arizona, USA 127 was heated to 95° f o r 36h with s t i r r i n g , cooled, neutralized (barium carbonate), and f i l t e r e d . The f i l t r a t e was concentra-ted to 500ml and was then dialyzed from water (7 x 3.5 l i t e r ) f o r one week, using Dennison #310 cellophane paper suspended i n a 4 l i t e r beaker. The water was s t i r r e d (magnetic s t i r r e r ) i n order to f a c i l i t a t e d i a l y s i s . The dialyzate was co l l e c t e d and evaporated i n order to obtain the crude arabinose (65g). The polysaccharide remaining i n the d i a l y s i s bag was concentrated to a volume of 300ml and methanol (300ml) was slowly added to i t while s t i r r i n g . A f t e r standing f o r l-2h, the pr e c i p i t a t e was f i l t e r e d and washed with methanol and ether. Drying at 4 5 ° overnight i n a vacuum oven at 15mm gave the dry polysa-ccharide J l (42g). ( i i ) Methanolysis of the polysaccharide J l . Methyl 4-0-methyl-D-glucuronic acid methyl ester 22 The polysaccharide obtained above (42g) was subjected to methanolysis i n 7$ methanolic hydrogen chloride (250ml) f o r I8h under r e f l u x . Examination by t . l . c . (solvent B) indicated that a f a i r amount of oligosaccharides was s t i l l present. An additio n a l portion of methanolic hydrogen chloride (150ml) was then added and refluxed for an additional 24hv The reaction mixture was cooled, neutralized with lead carbonate, f i l t e r e d and concentrated to a syrup. This was dissolved i n water (200 ml) and extracted overnight with chloroform (300ml) i n a c o n t i -nous extraction apparatus. Evaporation of the chloroform gave a syrup (7.7g) which contained methyl 4-0-methyl-D-glucuronoside 128 methyl ester j3J3t as compared with an authentic standard by paper chromatography. A small amount of methyl galactoside was also detected. The water la y e r contained methyl galac-toside and oligosaccharides. ( i i i ) Methyl 4-0-methyl-D-glucoside° J4 The ester 21 (7.7g) i n dry tetrahydrofuran (100ml) was added slowly with s t i r r i n g to a mixture of l i t h i u m alum-inum hydride (12g) i n tetrahydrofuran (400ml) at room tempera-ture. A f t e r l h excess hydride was destroyed by the dropwise addition of water u n t i l evolution of gas ceased. Evaporation gave a syrup which was dried by adding dry benzene (10 x 50ml) and evaporating. The mixture containing the reduced sugar and aluminum s a l t s was heated on a steam bath with acetic anhydride (200ml) and anhydrous sodium acetate (lOg) f o r 4h. The alumi-num s a l t s were separated by d i s s o l v i n g the mixture i n 4M hydro-c h l o r i c acid (300ml) and extracting with chloroform (3 x 2 0 0 m l ) . The organic layer was washed with water, saturated sodium hydrogen carbonate, and water. Removal of the chloroform gave a syrup 21 which was deacetylated i n ethanol (50ml) and 1M sod-ium hydroxide (50ml) under ref l u x f o r l h . Neutralization with Amberlite IR 120 H + r e s i n gave a syrup containing J4, 4 . 5 g . (iv) 4-0-Methyl-D-glucose 28 Methyl glycoside J 4 ( 4 . 5 g ) was refluxed i n 0.5M s u l f u r i c acid (150ml) f o r 1 5 h . Cooling, n e u t r a l i z a t i o n with Duolite A-4 anion exchange r e s i n , and concentration gave a syrup con-129 t a i n i n g 4-0-methyl-D-glucose 28 R g i u c o s e 2 » 6 5 (paper, solvent C). Acetylation as previously described gave the beta t e t r a -acetate, 4,0g, m.p. 100-102°. The p.m.r. spectrum of the t e -traacetate was as previously described. 2 ,3,6-Tri-0-acetyl -4-0-methyl-a-D-glucopyranosyl bromide 36 Tetraacetate ^0 (1.6g) was dissolved i n cold hydrogen bromide i n g l a c i a l acetic acid (32$, 28ml) and the solution was allowed to stand at room temperature f o r 45 min. Chloro-form (50ml) was added and the solution was shaken with cold water (50ml). The aqueous lay e r was extracted with chloroform (3 x 25ml) and the combined chloroform extracts were washed with cold saturated sodium hydrogen carbonate (2 x 50ral) and cold water (2 x 25ml). Drying (calcium sulfate) and evapora-t i o n of the chloroform below 35° gave bromide %6 as a syrup (1.8g)$ [<x]D +190° (c 4.2, chloroform)? R f 0.58 ( t . l . c . s o l -vent A. The workup described above should be done as fast as pos-s i b l e i n order to prevent hydrolysis of the product. Thus cold water i s used. The bromide i s r e l a t i v e l y unstable and cannot be kept f o r long without decomposition. 1 ,2»3,4-Di-O-isopropylidene-6-0-(4-0-methyl-g-D-glucopyranosyl a-D-galactopyranose 38 l,2« 3 l 4-Di-0-isopropylidene-a-D-galactopyranose 10 (600 mg, 2.5 mmol, d i s t i l l e d syrup), freshly prepared s i l v e r oxide (page 122,2g), and D r i e r i t e (2g, f i n e l y powdered, preheated 130 f o r 2h at 200° i n vacuo) were s t i r r e d f o r In i n absolute chloro-form (15ml) at room temperature. A solution of 2 , 3 , 6-tri-0-acetyl-4-0-methyl-a-D-glucopyranosyl bromide (1.5g» 3*7 mmol) i n absolute chloroform (8ml) was then added over 2h with con-tinuous s t i r r i n g . T . l . c . (solvent A) showed that the reaction was complete within 30 min a f t e r the f i n a l addition of the bro-mide, values were as followsi isopropylidene 10, 0.33$ bro-mide J6, 0.5^; a degradation product of bromide ^6, 0.33$ prod-uct, 0.44, corresponding to the f u l l y blocked disaccharide 37* The mixture was f i l t e r e d , the s o l i d residue was washed with chloroform, and the f i l t r a t e and washings were evaporated to a syrup, A small amount of the f u l l y blocked disaccharide 37 was i s o l a t e d by preparative t . l . c , and a p.m.r, spectrum (60 MHz) obtained. This showed (CDC13)i 6.58 (OCH^h 7.88, 7.93. 7.93. (0Ac*s), 8.50, 8.58, 8.68, 8.68, (isopropylidene CH^). The bulk of the syrup containing 2Z w a s treated with sodium methoxide i n anhydrous methanol (0.1M, 30ml) f o r 30 min at room temperature. Sodium ion was removed with Amberlite IR 120 H r e s i n and the methanol was evaporated to give a s o l i d product _3_8, m.p. 168-170° (680mg, 62$ ov e r a l l from 10). Re-c r y s t a l l i z a t i o n from absolute ethanol (4ml/g) gave pure l , 2 i 3,4-di-0-isopropylidene-6-0-(4-0-methyl-g-D-glucopyranosyl)-a-D-galactopyranose 38, m.p. 174-175°? M D -60 .5° (c 0.8, me-thanol) $ R f 0.50 ( t . l . c . solvent B)$ p.m.r. (CDCl^, 60 MHz)i 6.62 (0CH3), 8.45, 8.56, 8.63, 8.63 (isopropylidene CH 3) 4.42 ( p a r t i a l l y hidden doublet, J 1 2 » about 4 H z, H- l ) . Anal. Calcd. f o r C,QH.,90,.. « C, 51.88$ H, 7.35$ 0CH~, 6.93. 131 Foundi C, 5 2 . 2 5 ; H, 7 . 3 9 ; OCH3 7.11. The above condensations conditions were chosen when tes t runs (each run, 50mg bromide J3_6) indicated that other condi-tions ( s i l v e r oxide i n carbon tetrachloride or benzene, s i l v e r carbonate i n chloroform, and mercuric cyanide i n a c e t o n i t r i l e or nitromethane) were not as s a t i s f a c t o r y . 6-0-(4-0-Methyl-£-D-glucopyranosyl)-D-galactose 39 l,2«3»4-Di-0-isopropylidene-a-D-galactopyranose J 8 (600 mg) was heated on a steam bath f o r 40 min i n d i l u t e s u l f u r i c acid (0.025M, 3 0 m l ) . Neutralization with Duolite A-4 anion exchange r e s i n and concentration gave the free disaccharide 39 (486mg) as a syrup, [ c t ] n +4° (c 1.0 water). Paper chroma-tography showed .Bgiucose values of 0 .34 (solvent C) and 0.26 (solvent D). A small amount of monosaccharides were separated by passage of a lOOmg sample of J2 o n a Dowex 50W x2 ( L i + ) column (90cm x 2.3cm) (92). Fractions (1.4ml) were c o l l e c t e d at 15 min i n t e r v a l s . Fractions 87-97 contained the pure d i s -accharide (94mg), while f r a c t i o n s 99-103 contained monosac-charides. Pure J 2 had [ a ] Q about 0° (c 1.0,water). Anal. Calcd. f o r ^i^b0!!* °* ^3.82; H, 6 . 7 4 . Found (a f t e r drying over P 2 ° 5 ^ a t 2mm f o r three days); C, 4 3 . 6 3 ; H, 7 . 0 9 . The above hydrolytic conditions were chosen when other conditions ( l i s t e d below) were found to be somewhat l e s s sat-i s f a c t o r y . ( i ) Disaccharide J 8 (20mg) i n 2.0ml 0.005M s u l f u r i c acid was 132 heated on a steam bath« about 90$ of the isopropylidene groups were removed i n 60 min, a l l removed i n 90 min. About 5% gly-cosidic cleavage observed i n 90 min. ( i i ) Disaccharide J8 (lOmg) i n 1.0ml 0.05M s u l f u r i c acid was heated on a steam batht the acetals were hydrolyzed i n 20 min and about 10$ g l y c o s i d i c cleavage was observed i n t h i s time, ( i i i ) Compound J8 (lOmg) i n 0.05M s u l f u r i c acid at 80°i about 25$ of the acetals remained unreacted i n 40 min and about 5$ glycoside cleavage resulted. (iv) Compound J8 (lOmg) i n 1.0ml 0.25M s u l f u r i c acid at 80°« the isopropylidene groups were removed i n 15 min but about 10$ glycoside cleavage was observed. (v) Compound _3_8 (lOmg) i n 1.0ml 0.5M s u l f u r i c acid» the acetals were not completely removed at 50° f o r 30 min but they were removed at 80° f o r 10 min with concurrent glycoside cleavage of about 10$. (vi) Compound J8 (lOmg) i n 1.0ml 0.05M hydrochloric a c i d i gave more cleavage than 0.05M s u l f u r i c at 80° f o r 40 min and at steam bath temperature f o r 10 min. ( v i i ) Compound J8 (20mg) i n 300mg (wet) Amberlite IR 120 H + r e s i n and about Jml water heated on a steam bathi the acetals were completely removed i n 40 min but about 10$ glycoside hy-d r o l y s i s resulted. Removal of the isopropylidene groups was followed by t . l . c . using solvent B while glycoside cleavage 133 was monitored by paper chromatography using solvents C and D. Estimates of glycoside hydrolysis ($) were made by v i s u a l com-parison with standards of 4-0-methyl-D-glucose and galactose and are therefore only approximate. Reaction of 21 (5mg) i n pyridine ( 0 . 5 m l ) with c h l o r o t r i -methylsilane ( 0 . 1 m l ) and hexamethyldisilazane ( 0 . 2 m l ) at 50° f o r 2h (142A) and subsequent evaporation gave the correspon-ding per ( t r i m e t h y l s i l y l ) disaccharide as a syrup. Injection i n hexane on to column c at 260° (helium flow, 8? ml/min) gave one peak (15$) at 7 . 3 min and two unresolved peaks at 1 0 . 7 min ( p e r ( t r i m e t h y l s i l y l ) sucrose 5*5 min). Reaction of 21 with p_-nitroaniline. Disaccharide p_-nitroan-i l i d e 40 The disaccharide 39 (llmg) was dissolved i n dry methanol ( 1 . 0 m l ) . p_-Nitroaniline (13mg) and one drop g l a c i a l acetic acid were then added and the solution was refluxed f o r 2h under anhydrous conditions. The yellow c r y s t a l s obtained on cooling were washed with ether and a i r dried, m.p. 145-147°, R e c r y s t a l l i z a t i o n from absolute ethanol (1ml) and drying at 7 0 ° under vacuum gave c r y s t a l s that melted at 146-147°, R f values (solvent B) were as follows i p_-nitroaniline ( 0 , 7 6 , disaccharide £-nitroanilide 40 (0,18), disaccharide 21 ( 0 . 0 ) . Reaction of 21 with p_-nitrobenzoyl chloride A solution of 21 (12mg) i n pyridine (2ml) was reacted with p_-nitrobenzoyl chloride(5Qmg) at room temperature over-134 night. Workup (p.153) gave the disaccharide hepta-0-p_-nitro-benzoate 41 as amorphous c r y s t a l s , m.p. 1 5 3 - 1 5 6 ° . Treatment of 22 with 0-glucosidase Buffers (pH 5) « 1.96g trisodium c i t r a t e dihydrate and 0.7g c i t r i c a cid i n 100ml solution. Disaccharide (3mg) was dissolved i n 1ml buffer solution. To t h i s was added 3mg beta glucosidase i n 1ml buffer (143). A drop of toluene was added to the reaction i n order to pre-vent b a c t e r i a l growth, and the mixture was kept at 3 5 ° . Two blanks were run concurrently! i 3mg disaccharide i n 2ml buffer at 3 5 ° . i i 3mg enzyme i n 2ml buffer at 3 5 ° . The reaction was followed by paper chromatography (solvent C), but no hydrolysis of the disaccharide was observed i n two days. The blanks were also unchanged. Beta glucosidase a c t i v i t y was tested using a 3mg sample of cellobiose reacted under the above conditions, and e s s e n t i a l l y complete hydrolysis occurred in. 23. Methylation analysis of 39 Dried disaccharide (28mg) was dissolved i n dry methyl s u l -foxide (2.0ml) i n a f l a s k covered with a serum cap through which nitrogen was passed f o r 5 min (a hypodermic needle con-nected to a nitrogen supply tank i s inserted'through the serum cap. A second needle i s used as exit f o r the gas). Methy-135 s u l f i n y l anion (82) (.15M» 1.0ml) was then added with a sy-ringe and the reaction was kept f o r l h at room temperature. Ex-cess methyl iodide (6.0ml) was added dropwise i n the same man-ner with shaking while keeping the temperature at about 15°• After shaking f o r 2h at room temperature, the excess methyl iodide was removed by evaporation at 35°. Water (12ml) was added and the solution was extracted with petroleum ether (b.p. 65-110°, 6 x 15ml). Drying (calcium sulfate) and evap-oration gave a syrup 4_2 (53mg, contained some methyl sulfoxide) which showed no hydroxyl absorption i n the infrared. The syrup was then refluxed i n 3$ methanolic hydrogen chloride (5ml) for 4h and was neutralized with s i l v e r carbonate. Gas-l i q u i d chromatographic analysis using column b programmed at 2°/min from 130° to 190°at a helium flow rate of 85.?ml/min showed retention times i d e n t i c a l to those of authentic stan-dards of methyl 2,3,4,6-tetra-O-methyl-D-glucose 44 (5.1 min, 7.7 min) and methyl 2,3»^-tri-0-methyl-D-galactoside 4j5 (21.3 min). In addition, a peak of retention time 15.9 min was ob-served, probably due to methyl 2,3,5-tri-0-methyl-D-galacto-furanoside 46 (97). The r a t i o of 2,3,5 to 2,3,4 galactosides was about 2i3. Periodate oxidation of j>2 In duplicate determinations, the dry disaccharide was d i s -solved i n 20.0ml (100$ excess, pH 5.1, unbuffered) 0.015M sod-ium metaperiodate solution and allowed to stand at room tem-perature i n the dark. Portions (1ml) were withdrawn, were d i -136 luted to 250ml), and the absorptions at 222.5nm were taken on a Gary 15 U.V. spectrophotometer (104). The absorbance de-creased to a constant value i n about I8h and remained unchan-ged f o r about 4 days. The f i n a l absorbance was corrected f o r the absorbance of iodate, and the concentration of unreacted periodate i n solut i o n was then obtained by d i r e c t reading from a Beer-Lambert plot of absorbances at 222.5nm vs known concen-t r a t i o n s of sodium metaperiodate (ranging from 6 x 10"*% to 10"%. Run Milligrams F i n a l Corrected Moles lOjj," sample absorbance absorbance Consumed/mole sugar 1 10.50 .347 .307 5.0 a 10.40 .354 .314 4.9 Reduction and hydrolysis of the periodate oxidation products. Analysis by gas-l i q u i d chromatography Barium acetate was added to run 1 i n order to pr e c i p i t a t e unreacted periodate and iodate. The s a l t s were f i l t e r e d , were washed with a small amount of water, and the water solu-t i o n was concentrated to 2ml. Sodium borohydride (30mg) i n wa-te r (1ml) was then added and the mixture was l e f t standing overnight at room temperature. Excess borohydride was destro-yed with a few drops of g l a c i a l acetic acid and the mixture was passed through Amberlite IR 120 H + r e s i n . The water was evaporated and several portions of methanol were added and evaporated at 40° i n order to remove the v o l a t i l e methyl bor-1 3 7 ate. The r e s u l t i n g syrup was refluxed i n 0.5M s u l f u r i c acid (5«0ml) f o r 4 h , neutralized with barium carbonate, f i l t e r e d , evaporated to a syrup, and dried by adding portions of dry ben-zene and evaporating. The syrup was then converted to the t r i -m e t h y l s i l y l derivative (102) f o r g . l . c . analysis. The t r i m e t h y l s i l y l derivatives of 2 - 0-methyl-D-erythritol and of ethylene g l y c o l were used as standards. 2-0-Methyl D-ery-t h r i t o l (73) was obtained by periodate oxidation and subsequent borohydride reduction of a sample of 4-r'O-methyl-D-glucose which was i s o l a t e d from mesquite gum. G.l.c. analysis was done using column a at a helium flow rate of 87 ml/min and temperature pro-grammed from 1 2 0 ° to 1 9 5 ° at 2°/min. The retention times of the t r i m e t h y l s i l y l derivatives of the reduced-hydrolyzed oxidation products were i d e n t i c a l ( 7 3 * 1 ^ ) to those of the t r i m e t h y l s i l y l derivatives of ethylene g l y c o l (5*3 min) and 2-0-methyl-D-ery-t h r i t o l ( 2 7 . 2 min). Methyl 2 , 3 , 4-tri-O-methyl-D-galactoside 4j> This compound was prepared as a standard f o r chromatogra-phic analysis as followsi a mixture of l , 2 « 3 , 4 - d i - 0 - i s o p r o p y -lidene-a-D-galactopyranose 10 ( l g ) , ground potassium hydroxide (2g) and benzyl chloride (2ml) i n dry toluene (50ml) was refluxed f o r 4h under anhydrous conditions, cooled, and washed with water (20ml) i n a separatory funnel. Excess benzyl chloride was re-moved at 7 0 ° by successive evaporations with water (the mixture should be b a s i c ) . The remaining syrup of 6 - 0 - b e n z y l - l , 2 i 3 , 4 - d i -O-isopropylidene-a-D-galactopyranose j>l was dissolved i n chloro-form, washed with water, and the chloroform la y e r was concen-138 trated to a syrup %L which wast i subjected to methanolysis ( 3 $ HCl/MeOH, r e f l u x 1 day, neutralize with s i l v e r carbonate); t h i s gave a syrup containing methyl 6-0-benzyl-D-galactoside jj j which c r y s t a l l i z e d on stand-ing, mfp. 1 3 3 - 1 3 5 ° ; i i subjected to hydrolysis (0.025M HgSO^, ref l u x over-night, neutr a l i z e with barium carbonate); t h i s gave c r y s t a l l i n e 6-0-benzyl-D-galactose J5J2, m.p. 96-98°; ( l i t . ( 9 9 ) m.p. 9 6 - 9 8 ° ) . A portion of J£3_ was methylated by the Hakomori method and hydrogenated (10$ Pd/C, 5 0 p . s . i . , room temperature f o r 3 days) to give 4j>. Hydrolysis of ^  (0.5M H2S0^, ref l u x overnight) gave 2,3,4-tri-O-methyl-D-galactose 48 as a syrup, R g i u c o s e O.38. 6-0- (4-0-Methyl -e-D-glucopyranosyD-D-galaetitol 4-9 1-0-(4-0-Methyl-P-D-glucopyranosyl)-L-galactitol Disaccharide (180mg) was reduced with sodium borohydride ( 3 6 0 m g ) i n water (18ml) at room temperature f o r 4h. Excess bo-rohydride was neutralized by addition of a few drops of acetic acid. Passage through Amberlite IR 120 H + r e s i n , concentration and removal of the borate by the addition of several portions of methanol and evaporation gave a syrup 4£ which c r y s t a l l i z e d on standing f o r 1 year, m.p. 9 6 - 9 8 ° ; R g l u c o g e ° » 3 4 solvent C, O.33 solvent D. Acetylation of 4_£ i n acetic anhydride (10ml) and anhydrous sodium acetate gave a syrup $0 which c r y s t a l l i z e d on seeding with c r y s t a l s c o l l e c t e d from the g . l . c . (column c ) ; 139 crude y i e l d 346mg; m.p,; 95-100°. Two r e c r y s t a l l i z a t i o n s from ethanol (20ml/g) gave 6-O-(4-O-methyl-0-D-glucopyranosyl)-D-ga-l a c t i t o l octaacetate, j[0 m.p. 104-106°; [ a ] D -31.3° (c 2.2, , chloroform); R^ 0,35 (solvent A); retention time on column c at 2?5°» 8 min (helium flow, 86ml/min) t retention time of sucrose octaacetate 5«1 min. The p.m.r. spectrum of j>0 (CDCl^) did not show c l e a r l y the signal f o r the anomeric proton, but the free a l d i t o l 4_£ (DgO, external TMS) showed a doublet at 5»53» J i 2 = Hz» i n d i c a t i n g beta linkage (95»96). 140 Synthesis of a-D-glucosides Attempted use of a non-participating carbonate group Methyl 4 i r6-0-benzylidene-a-D-glucopyranoside jj2 ( i ) To a solution of anhydrous zinc chloride (15g» f r e s h l y fused) i n benzaldehyde (70ml, p r a c t i c a l grade) was added methyl a-D-glueopyranoside (28g) and the mixture was shaken at room temperature f o r l h (145). The reaction mixture was poured i n a t h i n stream with rapid s t i r r i n g into chloroform (700ml). The chloroform was decanted and the remaining gummy mass (containing zinc chloride and unreacted methyl glucoside) was washed with chloroform. The combined chloroform extracts were washed with cold water (2 x 100ml), and with saturated sodium hydrogen carbo-nate." ;Solid sodium hydrogen carbonate (ca lg) was added to the chloroform layer which was then evaporated under reduced pressure. Sodium hydrogen carbonate (ca 5g) i n water (100ml) was then added and the mixture was steam d i s t i l l e d u n t i l no more benzaldehyde ap-peared i n the d i s t i l l a t e (odor). The hot aqueous solution was transferred to a beaker and allowed to cool to room temperature, then to about 10° i n an ice bath. The c r y s t a l s were f i l t e r e d , washed with cold water and dried at 70° i n a vacuum oven at 10-15mm; y i e l d 21g, m.p. I6O-I63 0. R e c r y s t a l l i z a t i o n from water or chloroform-ether gave the pure products, m.p. 163-164°; [ a ] D +110° (c 2, chloroform). ( i i ) To a soluti o n of fr e s h l y fused zinc chloride (75g) i n 141 benzaldehyde (350ml, p r a c t i c a l grade) was added methyl a-D-glucopyranoside (120g) and the mixture was shaken at room tem-perature f o r l h . The mixture was poured i n a t h i n stream while s t i r r i n g into cold water (3 l i t e r s ) , seeded with an authentic sample, and allowed to c r y s t a l l i z e at about 10° f o r l h . Pet-roleum ether (150ml, b.p. 30-60°) was then added and the mix-ture was s t i r r e d f o r 30 min to remove benzaldehyde. The cry-s t a l s were f i l t e r e d , b y suction and washed with cold water (2 x 200ml), petroleum ether (2 x 200ml) and cold water (2 x 200ml). The product was a i r dried, then dried i n a vacuum oven at 70°, 10-15mm, y i e l d , 115g» m.p. 158-162°. I t was reery-s t a l l i z e d as shown under (a). Methyl 4,6-0-benzylidene-a-D-glucopyranoside 2,3-carbonate 60 To a solution of methyl 4,6-0-benzylidene-a-D-glucopyrano-side (59» 5g) i n p_-dioxane (25ml) was added ethyl chloroformate (50ml) and the reaction was cooled i n an ice-water bath. A solution of triethylamine (25ml) i n dry benzene (140ml) was added dropwise with s t i r r i n g over 35 min (inte r n a l temperature below 10°). The reaction mixture was kept at 5° f o r 5 min* and washed i n a separatory funnel with 2M hydrochloric acid (2 x 100ml) and water (3 x 100ml) u n t i l neutral. Drying (sod-ium s u l f a t e ) , f i l t r a t i o n , and evaporation gave c r y s t a l s of R f 0^65 (solvent A). R e c r y s t a l l i z a t i o n from ether-hexane gave * When the reaction mixture was allowed to stand f o r l h a f t e r the addition of the triethylamine solution was completed, part of the carbonate was removed by the base and s t a r t i n g material was obtained. Not a l l the triethylamine solution needs to be added, just enough to make the solution s l i g h t l y basic. 142 4.6g of product (86$) j m.p. 114-116°, [ a ] D +69° (c 2, chloro-form} ( l i t . (107) m.p. 1 1 5 - 1 1 7 ° , [ a ] D +69° (c 1, chloroform)). Acetolysis of methyl 4 ,6-0-benzylidene-a-D-glucopyranoside 2 ,3-carhonate ( i ) Methyl 4,6-0-benzylidene-a-D-glucopyranoside 2,3-carbonate 60 (400mg) was dissolved i n i$ (v/v) s u l f u r i c acid i n acetic anhydride (2ml). The reaction was allowed to stand at room temperature f o r 15h, and was subsequently poured into an i c e -cold solution of saturated sodium hydrogen carbonate i n water (20ml) and s t i r r e d u n t i l the evolution of gas ceased (ca 30 min). More sodium hydrogen carbonate was added i f necessary. The mixture was extracted with chloroform and the combined chloro-form extracts were washed with cold water and dried (sodium s u l f a t e ) . F i l t r a t i o n and evaporation then gave a syrup 63 of 0.45 (solvent A). The s t a r t i n g material 60 had an R f of O.65. P u r i f i c a t i o n (from benzaldehyde and traces of acetic anhydride) by preparative t . l . c . gave the pure syrup (360mg) §2\ [ a ] D - 3 3 ° (c 0.8, chloroform). ( i i ) Benzylidene compound 60 was reacted with a solution (2ml) of 2$ (v/v) s u l f u r i c acid In 1«1 acetic acid-acetic anhydride mixture. After standing f o r 15h at room temperature,the re-action mixture was worked up as i n ( i ) above. ( i i i ) Compound 60 (200mg) was reacted with 2$ s u l f u r i c acid in acetic anhydride (2ml) f o r 5h at room temperature. Worked up as ( i ) . 143 (iv) Compound 60 (200mg) i n 2$ s u l f u r i c acid i n acetic anhy-dride (2ml) f o r 15h at room temperature. Worked up as ( i ) . (v) Compound 60 (200mg) i n 4$ s u l f u r i c acid i n acetic anhydride (2ml) f o r 15h at room temperature. (vi) Compound 60 (lOOmg) i n acetic anhydride (4ml), d i s t i l l e d boron t r i f l u o r i d e etherate (3 drops) was then added and the mixture was allowed to stand f o r 15h at room temperature. The reaction was worked up as described for ( i ) . ( v i i ) As ( v i ) , except that 10 drops of boron t r i f l u o r i d e etherate were added. ( v i i i ) As ( v i ) , except that the reaction was conducted at 40°. (ix) Compound 60 (200mg) i n 10$ s u l f u r i c acid i n acetic an-hydride (15ml) reacted f o r 15h at room temperature and worked up as ( i ) . (x) 60 (200mg) i n 2$ s u l f u r i c acid i n acetic anhydride was heated on a steam bath f o r 15h and worked up as i n ( i ) . Summary of r e s u l t s i Acetolysis conditions ( i ) to ( v i i i ) gave e s s e n t i a l l y one spot on t . l . c , the same as described f o r ( i ) . The p&m.r. spec-t r a (CDCl-j) of the isola t e d products indicated the presence of acetates (7.86, 7.86, 7.89,7.95, 3H singlets) and methoxy (6.45, 6.48 l£H singlets) signals i n the r a t i o of 4 i l . In ad-d i t i o n , two anomeric signals centered at 4.02 (fH, J 1 2 = 3.1 H ) and 4.10 (iH, J n 0 = 4.0 H ) were observed. Deacetylation Z » z 144 -(0.1M sodium methoxide i n methanol) of the product from ace-t o l y s i s and subsequent analysis by paper chromatography ( s o l -vent C) showed the presence of glucose only, i n d i c a t i n g that the methoxy i s present as a hemiacetal linkage. These data, and the p.m.r. data above, indicate that 6J3 i s an a c y c l i c hemiacetal which may have the structure shown i n 6^ (P» 5*0* Acetolysis conditions (ix) removed most of the methoxy, but a small amount remained. Conditions (x) however, removed a l l the methoxy, but the carbonate group was also replaced by acetates. Syntheses of a-D-glucosides by the 2 , 3-dibenzyl method Methyl 2 , 3-di - 0-benzyl - 4 , 6 - 0-benzylidene«a i-D-glucopyranoside 66 A mixture of methyl 4 , 6 - 0-benzylidene -a-D-glucopyranoside 59 ( l 4 g , 0 . 0 5 mole), ground potassium hydroxide (35g) and benzyl chloride ( 2 5 m l ) , 0 . 2 2 mole) i n xylene (225ml) was heated under refl u x f o r about 2 h . The reaction was followed by t . l . c . (solvent A, R f of j5£ 0 . 0 9 ; R f of product 8£, 0 . 7 9 ) . The mixture was c o-o l e d and decanted from the bulk of the potassium s a l t s . The s o l i d remaining i n the bottom of the f l a s k was washed with chloroform and the chloroform wash was combined with the xylene solution. The solution ( s t i l l basic) was ev-aporated at 7 0 ° and water (4 x 350ml) was added and evaporated at t h i s temperature (to remove benzyl alcohol). The product 66, which c r y s t a l l i z e d on concentration of the solution, was Ik 5 dissolved i n chloroform (300ml) and washed with water (3 x 75ml). Drying (sodium s u l f a t e ) , f i l t r a t i o n and concentration gave the c r y s t a l l i n e compound. R e c r y s t a l l i z a t i o n from hot absolute ethanol (100ml) gave 66, 14g, m.p. 97-98?. Processing of the ethanol f i l t r a t e gave an additional 2g of product. Methyl 2,3-di-0-benzyl-cuD-glucopyranoside'62 A mixture of methyl 2,3-di-0-benzyl-4,6-0-benzylidene-a-D-glucopyranoside 66 (l4g), acetone (180ml), water (75ml) and 1M hydrochloric acid (25ml) was heated under r e f l u x f o r about 2h (monitored by t . l . c ) . N eutralization ( s i l v e r carbonate), f i l t r a t i o n through carbon-Celite, and concentration, gave a syrup which c r y s t a l l i z e d from petroleum ether (250ml, b.p. 65-110°), y i e l d , lOg; R f 0.19 (solvent A): m.p. 77-78°. l,4,6-Tri-0-acetyl-2,3-di-0-benzyl-a-D-glucopyranose 68 Methyl 2,3-di-0-benzyl-a-D-glucopyranoside 62 (10g) was dissolved i n a mixture of acetic acid (100ml) and acetic an-hydride (25ml). The mixture was cooled to 0° and concentrated s u l f u r i c acid (1.5ml) i n acetic acid (15ml) was added drop-wise. A f t e r standing at room temperature overnight, the rea-t i o n mixture was poured into ice-water (250ml) and was extrac-ted with ether (3 x 150ml). The combined ether extracts were washed with water (3 x 100ml) saturated sodium hydrogen car-bonate ( u n t i l the acid was neutralized) and water (2 x 100ml). Drying, f i l t r a t i o n , concentration, and r e c r y s t a l l i z a t i o n from methanol gave 6.5g (50$) of product, m.p. 123-125°; ( l i t . (23) m.p. 125-126°). 146 2 , 3 - D i - O - b e n z y l - a - D - g l u c o s e 69 The t r i a c e t a t e 68 ( l O g ) i n d r y m e t h a n o l (30ml) was t r e a t e d w i t h 0.2M s o d i u m m e t h o x i d e i n m e t h a n o l (30ml) a t room t e m p e r a -t u r e f o r 45 m i n . N e u t r a l i z a t i o n ( A m b e r l i t e IR 120 H + r e s i n ) a n d e v a p o r a t i o n g a v e t h e p r o d u c t a s a s o l i d , w h i c h was r e c r y -s t a l l i z e d f r o m c h l o r o f o r m - e t h e r , y i e l d 5«0g (67$), m.p. 113-114°1 ( l i t . (23) m.p. 114 - 1 1 5 . 5 ° ) . 2 , 3 - D i - 0 - b e n z y l - a - D - g l u c o s e 6£ was a l s o p r e p a r e d b y h e a t i n g m e t h y l 2 , 3 - d i - 0 - b e n z y l - a - D - g l u c o p y r a n o s i d e 6£ (3g) a s f o l l o w s 1 i i n 1.5M s u l f u r i c a c i d (300ml) o n a s t e a m b a t h f o r 4hi most o f t h e s t a r t i n g m a t e r i a l d i d n o t r e a c t ; i i i n 0.5M s u l f u r i c a c i d h e a t e d u n d e r r e f l u x f o r 12h» some s t a r t i n g m a t e r i a l was l e f t u n r e a c t e d a n d some d e g r a d a -t i o n o f t h e p r o d u c t r e s u l t e d ; t h e p r o d u c t 6£ was i s o l a t e d i n p o o r y i e l d ( c a 25$). 2 , 3 - D i - O - b e n z y l - l , 4 , 6 - t r i - O - p _ - n i t r o b e n z o y l - 0 - D - g l u c o p y r a n o s e 70 2 , 3 - D i - 0 - b e n z y l - a - D - g l u c o s e 6£ (4.0g) was d i s s o l v e d i n d r y p y r i d i n e (40ml) and t o t h e c o o l e d s o l u t i o n d i s t i l l e d p.-n i t r o b e n z o y l c h l o r i d e (7.4g) was a d d e d w i t h s t i r r i n g . The m i x t u r e was a l l o w e d t o s t a n d a t room t e m p e r a t u r e o v e r n i g h t a n d was t h e n p o u r e d i n t o a s t i r r e d i c e - w a t e r m i x t u r e (300ml). The r e s u l t i n g p r e c i p i t a t e was f i l t e r e d , d i s s o l v e d i n e t h e r , a n d t h e e t h e r s o l u t i o n was s u c c e s s i v e l y w a s h e d w i t h w a t e r , s o d i u m h y -d r o g e n c a r b o n a t e - a n d w a t e r . F i l t r a t i o n and e v a p o r a t i o n g a v e a n a m o r p h o u s s o l i d w h i c h was r e c y r s t a l l i z e d f r o m e t h y l a c e t a t e -147 petroleum ether (b.p. 6 5 - 1 1 0 ° ) , y i e l d 6.5g, m.p. 1 7 3 - 1 7 6 ° ; [ a ] D -18° (c 2.3, dichloromethane); ( l i t . (23) m.p. 1 7 7 - 1 7 7 . 5 ° , [ a ] D -18 .3° (c 2, dichloromethane)).! 2,3-Di-0-benzyl-4,6-di-O-£-nitrobenzoyl-0-D-glucopyranosyl bromide 71 Saturated hydrogen bromide i n dichloromethane; t h i s was prepared by passing the gas through drying tubes containing magnesium perchlorate and D r i e r i t e connected to an empty trap (magnesium perchlorate i n contact with dichloromethane may re-act v i o l e n t l y ) which was i n turn connected to a 3-necked fl a s k containing the d r y - ( d i s t i l l e d from CaH 2) dichloromethane. The gas exit was protected by a drying tube containing D r i e r i t e . The £-nitrobenzoate £0 (2g) i n saturated hydrogen bromide i n dichloromethane (150ml) was allowed to stand at room tempera-ture f o r 2|h. The pre c i p i t a t e d p_-nitrobenzoic acid was f i l t e r -ed and the f i l t r a t e was evaporated below 35° (a drying tube con-t a i n i n g D r i e r i t e was inserted between the rotary evaporator and the remaining syrup was dried on an o i l pump at 0.5mm, a f t e r which the product was obtained as an amorphous s o l i d ; [°-] D (of 2 runs) + 1 7 ° , +26° (c 1.0, 0.7, dichloromethane); ( l i t . (23) [ a ] D + 1 0 . 2 ° (c 2.24, dichloromethane). In a separate experiment, the £-nitrobenzoate £0 (2g) was brominated as previously described. The mixture was concentra-ted, and portions of dichloromethane (10 x 20ml) were added and removed below 3 5 ° . F i l t r a t i o n of the o-nitrobenzoic acid and concentration gave a syrup; [cc]n +76° (c 1.6, dichlorome-148 thane). The high o p t i c a l r o t a t i o n of t h i s syrup £2 suggests that t h i s compound i s 2,3-di-0-benzyl-4,6-di-0-p_-nitrobenzoyl-a-D-glueopyranosyl bromide. 1,2, 3,4-Tetra-O-acetyl -6-O-trityl -0-D-glucopyranose 73 Compound 22 w a s prepared e s s e n t i a l l y as described by Rey-nolds and Evans (119). A mixture containing anhydrous D-glu-cose (18g, f i n e l y powdered), freshly prepared t r i t y l chloride (148) (29g) and anhydrous pyridine was heated on a steam bath under anhydrous conditions with occasional shaking u n t i l the sugar dissolved. I f a l l the sugar did not dissolve within a reasonable time (2h), the mixture was f i l t e r e d hot using a si n -tered glass funnel with suction. The f i l t r a t e was again heated on the steam bath f o r an additional 15 min. Without cooling, acetic anhydride (54ml) was added i n one portion (acetylation while hot favors the beta anomer) and the mixture was allowed to stand at room temperature overnight. The reaction mixture was slowly poured, i n a fin e stream, into a vigorously s t i r r e d solution of ice-water (2 l i t e r s ) and g l a c i a l acetic acid (? 5ml). The r e s u l t i n g mixture was mechanically s t i r r e d f o r 2h, keeping the temperature below 5 ° . The pr e c i p i t a t e was f i l t e r e d , then was vigorously s t i r r e d i n ice water (2 l i t e r s ) f o r 30 min. The granular p r e c i p i t a t e was f i l t e r e d , washed well with cold water, and a i r dried. The dried material was digested i n di e t h y l ether (75ml) to dissolve the alpha anomer and the insoluble portion (the beta anomer) was removed by f i l t r a t i o n . I f a l l of the s o l i d dissolved, the solut i o n was cooled i n order to 149 induce c r y s t a l l i z a t i o n . The 0-anomer was dissolved i n hot 95$ ethanol (ca400ml), decolorized with activated charcoal, and f i l t e r e d while hot. Compound 22 c r y s t a l l i z e d on cooling, and 17g (30$) pure product was obtained? m.p. 165-166°; ( l i t . (119) m.p. 166-166.5°). Workup of the ethanol f i l t r a t e gave an additional l-3g product. 1,2,3,4-Tetra-O-acetyl-0-D-glucopyranose • 74'.' Compound 22 (9g) was dissolved i n g l a c i a l acetic acid (40 ml) by warming on a steam bath and the solution was cooled to about 10°. Hydrogen bromide (32$) i n acetic acid (3.6ml) was then added and the mixture was shaken about 45 sec. The precip i t a t e d t r i t y l bromide was removed at once by f i l t r a t i o n and the f i l t r a t e was immediately poured into ice cold water (200ml). This was extracted with chloroform (3 x 30ml) and the combined chloroform extracts washed with water (4 x 30ml) and dried (sodium s u l f a t e ) . F i l t r a t i o n , evaporation and d i -l u t i o n with anhydrous ether gave the c r y s t a l l i n e product. Re-c r y s t a l l i z a t i o n from chloroform-anhydrous ether gave pure 74, (3g, 56$); m.p. 127-128°; [<x]D +12° (c 2.0, chloroform); ( l i t . (119) m.p. 128-129°, [a]D +12.1° (chloroform)). 6-0-a-D-Gluc opyrano syl-D- glucose (isomaltose) 22 A mixture of l,2,3,4-tetra-O-acetyl-0-D-glucopyranose 74 (lOOmgi 0.29 mmol) and mercuric cyanide (125mg, 0.49 mmOl) i n absolute nitromethane (5ml) was s t i r r e d at room temperature with the exclusion of moisture. Excess bromide 71 (crude, 150 ca 500mg, 0.5 mraOl) was added i n 3 portions and the mixture was s t i r r e d f o r 5h. Workup and deacylation gave a product of R f 0.26 ( t . l . c . solvent B) corresponding to 6-0-(2,3-di-0-benzyl-a-D-glucopyranosyI)-D-glucose P u r i f i c a t i o n hy pre-parative t . l . c . gave a syrup (58mg, 3 9 $ from 2ih)» Hydrogena-t i o n of the syrup (50 p . s . i . , 10$ Pd/C (lOOmg), ethanol, room temperature overnight) gave a syrup (40mg) containing isomaltose (paper chromatography, £ g i u c o s e 0.14, solvent C), gentiobiose ( £ g i u c o s e 0»°3» estimated by paper chromatography to be l e s s than 5$)i and a small amount of unreacted £6 ( t . l . c . solvent B). Isomaltose (lOmg) was acetylated (sodium acetate catalyst) to give a syrup which on seeding gave beta isomal-tose octaacetate 2§» m.p. 143-145°$ [ct] D +94° (c 0.8, chloro-form)} ( l i t . (16) m.p. 144-145°, [ a ] D +95° (c 1.0, chloroform)) 6-0-a-D-Glue opyrano syl-D-galac to se 80 l,2i3,4-Di-0-isopropylidene-a-D-galactopyranose 10 (100 mgj 0.38 mmol), mercuric cyanide (200mg, 0.8 mmol) and excess bromide 7jL ( c a 800mg, 0.8 mmol) were s t i r r e d i n absolute n i t r o -methane (8ml) at room temperature f o r 5h. The mixture was worked up (p. 164), p u r i f i e d by preparative t . l . c , deacyla-ted (0.2M sodium methoxide), the isopropylidene groups re-moved (0.025M s u l f u r i c acid at 100° for 30 min), and hydroge-nated (50 p . s . i . , 10$ Pd/C, ethanol, overnight at room tempera-tu r e ) . The r e s u l t i n g mixture containing 80 was p u r i f i e d by preparative paper chromatography (solvent C) giving 80, 32mg; ^glucose 0 , 2 3 5 £ a - t ) + 1 1 2 ° ( ^ °«3,water); ( l i t . (25) [ a ] D + 1 2 3 ° 151 (c 1.0, water ) ) • In a separate experiment, 2 ,3-di-0-benzyl-4 ,6-di-0-p_-ni-trobenzoyl-a-D-glucopyranosyl bromide £2 (900mg) was condensed with 10 (260mg) as described above, except that the reaction was c a r r i e d out f o r 3 days. Workup gave a mixture containing the alpha and beta forms of 6-0-(2,3-di-0-benzyl-4,6-di-0-JJ-nitrobenzoyl-D-glucopyranosyl)-l , 2.0»4-di - 0-isopropylidene-a-D-galactose. The two products were separated by preparative t . l . c . using benzene-ether (4«l) and the p.m.r, spectra ob-tained confirmed that they were the disaccharide products, a-Anomer (71/0? R f 0.^9; [ a ] D +27° (c 1.9, chloroform); 0-anomer (29$) i Rj. 0,40; [ a ] D - 3 ° (c 1.3, chloroform);other components of Rf, 0.19, 0.25, and O.65 were also detected. The y i e l d of a-anomer was 270mg. Syntheses of a-D-glucosides by the 2-benzyl method 3,4 , 6-Tri-O-acetyl-l-deoxy-l-piperidino-0-D-glucopyranose 81 Penta-O-acetyl-0-D-glucopyranose 2 (117g, 0,3 mole) and piperidine (90ml, dried and d i s t i l l e d from sodium hydroxide p e l l e t s , b.p. 1 0 5 - 1 0 6 ° ) were s t i r r e d i n a fl a s k . In about 5 min, when warming was detected, the mixture was cooled i n an ice-water bath at in t e r v a l s , i n order to keep the internal tem-perature at 25± 5 ° . S t i r r i n g was continued u n t i l a l l the sug-ar dissolved and the mixture became f l u i d (ca 10 min). Minutes l a t e r , when c r y s t a l l i z a t i o n began, d i e t h y l ether (100ml) was added with good s t i r r i n g . The reaction mixture was then cool-152 ed f o r l h at 0 ° . The c r y s t a l s were f i l t e r e d , a i r dried f o r 5 min, s t i r r e d i n absolute ethanol (150ml, to remove piperidine acetate) f o r 10 min, f i l t e r e d again, and washed with absolute ethanol (75ml) and with ether (50ml); y i e l d 33g. The o r i g i n a l ether f i l t r a t e (ca 100ml) was kept overnight at 0 ° , f i l t e r e d and the s o l i d was s i m i l a r l y s t i r r e d i n absolute ethanol, f i l -tered, and washed with absolute ethanol and ether; y i e l d 5 - 1 ° g. The combined product (38-43g) had m.p. 124 -125° ; [ a ] Q +31° (c 2.0 chloroform); ( l i t . (Ilk) m.p. 1 2 5 ° , [<x]n + 3 1 . 6 ° (c 4.0, chloroform)). 3 , 4 , 6-Tri-O-acetyl - 2-O-benzyl-l-deoxy-l-piperidino-0-D-gluco-pyranose 82 3 , 4 , 6-Tri - 0-acetyl-l-deoxy-l-piperidino-g-D-glucopyranose 81 (35g), f r e s h l y prepared powdered dry s i l v e r oxide (40g) and ground D r i e r i t e (30g) were s t i r r e d i n dry benzene (250ml) f o r 30 min i n the dark at room temperature with exclusion of mois-ture. The mixture was cooled to about 15° and benzyl bromide (13ml, 0.2 molar excess) was added. After s t i r r i n g at room temperature for 3-§h t . l . c . (solvent A) indicated that the re-action was complete. The mixture was f i l t e r e d , the s a l t s were washed with benzene and the combined solvents were evaporated, a f t e r which the product 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 hot methanol (3ml/g) gave pure 3 , 4 , 6-tri - 0-acetyl - 2 - 0-benzyl-l-deoxy-l-piperidino-0-D-glucopyranose, 38.7g (89$), m.p. 99-1 0 0 ° ; [ a ] D +42° (c 2.2, methanol);(lit. (115) m.p. 1 0 0 ° , [ a ] D +41.5° (c 0.9, methanol)). The p.m.r. (CDCl^) spectrum 153 included aromatic (2.78, 5H-singlet) and acetates (8,01, 8,07. 8,15) signals. 2-0-Benzyl-D-glucose 84 Compound 82 (35g) was treated with 2$ sodium methoxide i n anhydrous methanol (300ml) at room temperature. T . l . c . (solvent A) showed that the reaction was complete within 30 min. Sulfu-r i c acid (2M, about 100ml) was added u n t i l the a c i d i t y of the solution was pH 3 and the mixture was refluxed f o r l h (116). A f t e r n e u t r a l i z a t i o n (barium carbonate) and concentration 2-0-benzyl-D-glucose 84 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 hot methanol (lOml/g) gave pure 84 (l6g), m.p. 1 7 6 - 1 7 7 ° , Of] + 4 7 ° (c 2, methanol); ( l i t . (115,116) m.p. 176-177? 0 ] D + 4 7 ° (c 1.0, methanol)). 2-0-Benzyl-l.3»4.6-tetra-0-£-nitrobenzoyl-a-D-glucopyranose 87 Ground p_-nitrobenzoyl chloride (37g, 0.2 moles, prepared from the acid by the thion y l chloride method (147), then d i s t i l l e d at 1 9 5 ° , 15mm, dried i n vacuo over phosphorus pentoxide) was & added to cooled pyridine (200ml, dried and d i s t i l l e d from po-tassium hydroxide p e l l e t s ) with good s t i r r i n g . 2-0-Benzyl-D-glucose (lOg, 0.037 moles) was slowly added with s t i r r i n g while keeping the temperature below 2 0 ° . T . l . c . (solvent B) showed that the reaction was complete within 2h, the product having an Rj, of 0.90, the s t a r t i n g material 0.34. The mixture was poured i n a fine stream into a s t i r r e d , cold solution of 50$ sodium hydrogen carbonate (1200ml). The pre c i p i t a t e was f i l t e r e d , washed with water, dissolved i n chloroform, and dried (sodium s u l f a t e ) . F i l t r a t i o n , concentration and d i l u t i o n with petroleum ether 154 (b.p. 30-60°) gave the crude product (30g) which was recry-s t a l l i z e d from chloroform-ethyl acetate? 94$ m.p. 225-227°; [ a ] D +59° (c , 1 . 5 , dichloromethane); ( l i t . (23) m.p. 227-228°, [cf ] D + 6 0 . 1 ° (c 1.43, dichloromethane)). The p.m.r. spectrum ( 6 0 M H z , CDCl^) included a p_-nitrobenzoate multiplet (1.6-2 .1, l 6 H * s ) , a benzyl si n g l e t (2.88, 5H*s) and an anomeric doublet (3.23, 1H, J l f 2 = 3.2). 2-0-Benzyl-3»4 ,6-tri-O-p_-nitrobenzoyl-0-D-glucopyranosyl bromide 88 Compound 8£ (15g) was dissolved i n a saturated solution of hydrogen bromide i n absolute dichloromethane ( 7 0 0 m l ) and was allowed to stand under anhydrous conditions f o r 6h at room temperature. The mixture was f i l t e r e d and the f i l t r a t e was evaporated at 3 5 ° , then dried on a vacuum pump for 30 min, during which time c r y s t a l l i z a t i o n occured. The product was r e c r y s t a l l i z e d from dry dichloromethane-anhydrous ether; y i e l d , 4.2g; m.p. 142-143°; [ a ] D +3° (c 2 . 0 , dichloromethane); ( l i t . (23) m.p. 143-144°, [ a ] D + 2 . 4 ° (c 2 . 1 , dichloromethane)). On some occasions 88 c o - c r y s t a l l i z e d with an impurity (pro-bably the alpha anomer) which was hard to separate by crysta-l l i z a t i o n . This impurity however, did not in t e r f e r e with the condensation step. The bromide was stored at room temperature i n a vacuum desiccator over phosphorus pentoxide, or at 0 ° i n a stoppered f l a s k . 6-0-(2-0-Benzyl-ct-D-glucopyranosyl)-D-glucose £1 155 To a soluti o n of l,2 , 3 » 4-tetra-O-acetyl - 0-D-glucopyranose 74 (lg» 2 .87 mmol) and mercuric cyanide (1.3g» 5.1 mmol) i n absolute nitromethane (50ml, d i s t i l l e d from calcium hydride) was added excess 2 - 0-benzyl - 3 » 4 , 6-tri - 0-p_-nitrobenzoyl-g-D-glucopyranosyl bromide 88 (4g, 5.1 mmol). The reaction mix-ture was s t i r r e d under anhydrous conditions at 40-45° f o r 3 days. Examination of the mixture by t . l . c . (solvent F) showed the following values 1 tetraacetate 74 0 . 0 3 , bromide 88 0.16, product 90 0 . 1 9 . A small amount of tetraacetate 74 remained unreacted. The solvent was removed and the syrup obtained was diluted with chloroform (100ml). The chloroform solution was washed successively with water ( 2 x 100ml) saturated sodium hydrogen carbonate 3 x 100ml) and water ( 2 x 100ml), then dried (sodium s u l f a t e ) . F i l t r a t i o n and evaporation gave a syrup, a small portion (60mg) of which was p u r i f i e d by preparative t . l . c . using solvent F. The p.m.r. spectrum of the pure material i n -dicated the presence of p_-nitrobenzoates (1.60-2.65, 12H mul-t i p l e t ) , benzyl (2 , 8 3 , 5H s i n g l e t ) , and acetates (7.97, 7.98, 8.01, 8.02, 3H s i n g l e t s ) . The bulk of the condensation mix-ture was dissolved i n dichloromethane (25ml) and 0.2M sodium methoxide i n anhydrous methanol (50ml) was added to i t . Neu-t r a l i z a t i o n (Amberlite IR 120 H + resin) a f t e r 30 min and con-centration gave a syrup containing ( t . l . c , solvent B) £1 (R f 0 .06), 2 -0-benzyl-D-glucose (R f 0.34) and glucose (R f 0 . 0 ) . Methyl p_-nitrobenzoate was separated by dis s o l v i n g the syrup i n water (30ml) and washing with chloroform (4 x 20ml). The water layer (containing the disaccharide) was then evaporated 156 under reduced pressure. The r e s u l t i n g mixture was chromato-graphed on a short column (120) (4cm x 25cm, l60g s i l i c a gel G, 20ml f r a c t i o n s c o l l e c t e d every 4 to 5 min) using solvent B. Fractions 21-60 were heterogeneous,61-180 contained the d i -saccharide intermediate 9_1 (600mg) 48$ from ?4], 1,2,3,4-Tetra-O-ac etyl-6 - 0 - ( 2 - 0-benzyl-3 »b , 6 -tri - 0-ac etyl-a-D-glucopyrano syl) -D-glucopyranose 92 Compound 9_1 (50mg) was treated with acetic anhydride (3ml) and anhydrous sodium acetate (lOOmg). After heating on a steam bath f o r 2h, the mixture was cooled and s t i r r e d i n ice water (20ml) f o r 3° min. The aqueous mixture was extracted with chloroform (3 x 20ml) and the combined chloroform extracts were washed with water (2 x 20ml), 50$ sodium hydrogen carbo-nate (2 x 20ml) and water. Drying (sodium sulfate) of the chloroform solution, f i l t r a t i o n and concentration gave a syrup. Traces of acetic anhydride were eliminated by adding 10ml por-tions of ethanol and removing. The product 92 was p u r i f i e d by preparative t . l . c . using solvent A (R f 0.35); C°0TJ +66° (c 1 .5t chloroform). The p.m.r. spectrum of 92 showed the pre-sence of one benzyl group and seven acetates. 6-0-a-D-Glucopyranosyl-D-glucose 77 (isomaltose) 6-0-(2-0-Benzyl-2-D-glucopyranosyl)-D-glucose 9_1 (475mg) i n absolute ethanol (15ml) was hydrogenated using 5$ palladium on carbon (lg) as ca t a l y s t and a hydrogen pressure of 50 p . s . i . A f t e r shaking at room temperature overnight the reaction mixture 157 was f i l t e r e d and concentrated to give 72 as a syrup, 390mg„ Paper chromatographic analysis showed a large spot correspond-ing to isomaltose ( £ g i u c o s e 0.14, solvent C), and a f a i n t spot (le s s than 5$» which could not be detected by p.m.r.) corres-ponding to 6-0-g-D-glucopyranosyl-D-glucose (gentiobiose). After drying ( 6 5 ° , 1mm, J>2 Q^  the syrup gave Lalj) + 9 5 ° (c 1.0, water)? ( l i t . (149,150) [ a ] D +120° (c 1.2, water). The p.m.r. spectrum (DgO, external TMS) of 22 showedt 4.74 (doublet, 2 * 3 . 5 H , a form of reducing end), 5.29 (doublet, J, 0 = 7.2 H , 0 form of reducing end} the r a t i o of beta to alpha form was about 2), 5.02 (IH doublet, J 1 2 = 3 . 5 H^, H-l of non-reducing end, alpha form). g-Isomaltose octaacetate £8 A mixture of isomaltose (50mg), anhydrous sodium acetate (lOOmg) and acetic anhydride (4ml) was heated on a steam bath fo r 2h. The mixture was worked up as described f o r £2 and puri-f i e d by preparative t . l . c . using solvent A (R f 0.31) or solvent B (R f O . 7 6 ) , to give a syrup (84mg) having [ a ^ +80° (c 4.0, chloroform). This c r y s t a l l i z e d from ethanol on seeding with authentic c r y s t a l s of £8 and was r e c r y s t a l l i z e d from the same solvent; m.p, and mixed m.p. 145-146°; [ a ] Q +95° (c 1 . 3 , c h l -oroform); ( l i t . (16) m.p. 144-145°, [ a ] D +95° (c 1.0, chloro-form) ). 1,2,3»^-Tetra-0-acetyl-6-0-trityl-@-D-mannopyranose £ 2 F i n e l y ground D-mannose (12g), f r e s h l y prepared t r i t y l chloride (19.3g) and dry pyridine (50ml) were heated on a steam 158 bath u n t i l the sugar dissolved (15 min) and f o r an additi o n a l 45 min. Without cooling, acetic anhydride (36ml) was added at once and the reaction was allowed to stand at room tempera-ture overnight. The mixture was slowly poured (ca 45 min) into ice-water (1 l i t e r ) which was vigorously s t i r r e d (mechanical s t i r r e r ) . The mixture was s t i r r e d f o r l i h , keeping the tem-perature below 5 ° . The pr e c i p i t a t e was f i l t e r e d , s t i r r e d for 45 min i n i c e - c o l d water (1 l i t e r ) , and again f i l t e r e d . The s o l i d product was s t i r r e d f o r a t h i r d time with ice water (1 l i t e r , 45 min), f i l t e r e d , washed with cold water, and a i r dried (ca 45 min). I t was then dissolved i n hot 95$ ethanol (ca 300ml), passed through charcoal, and allowed to c r y s t a l l i z e . The f i l -tered product was f i r s t a i r dried, then dried i n a vacuum desi-ccator over phosphorus pentoxide, y i e l d , l8g. R e c r y s t a l l i z a t i o n from ethanol gave the pure compound, m.p. 202-204°; ( l i t . (121) m.p. 204 - 2 0 6 ° ) . 1 *2,3»4-Tetra -0-acetyl-g-D-mannopyranose £4 This was prepared from £2 as described f o r the glucose analog, lOg £2 gave 4g 1,2,3t4-tetra-O-acetyl-0-D-mannopyranose, m.p. 133-135°$ ( l i t . (121) m.p. 1 3 5 . 5 - 1 3 6 . 5 ° ) . 6-0-(2-0-Benzyl-a-D-glucopyranosyl)-D-mannose £6 A mixture of 1,2,3,4-tetra-O-acetyl-g-D-mannopyranose £4 ( l g , 2.87 mmol), mercuric cyanide (1.3g, 5.1 mmol), and bromide 88 (4g, 5.1 mmol) i n absolute nitromethane (50ml) was s t i r r e d under anhydrous conditions at 40° f o r two days. T . l . c . ( s o l -vent F) gave R.p values as follows; tetraacetate £4 0.03, bro-159 mide 88 0.16 (main spot) and 0.70, 1,2,3.4-tetra-0-acetyl -6-0-(2@0-benzyl -3,4 ,6-tri-0-p_-nitrobenzoyl-a-D-glucopyranosyl)-3 -D-mannopyranose 21 °»19. The reaction mixture was worked up as described under £1. A small portion (40mg) of 21 w a s is o -l a t e d by preparative t . l . c . and the p.m.r. spectrum indicated the presence of benzyl, p_-nitrobenzoate and acetate groups, The bulk of the condensation mixture was deacylated (0.2M sodium methoxide i n methanol), neutralized (Amberlite IR 120 H + r e s i n ) , concentrated, dissolved i n water (30ml) and washed with chloroform (4 x 20ml). The syrup obtained a f t e r concentra-t i o n of the aqueous layer was again chromatographed on a short column (170g s i l i c a ) but using chloroform-methanol(3il) as s o l -vent (faster than solvent B). Fractions (20ml) were c o l l e c t e d every 5 min, f r a c t i o n s 21-70 contained impurities, 71-80 con-taminated £6 and 81-140 contained £ 6 (550mg, 44$ from 94). 1.2,3,4-T etra-0-ac etyl-6-0-(2-0-benzyl-3,4,6-tri-0-ac etyl-a-D-glucopyranosyl)-D-mannopyranose 92 A portion (40mg) of the i s o l a t e d 6-0-(2-0-benzyl-a-D-glucopyranosyl)-D-mannose £6 was acetylated as described for the isomaltose analog £2, Workup and p u r i f i c a t i o n by preparative t . l . c . (solvent A R f 0.35) gave a syrup with [ a ] D +69° (c 2 .3, chloroform). The p.m.r. spectrum (CCl^) of £ 2 showed: 2.73 (5H sin g l e t , from benzyl), 4.00 (doublet, J, 0 = 2.2 H , H-l of a-D-form of the reducing mannose residue), 4.17 (doublet, J, 1.7 H , H-l of P-D form of the reducing mannose residue, the ra t i o of alpha to beta form of the mannose residue was about 160 3 ) , 7.80-8.10 (21H's, ©Ac's). 6-0-a-D-Glucopyranosyl-D-mannose 98 6-0-(2-0-Benzyl-a-D-glucopyranosyl)-D-mannose (197mg) i n absolute ethanol (10ml) was hydrogenated at 50 p . s . i . at room temperature f o r 2 days using 5$ palladium on carbon (lg) as cat a l y s t . F i l t r a t i o n and concentration gave a syrup (154mg) O F M D + ? I * ' 0 (£ °«5» water); B G L U C O S E ° « 2 1 (solvent C); p.m.r. (D 20, external TMS) 4.83 (doublet, J l j 2 - 1.7 H z, H-l of a-D form of the reducing mannose residue), 5.H (doublet, 2 1.0 H , H-l of 0-D form of the reducing mannose residue), 5-05 z (1H doublet, 2 » 3 . 2 H 2, H-l of a-D form of the non-re-ducing glucose residue). 112,3,4-Tetra-0-ac etyl-6-0- (2 ,3 ,4 ,6-tetra-O-acetyl-a-D-glucopy-ranosyl)-D-mannopyranose 99 6-0-a-D-Glucopyranosyl-D-mannose £8 (50mg) was heated on a steam bath for 2h i n acetic anhydride (4ml) and anhydrous sodium acetate (lOOmg). Workup and p u r i f i c a t i o n by prepara-t i v e t . l . c . using solvent A (R^ , 0.31) gave a syrup (88mg), [ a ] D +74° (c 3.1, chloroform); p.m.r. (CDCl^) 3.99 (doublet, Jlf2 = 2 , 0 H z ' H"" 1 o f a " D f o r m o f t h e m a n n o s e residue), 4.19 (doublet, J, 0 - 1.0 H , H-l of the 0-D form of the mannose X f c Z residue, the r a t i o of alpha to beta form was about 3), 7.85-8.04 (24H's, O A C ' S ) . Methyl a-L-Rhamnopyranoside 100 L-Rhamnose monohydrate (30g) was dissolved i n 1.5$ 161 (300ml, prepared by addition of 7.5ml of acetyl chloride to 300ml dry methanol) methanolic hydrogen chloride and the so-l u t i o n was refluxed f o r 2h on a steam bath. The mixture was cooled, neutralized with lead carbonate, f i l t e r e d , and eva-porated to a syrup. Residual methanol was removed at 1mm or by adding portions of ethyl acetate (3 x 25ml) and evaporating on a rotary evaporator. The remaining syrup was dissolved i n ethyl acetate (45-50ml), seeded, and l e f t overnight. The cry-s t a l s were f i l t e r e d , washed with 2-4ml of cold ethyl acetate, and a i r dried; y i e l d , l4g. The f i l t r a t e was concentrated to about h a l f volume and seeded. F i l t r a t i o n a f t e r 24h gave an additional 4-6g of product. R e c r y s t a l l i z a t i o n of the crude product (20g) from hot ethyl acetate (80ml) with seeding gave the pure compound (ca I8g 61$), m.p. 108-109°; [<*]D -60° (c 8.2, water); ( l i t . (127) m.p. 108-109°, [a]D -62.5° (c 9.1 water)). The p.m.r. spectrum included a one hydrogen doublet at 5.35 ( J i t 2 " H z ^ ' a m e " t n o x y singlet at 6,63 and a methyl doublet at 8.74 ( J ^ 6 = 6 H 2). Methyl 2,3-0-isopropylidene-a-L-rhamnopyranoside 101 Methyl a-L-rhamnopyranoside (lOg), dry acetone (200ml), anhydrous cupric sulfate (20g, dried at 150° overnight at 10mm) and concentrated s u l f u r i c acid (0.2ml) were s t i r r e d at ambient temperature (about 32°, due to warming by the magnetic s t i r r e r ) f o r 20-24 hours under anhydrous conditions. The mixture was neutralized with calcium oxide, f i l t e r e d , and evaporated to a syrup under reduced pressure. Attempted p u r i f i c a t i o n by d i s -162 t i l l a t i o n at 110-115° and 1mm pressure as described by Levene and Muskat (12?) (but adding about 50mg of sodium hydrogen carbonate i n order to prevent deacetalation) gave 101 contami-nated with methyl rhamnoside. Pure 101 was obtained without d i s t i l l i n g by deacetylation of the c r y s t a l l i n e 4-acetate (125) described below. The mixture containing methyl 2,3-0-isopropylidene -a-L-rhamnopyranoside was acetylated i n pyridine (35ml) and acetic anhydride (35ml) at room temperature f o r 2h. The mixture was poured into ice water, s t i r r e d , and extracted with ether (ca 300ml) . The combined ether extracts were washed with cold water (3 x 40ml) and dried (sodium s u l f a t e ) . F i l t r a t i o n and evaporation gave c r y s t a l l i n e methyl 4-O-acetyl-2,3r0-isopropy-lidene-a-L-rhamnopyranoside 102 which was dissolved i n hot ethanol and f i l t e r e d ( i f necessary) through activated charcoal. On cooling, c r y s t a l l i z a t i o n occurred. The c r y s t a l s were f i l -tered and washed with a small amount of petroleum ether (b.p. 30-60°), y i e l d 8g. Processing of the ethanol f i l t r a t e gave an additional 2g. The product was dried i n a vacuum over phosphorus pentoxide, R f O.69 (solvent B); m.p. 66-67° ; [ a ] D -16.4° (c 2.0, chloroform); ( l i t . (125,127) m.p. 66-67° , [a]D -16 .5° (c 2.4, chloroform); p.m.r. (CDCl-j): 5.10 (IH s i n g l e t , H-l of a-L«form), 6.62 (3H s i n g l e t , 0CH 3), 7.92 (3H s i n g l e t , OAc), 8.44, 8 .66 (3H s i n g l e t s , isopropylidene CH^), 8.83 (3H doublet, J - A - 6 H C H J . A l t e r n a t i v e l y , the mixture containing methyl 2 , 3-0-iso-propylidene-a-L-rhamnopyranoside (as above, obtained from lOg 163 of methyl rhamnoside) was also acetylated i n acetic anhydride (35ml) and anhydrous sodium acetate (4g) "by heating on a steam bath f o r 3h. The reaction mixture at the end of t h i s period was considerably darker than that obtained by the pyridine method, probably due to the presence of small amounts of copper ions. The cooled mixture was dilu t e d with chloroform (100ml) and washed with water (2 x 25ml, to remove sodium acetate and any remaining copper s u l f a t e ) . Ethanol (25ml) was added to the chloroform layer and the mixture was evaporated. Excess acetic anhydride was then removed (as ethyl acetate) by evaporation with portions of ethanol. Methyl 4 - 0-acetyl-2 , 3 - 0-isopropyli-dene-a-L-rhamnopyranoside 102 c r y s t a l l i z e d spontaneously and was r e c r y s t a l l i z e d from hot ethanol j y i e l d , 9.6g; m.p. 66-67°. The acetate 102 obtained above (1.3g) i n dry methanol (10ml) was deacetylated with 0.2M sodium methoxide i n anhydrous meth-anol (20ml) at room temperature f o r 30 min. The reaction was followed by t . l . c . (solvent A). The mixture was cooled and c a r e f u l l y neutralized with Amberlite IR 120 H + r e s i n , (pre-washed with methanol) which was immediately f i l t e r e d as soon as (or just before) n e u t r a l i z a t i o n was reached (or hydrolysis of the l a b i l e isopropylidene group may occur). Evaporation of the methanol gave methyl 2 ,3-0-isopropylidene-a-L-rhamnopyr-anoside 101 as a syrup, l . l g ; R^ (solvent A) 0.53; C°0D - 1 6 ° (c 2.0, acetone); ( l i t . (12?) [ a ] D - 1 5 . 9 ° (c 1.6, acetone)); p.m.r. (CDCl^Ji 5.14 (1H sin g l e t , H - l ) , 6.62 (3H s i n g l e t , 0 C H 3 ) , 8.48, 8.65 (3H s i n g l e t s , isopropylidene C H 3 ) , 8.69 (3H doublet, J 5 ( $ " 6 Hz, CH3). 164 Methyl 4-0-(2-0-benzyl-3,4,6-tri-0-jj-nitrobenzoyl-a-D-gluco-pyrano syl)-2,3-0-isopropylidene-a-L-rhamnopyranoside 103 To a solution of methyl 2,3-0-isopropylidene-a-L-rhamnopy-ranoside 101 (0.78g, 3.58 mmol) and mercuric cyanide (1.08g, 4.3 mmol) i n nitromethane (50ml, d i s t i l l e d from calcium hydride) was added 2-0-benzyl-3,4,6-tri-O-p_-nitrobenzoyl-0-D-glucopyrano-syl bromide 88 (3.^g k.3 mmOl) and the mixture was s t i r r e d at 40° with exclusion of moisture. A f t e r 5h t . l . c . (benzene ether ( 9 s i ) ) showed a small amount of unreacted 101 (R^ 0.06, did not decrease sub s t a n t i a l l y on the addition of more bromide and cyanide) and a large yellow-brown spot (R^ 0.33) corres-ponding to the f u l l y blocked disaccharide 103. In addition, a f a s t e r moving component 104 (R f O.56) was detected. The p.m.r. spectrum of a sample obtained by preparative t . l . c . i n -dicated the presence of one benzyl group and three p_ - n i t r o -benzoate groups., The reaction solvent was removed and the re-maining syrup was dissolved i n benzene (100ml) and washed suc-cessively with water, sodium hydrogen carbonate and water, then dried (calcium s u l f a t e ) . F i l t r a t i o n and evaporation of the solvent gave a syrup which was p u r i f i e d on a 60cm x 7cm s i l i c a gel pressure column (120) (I80g s i l i c a gel, 20ml fractio n s c o l l e c t e d at 90 sec intervals) using benzene-ether (9«D as solvent. Fractions 50-70 contained the disaccharide 103» The combined fracti o n s gave a syrup (2.65g, 80$) which was homoge-neous on t . l . c , [ a ] D +65° (c 2.0, chloroform)? p.m.r. (CCl^)j 1.6-2.1 (12H, p.-nitrobenzoates), 2.8-3.0 (5H, phenyl), 6.65 (3H s i n g l e t , 0CH ?), 8.43, 8.67 (3H s i n g l e t s , isopropylidene 165 CH,), 8.70 (3H doublet, J w = 6 H . CH,). 3 5t & z 3 Methyl 4-0-(2-0-benzyl-a-D-glucopyranosyl)-2,3-0-isopropyli-dene-a-L- rhamnopyranoside 105 The f u l l y blocked disaccharide 103 (1 .5g) was refluxed i n a mixture of potassium hydroxide (6g) i n water(15ml) and ethanol (60ml). Afte r about 90 min ( t . l . c . solvent B) the spot corresponding to s t a r t i n g material (R f 0.84) was replaced by a large product spot (R f 0 .74) corresponding to 105 and a f a i n t spot moving s l i g h t l y f a s t e r (R f 0 . 7 6 ) ; t h i s was more conveniently i d e n t i f i e d a f t e r hydrogenolysis. The cooled s o l -ution was neutralized with dry ice and the solvent was removed by evaporation. Residual water was removed by azeotroping with dry benzene or ethanol. The disaccharide was then extracted from the insoluble s a l t s by r e f l u x i n g with d i e t h y l ether (3 x 3 0 0 m l ) . The syrup obtained c r y s t a l l i z e d a f t e r seeding with a sample obtained by preparative t . l . c , y i e l d 565mg (75%)• A l -t e r n a t i v e l y 105 was obtained by d i s s o l v i n g the f u l l y blocked disaccharide 103(lg) i n chloroform (50ml) and adding 0.2M sod-ium methoxide i n anhydrous methanol (40ml). After l h at room temperature the solution was neutralized with dry ice or, while cold, with Amberlite IR 120 H + r e s i n . In the l a t t e r case, the r e s i n must be removed as soon as the mixture i s neutral i n order to avoid deacetalation. P u r i f i c a t i o n by preparative t . l . c gave c r y s t a l l i n e 105 which was r e e r y s t a l l i z e d from 2-propanol (12ml/g), m.p. 158 - 1 5 9 ° ; [a]D +71.5° (c 1.8, methanol); p.m.r. (CDC1~« 2 .68 (5H, phenyl), 5 .04 (1H doublet, J. 0 - 3 . 7 , H-l of 166 a-D form of the glucose u n i t ) , 5.18 (1H sing l e t , H-l of a-L form of the rhamnose u n i t ) , 6.6? (3H s i n g l e t , QCH^), 8.50, 8.68 (3H s i n g l e t s , isopropylidene CH^), 8.70 (3H doublet, J<- g = 6 H z, rhamnose CH^). Anal. Calcd. f o r C^H^O^: C, 58.69: H, 7.29. Foundi C, 58.36; H, 7.34. Methyl 4-0-a-D-glucopyranosyl-2,3-0-isopropylidene-a-L-rhamno-pyranoside 107 Methyl 4-0-(2-0-benzyl-a-D-glucopyranosyl)-2,3-0-isopro-pylidene-a-L- rhamnopyranoside 105 (400mg, m.p. 158 -159°) was hydrogenated with 5% palladium on carbon (lg) i n absolute ethanol (15ml) at 50 p . s . i . at room temperature f o r l6h. A f t e r f i l t r a t i o n , washing of the carbon with ethanol and evaporation of the solvent, a chromatographically pure syrup 107 (320mg) was obtained; [ a ] Q +73° (c2.0, methanol); p.m.r. 6.64 (3H s i n g l e t , 0CH 3),8 .50, 9,68 (3H s i n g l e t s , isopropylidene CH^). A portion (30mg) of 107 i n pyridine (2ml) was treated with hexamethyldisilazane (0.2ml) and chlorotrimethylsilane (0.1ml) (100). A f t e r standing f o r 5 min, the solvent and excess rea-gents were removed. Traces of reagents were removed by ad-ding portions of dry hexane and evaporating. The p.m.r. spec-trum of the t r i m e t h y l s i l y l derivative 109 (131) thus obtained showed (benzene -dg, external TMS)1 5.03 (1H doublet, 2 -4 H , H-l of the glucose u n i t ) , 5.11 (1H ;singlet, H-l of the rhamnose u n i t ) , 7,.0 (3H s i n g l e t , OCH^), 8.40, 8.76 (3H s i n g l e t s , isopropylidene CH,), 8.56 ( 3 H doublet, J, 0 • 6 H , rhamnose 167 CH 3), 9.65, 9.68, 9.78, 9.90 (9H s i n g l e t s , 4 t r i m e t h y l s i l y l groups). S i m i l a r l y , a syrupy sample (30mg) of methyl 4-0-(2-0-benzyl-a-D-glueopyranosyl) -2*3-0-isopropylidene-a-L-rhamnopyra-noside 105 containing a small amount of a fas t e r moving com-pound was hydrogenolyzed giving 107 (Rf 0.34, solvent B) and a second component 108 (R f 0.43), which was isolat e d hy pre-parative t . l . c . (solvent B) (lmg of 108 was obtained). The p.m.r. spectrum (Fourier transform) of 108 was sim i l a r t o ; that of 10?, but both spectra were unclear except f o r the me-thoxy and isopropylidene signals. However, the p.m.r. spectrum (Fourier transform) of the t r i m e t h y l s i l y l derivative of 108 (compound 110) showed (benzene-d^, external TMS)i 4.86 (IH doublet, J 1 > 2 - 7.3 H z, H-l of the glucose u n i t ) , 5.06 (IH sin g l e t , H-l of the rhamnose u n i t ) , 6.92 (3H s i n g l e t , oCH^), '4 8.43 (3H doublet, J ^ 6 - 6 H z, rhamnose CH^), 8.46, 8.76 (3H s i n g l e t s , isopropylidene CH^), 9.70, 9.71, 9.75. 9.84 (9H singl e t s , 4 t r i m e t h y l s i l y l groups). This spectrum was de-f i n i t e l y d i f f e r e n t from the one already described f o r 109, but was i d e n t i c a l to the p.m.r. spectrum of the t r i m e t h y l s i l y l de-r i v a t i v e of an authentic sample of methyl 4-0-g-D-glucopyranosyl-2-3-0-isopropylidene-a-L-rhamnopyranoside which was obtained by deacetylation of the c r y s t a l l i n e tetraacetate (125). Methyl 4-0-a-D-glucopyranosyl-a-L-rhamnopyranoside 111 Methyl 4 -0-a-D-glucopyranosyl -2 ,3-0-isopropylidene-a-L-rhamnopyranoside 107 (250mg) i n chloroform (10ml) was treated 168 at room temperature f o r 2fh with t r i f l u o r o a c e t i c acid (TFA, lml) containing 1-2$ water< The mixture was concentrated and small amounts of t r i f l u o r o a c e t i c acid were removed by eva-poration with toluene or benzene, or by treatment with Duolite A-4 (OH") anion exchange r e s i n . A f t e r f i l t r a t i o n and evapora-t i o n , 111 was obtained as a syrup (211mg, 94$); [a]D +43° (c 1.2, methanol); R g ] _ u c o s e (paper, solvent C) 2.1. The p.m.r. spectrum (DgO, external TMS) showed8 4.97 (IH doublet, J 1 2 • 3.3 H z, H-l of the glucose residue), 5.35 (IH doublet, 2 -1.7 H . H-l of the rhamnose residue), 8.64 (3H doublet, J c , = z j, o 6 H . rhamnose CH-,), 6.64 (3H s i n g l e t , 0CH-). z j • j Methyl 2,3-di-0-acetyl-4-0-(2 ,3,4 ,6-tetra-0-acetyl-a-D-gluco-pyranosyl)-a-L-rhamnopyranoside 112 Methyl 4-0-a-D-glucopyranosyl-a-L-rhamnopyranoside 111 (200mg) was acetylated i n pyridine (5ml) and acetic anhydride (5ml) at room temperature overnight. Excess reagents were re-moved by successive evaporations f i r s t with ethanol and then with water. The product c r y s t a l l i z e d on standing from ethanol (ca lml) and was r e c r y s t a l l i z e d from the same solvent (6ml/g), y i e l d 290mg (83$): R f 0.4l (solvent A); m.p. 136-137°; [ a ] Q +62.3° (c 2.6, chloroform); p.m.r. (CDCl^) 1 6.62.(3H sin g l e t , OCH 3), 7.90-8.02 (18H, OAc's), 8.60 ( 3 H doublet, 3^ 6 - 6 H z, rhamnose CH^). Anal. Calcd. f o r C25 H36°l6 l C ' 50.65; H, 6.I3. Found 1 C, 50.45; H, 6.11. 169 l,2,3-Tri-0-acetyl-4-0-(2,3»4,6-tetra-0-acetyl-a-D-glucopyra-nosyl)-L-rhamnopyranose 113 To the hexaacetate 112 (80mg) i n acetic anhydride (1ml) was added a solution of 2$ (v/v) s u l f u r i c acid i n acetic an-hydride (2ml). After standing at room temperature f o r 3h, the mixture was poured into cold saturated sodium hydrogen carbo-nate (15ml) and the mixture was s t i r r e d f o r 35 min (more sod-ium hydrogen carbonate was added i f necessary, to neutralize the acetic a c i d ) . The product was extracted with chloroform (2 x 15ml) which was washed with water (2 x 5ml) and dried (calcium s u l f a t e ) . T . l . c , (solvent A) showed one major spot (R f 0,41, indistinguishable from s t a r t i n g material) and a small amount of cleavage products. Preparative t . l . c . gave chromatographically pure heptaacetate 113 as a syrup, y i e l d , 78g (93$) i l > ] D 55° (c 2.0, chloroform)? p.m.r. (CDCl 3 ) i 4.00 (1H doublet, 2 ~ H z, H-l of the rhamnose residue). 7.82 8.02 (21H, OAc's), 8.62 (3H doublet, 6 = 6 H z, rhamnose CH 3). 4-0-a-D-Glue opyrano syl-L-rhamnopyrano se 114 The heptaacetate 113 (70g) was deacetylated using 0.2M sodium methoxide i n anhydrous methanol (5ml) f o r l h at room temperature. Passage through Amberlite IR 120 H + r e s i n and evaporation of the solvent gave the free disaccharide 114 as a syrup, 35mg (95$) M D +10° (c 1.0, water). Paper chro-matography gave one component with E g i u c o s e 0.68 (solvent C). The p.m.r. spectrum included (D 90, external TMS)i 4.88 (doub-170 l e t , J, , = 1.3 H , H-l of a-L form of the rhamnose residue), 5.14 (doublet, J, , • 0,8 H., H-l of 0-L form of the rhamnose 11 c Z residue), 4.94 (1H doublet, J. 0 - 3.8 H , H-l of a-D-form of 1»& z the nonreducing glucose residue), 8.64 (3H doublet, ^ = 6 H z, rhamnose CH^). T r i m e t h y l s i l y l derivative- The free disaccharide 114 (5mg) i n dry pyridine (1ml) was treated with hexamethyldisilazane (0.2ml) and chlorotrimethylsilane (0.1ml). Evaporation of the reagents gave a syrup. Gas-liquid chromatography analysis (column a, 2 5 0 ° ) gave one peak (77$) at 6.4 min and a second peak at 8.7 min, ( p e r ( ( t r i m e t h y l s i l y l ) sucrose, 9.0 min). Enzymatic hydrolysis- The disaccharide 114 (2mg) i n pH 6.5 buffer (1ml) was incubated with maltase (2mg) at 3 7 ° . Over 90$ cleavage occurred i n 30 min, as shown by paper chromato-graphy using solvent C. Incubation with beta glucosidase (143) at pH 5.1 gave only trace cleavage i n l6h. 4 -0-a-D-Glucopyranosyl-L-rhamnitol 115 The disaccharide 114 (15mg) i n water (2ml) was reduced with sodium borohydride (30mg) at room temperature overnight. The reaction mixture was s t i r r e d with Amberlite IR 120 H + r e s i n , f i l t e r e d and concentrated. Several portions (10 x 15ml) of methanol were then added and evaporated to remove methyl borate. A syrup 115 (l4mg) was obtained, £ g i u c o s e 0.58 ( s o l -vent C). The p.m.r. spectrum (DgO, external TMS) included i 4.76 (1H doublet, ^ 2 = 3.8 H z, H-l of a-D form of the non-reducing glucose u n i t ) , 8.70 (3H doublet, J . / « 6 H , CH,). 171 Gas-liquid chromatography of the per ( t r i m e t h y l s i l y l ) a l d i t o l on column a at 250° gave one peak at 9.6 min, (per (trimethyl-s i l y l ) sucrose, 9.0 min). Acetylation of the a l d i t o l 115 (12 mg) i n pyridine (2ml) and acetic anhydride (2ml) at 100° f o r 30 min and subsequent workup gave a syrup 116 which was p u r i -f i e d by preparative t . l . c . (R f O.38 solvent A); [ a ] D +56° (c 1.25, chloroform)? p.m.r. (CDCly 7.90-8.02 (24H, OAc's), 8.66 (doublet, g = 6 H z, CH^). G l l . c . of the a l d i t o l ace-tate 116 using column a at 275° gave one peak of retention time of 5*8 min (sucrose octaacetate, 7.2 min). Methylation analysis Methyl 4-0-a-D-glucopyranosyl-a-L-rhamnopyranoside 111 (40mg) i n methyl sulfoxide was methylated (82) by reaction with methyl s u l f i n y l anion (2M, 2ml) for 3h and then shaking with methyl iodide for 3h. Water (5ml) was added and the pro-duct was extracted with petroleum ether (b.p. 65-70°, 10 x 20ml). Removal of the petroleum ether gave the f u l l y methy-lated disaccharide 117 as a syrup which gave one spot on t . l . c . ( E f 0.13, solvent A; 0.61, solvent B). P u r i f i c a t i o n (from me* t h y l sulfoxide) by preparative t . l . c . gave 40mg of product; [<x]D +80° (c 0.9, methanol); p.m.r. (CDCl^)« 4.97 (IH doublet, J l , 2 = 3 , 7 H z ' H - 1 ° f t h e a"" D f o r m o f * h e £ l u c o s e residue), 5.27 (IH doublet, J ~ l j 2 = 1.8 H z, H-l of the a-L form of the rhamnose residue), 6.38, 6.44, 6.50, 6.52, 6.61, 6.63, 6.67 ( 3 H s i n g l e t s , seven OCH^ groups), 8.65 (3H doublet, J^ ^ = 6 K% rhamnose CH-). 172 The f u l l y methylated disaccharide (lOmg) was subjected to methanolysis i n 3$ hydrogen chloride i n methanol (3ml) un-der reflux overnight. Neutralization ( s i l v e r carbonate or lead c a r b o n a t e ) . f i l t r a t i o n , and removal of the solvent below 35° (high temperatures may r e s u l t i n loss of v o l a t i l e products) gave a syrup. This was converted to the t r i m e t h y l s i l y l d e r i -vatives f o r subsequent analysis by g . l . c . Injection on column b programmed from 1 1 0 ° to 1 8 0 ° at 2°/min at a helium flow of 60ml/min gave peaks corresponding to standards of methyl 2 , 3 -di - 0-methyl - 4 - 0-trimethylsilyl-L-rhamnopyranosides ( 4 . 4 , 6 . 6 min) and methyl 2 , 3 , 4 , 6-tetra - 0-methyl-D-glucopyranosides ( 1 1 . 2 , 15.4 min). The f u l l y methylated disaccharide 117 (30mg) i n 2M t r i -f l uoroacetic acid (5ml) was hydrolyzed on a steam bath f o r l 6 h . The solvent was removed and traces of acid were e l i m i -nated by c o - d i s t i l l a t i o n with benzene under reduced pressure. The r e s u l t i n g syrup gave spots on paper (solvent E) corres-ponding to standards of 2 ,3,4 ,6-tetra -0-methyl-D-glucose (R 1 .0) and 2,3-di-0-methyl-L-rhamnose (R 0.84). A portion of 6 the hydrolysate (lOmg) was acetylated i n acetic anhydride ( 0 . 5 m l ) and pyridine ( 0 , 5 m l ) . Injection onto column b pro-grammed from 1 1 0 ° to 210° at 2°/min (flow rate 60ml/min) gave two peaks f o r l - 0-acetyl - 2 , 3,4 , 6-tetra - 0-methyl-D-glucose (24 . 5 , 26.7 min), and two peaks f o r l,4-di - 0-acetyl - 2 , 3-di - 0-methyl-L-rhamnose (33.3» 38.2 min). A second portion of the hydrolysate (about 15mg) was reduced with sodium borohydride (30mg) i n water overnight. Workup and ace t y l a t i o n (pyridine-acetic an-173 hydride) gave the corresponding a l d i t o l acetates. Injection onto column d at 225° gave two peaks, corresponding to stand-ards of l,4,5-tri-0-acetyl-2,3-di-O-methyl-L-rhamnitol (21.7 min) and to l,5-di-0-acetyl-2,3,4,6-tetra-0-methyl-D-glucitol (26.0 min). Samples were c o l l e c t e d from the gas chromatograph and gave mass spectra i d e n t i c a l to authentic standards (125). Periodate oxidation Methyl 4-0-a-D-glucopyranosyl-a-L-rhamnopyranoside 111 (lOmg) was reacted with sodium metaperiodate (0.015M, 10ml) at room temperature i n the dark. The periodate uptake was moni-tored by the u l t r a v i o l e t absorption at 222.5nm as described. After standing f o r 36h, the uptake of periodate was 3.0 moles/ mole sugar and remained unchanged. Iodate and excess periodate were removed by addition of barium acetate, the s a l t s were f i l -tered and washed with water. The f i l t r a t e was concentrated to about 4ml and treated with sodium borohydride (40mg) overnight. The polyalcohol obtained a f t e r deionization, d i s t i l l a t i o n with methanol and concentration was subjected to methanolysis (3$ HC1 i n methanol, 5ml, re f l u x f o r 5h), neutralized (Duolite A-4 (OH") resin) and concentrated. Paper chromatography ( s o l -vent G) showed two major spots corresponding to standards of 1-deoxy-D-erythritol (R f 0.57) and g l y c e r o l (R f 0.47). Acety-l a t i o n i n pyridine (0.5ml) and acetic anhydride (0.5ml) and subsequent i n j e c t i o n i n hexane onto column b at 140° gave peaks i d e n t i c a l to the peracetylated standards of 1-deoxy-D-e r y t h r i t o l (5.0 min) and g l y c e r o l (7.0 min). 174 APPENDIX 175 APPENDIX I Proton magnetic resonance spectroscopy ' of the t r i m e t h y l s i l y l ethers  of some disaccharide derivatives Per-O-trimethylsilyl ethers (-OSiMe^) are widely used for the analysis of carbohydrates by gas-liquid chromato-graphy following the introduction of t h i s technique by Sweeley and co-workers (102). These applications have been reviewed by Dutton (152). Recently per - O - t r i m e t h y l s i l y l ethers have been used as convenient derivatives f o r the p.m.r. spectro-scopic determination of the geometry of the anomeric proton i n mono-and oligo-saccharides (153il54,155)» but i n these studies, attention was concentrated on the region of the spectrumT=5 and the signals due to the O - t r i m e t h y l s i l y l ethers were dismissed as having z^lO. Gibney (9*0 has shown that the number of hydroxyl groups i n monosaccharides can be conveniently monitored by the p.m.r. spectra of the t r i m e t h y l s i l y l d e r i v a t i v e s . This technique can be applied to small amounts of sample because each -OSiMe^ i n -troduced, increases the number of protons on the hydroxyl group by a factor of nine. In the course of the present work, t h i s technique was use-f u l i n i d e n t i f y i n g the number of free hydroxyl groups of un-known sugars and i n addition, i t was found that i n most of the disaccharide spectra obtained, the introduction of a trimethyl-s i l y l group usually s i m p l i f i e d the anomeric region of these 176 disaccharides, thus f a c i l i t a t i n g the determination of the anomeric form (a or g). In addition, a large solvent depen-dence i n the chemical s h i f t s of the t r i m e t h y l s i l y l ethers was observed, a s i t u a t i o n s i m i l a r to that which exists f o r 0-methyl ethers (156). Figure 10 i l l u s t r a t e s the p.m.r. spectrum of the trimethyl-s i l y l (tms) ether of l,2i3,4-di-0-isopropylidene-6-0-(4-0-methyl-g-D-glucopyranosyl)-D-galactose j>8 i n carbon t e t r a c h l o r i d e . The spectrum, taken at sweep width (s.w.) 1000, shows one un-resolved peak fo r the three tms groups, but when the tms re-gion i s expanded at s.w. 100, the three tms signals are r e s o l -ved. As noted e a r l i e r , the p.m.r. spectra of methyl 4-0-ct-D-glucopyranosyl-2,3-0-isopropylidene-a-L-rhamnopyranoside 107 and of the corresponding g-linked disaccharide 108 ( i s o l a t e d i n small amounts) could not d i s t i n g u i s h these two compounds. However, when the p.m.r. spectra ( i n benzene) of the corres-ponding tms ethers 109 and 110 were obtained, the alpha linkage i n 109 (Figure 11) and the beta linkage of 110 (Figure 12) were c l e a r l y demonstrated by the coupling constants. The tms s i g -nals were also well separated from each other and from t e t r a -methylsilane (TMS) standard which was added. F i n a l l y , Figure 13 shows the p.m.r. spectrum of the tms derivative of l,2i3,4-di-0-isopropylidene-6-0-g-D-glucopyranosyl-D-galactose 12 (CCl^, s.w. 1000). Figure 14 i l l u s t r a t e s an expanded (s.w. 100) spectra of the tms region of the same com-pound i n two solvents, A (carbon tetrachloride) and B (benzene). 177 T h i s F i g u r e c l e a r l y i n d i c a t e s a l a r g e c h a n g e i n t h e c h e m i c a l s h i f t o f t h e tms s i g n a l s i n b e n z e n e a s c o m p a r e d t o c a r b o n t e t r a c h l o r i d e , t h e s p e c t r u m i n b e n z e n e b e i n g more h i g h l y r e s o l -v e d t h a n i n t h e c o r r e s p o n d i n g s p e c t r u m i n c a r b o n t e t r a c h l o r i d e . E x p e r i m e n t a l D i s a c c h a r i d e (50mg) i n d r y p y r i d i n e (3ml) was t r e a t e d w i t h h e x a m e t h y l d i s i l a z a n e (1ml) a n d c h l o r o t r i m e t h y l s i l a n e (0.5ml) (102). A f t e r 30 m i n , e x c e s s r e a g e n t s w e r e e v a p o r a t e d a n d p o r -t i o n s o f d r y h e x a n e (5 x 10ml) w e r e a d d e d a n d e v a p o r a t e d . The r e s u l t i n g s y r u p was t h e n d r i e d a t 0.1mm f o r l h . P.m.r. s p e c t r a (100 MH ) w e r e r e c o r d e d u s i n g b e n z e n e l o c k ; t h r e e d r o p s o f b e n -z e n e w e r e a d d e d t o t h e c a r b o n t e t r a c h l o r i d e s o l u t i o n s . •| I i I | l M I M I I I I I M I.I I I I I I I I I I I I M I I M . I. i ' 10.0 Figure 10. P.M.R. Spectrum (CCl^) of the t r i m e t h y l s i l y l ether of compound J3J3 M 00 Standard r 5To 6i6 1 y:a 8 : 0 1 • 1 1 • 1 9 : 0 ' ' ' ' luJu Figure 11. P.M.R. Spectrum ( C ^ ) of the t r i m e t h y l s i l y l ether of compound 10? 400 300 200 TMS Standard 4;: .M: f ' " ^ • w ' 6 . 6 7 . 0 8.0 9.0 10.0 Figure 12. P.M.R. Spectrum (CgH^) of the t r i m e t h y l s i l y l ether of compound 108 M l • - 1 CO O T D T D ~ Figure 1 3 . P.M.R. Spectrum (CCl^) of the t r i m e t h y l s i l y l ether of compound 1 2 CO 00 Figure 14. P a r t i a l P.M.R. Spectrum of the t r i m e t h y l s i l y l region of compound 12, A (CClj^) and B (CgHg), s.w. 100, 183 APPENDIX II Synthesis of monosaccharide esters  having one free hydroxyl group H e l f e r i c h and Zirner (13) have shown that the 1,3,4,6-tetraacetates of glucose and galactose may be re a d i l y obtained by the controlled hydrolysis of the corresponding acetobromo sugars. These two tetraacetates, as well as the corresponding mannose analog (157)» were prepared i n the laboratory without much d i f f i c u l t y , except that the glucose compound did not cry-s t a l l i z e spontaneously and seeding with an authentic sample was eventually required. The tetraacetates were l a t e r used f o r the synthesis of the corresponding 3 , 4 , 6-trimethyl sugars (Appendix IV). With the intention of preparing 1 , 3 , 4-tri-O-acetyl-L-rhamnose 126 i n a si m i l a r manner, L-rhamnose monohydrate was converted to the corresponding 2 ,3 .4-tri-O-acetyl-L-rhamnopyr-anosyl bromide and t h i s compound was subjected to three d i f -ferent c o n t r o l l e d hydrolysis conditions using aqueous sodium acetate. This, however, consistently gave two d i f f e r e n t t r i -acetates, both c r y s t a l l i n e . The t r i a c e t a t e of lower melting point was the main pro-duct, but preliminary analysis by p.m.r. spectroscopy of the t r i a c e t a t e and of i t s t r i m e t h y l s i l y l derivative suggest that t h i s i s probably the 2,3,4 t r i a c e t a t e 12?, not the expected 1,3.4. The higher melting, minor component (about 20$ of the t o t a l ) may i n fact be the expected product, but conditions which w i l l maximize the y i e l d of t h i s component s t i l l have to be found. The p o s i t i o n of the free hydroxyl group i n both these acetates i s under further investigation. A d i f f e r e n t approach was used f o r the synthesis of ano-ther L-rhamnose ester having one free hydroxyl namely, methyl 4-0-acetyl-O-benzoyl-a-L-rhamnopyranoside 129. This was pre-pared s t a r t i n g from methyl 4-0-acetyl-a-L-rhamnopyranoside 128, which was obtained by hydrolysis (125) of the acetal group i n methyl 4-0-acetyl-2,3-0-isopropylidene-a-L-rhamnopyranoside 102. Selective benzoylation of 128 with one molar equivalent of benzoyl chloride i n cold pyridine gave one major product 129. The p.m.r spectrum of 129 and of i t s t r i m e t h y l s i l y l ether c l e a r l y indicated one free p o s i t i o n . Assuming that no acetate migration occurred during the reaction, and depending on which p o s i t i o n i s free, compound 129 (and 126) should he a good intermediate f o r the synthesis of disaccharides linked at positions 2 or 3 of rhamnose, which at the present time cannot be prepared because no suitable aglycons are available.-The p o s i t i o n of the free hydroxyl group i n 129 i s also under further in v e s t i g a t i o n . Experimental 1,3»4,6-Tetra-0-acetyl-a-D-glucopyranose 123 The procedure of H e l f e r i c h and Zirner (13) was used f o r the preparation of 123. Acetic anhydride (100ml) was placed i n a 3-necked f l a s k equipped with mechanical s t i r r i n g and a thermometer. A spatula of D-glucose monohydrate was added with 185 s t i r r i n g , followed by 70$ perchloric acid (10 drops). D-Glucose monohydrate (26g) was then added i n portions while keep-ing the i n t e r n a l temperature between 40 and 45° with an ice water bath. A f t e r standing f o r l h at room temperature, the solution was cooled to 15° and phosphorus tribromide (17.2ml) was added keeping the temperature below 25°. Water (9.2ml) was then added dropwise keeping the temperature between 25 and 30° (temperature control i s d i f f i c u l t i f the water i s added faster) and the reaction mixture was l e f t standing at room temperature f o r l ^ h . The mixture was cooled to 10° and with good s t i r r i n g and cooling, a solution (at 5°) of sodium acetate trihydrate (80g) i n water (100ml) was added (at f i r s t dropwise) keeping the i n t e r n a l temperature between 45 and 50°. The mixture was kept at 40 to 45° f o r 45 min. Chloroform (75ml) was added and the solu t i o n was poured into ice (ca 500ml), shaken, and the chloroform layer drawn o f f . The aqueous layer was extrac-ted with chloroform (2 x 50ml) and the combined chloroform extracts were washed with ice water (2 x 50ml), cold saturated sodium hydrogen carbonate solution (3 x 50ml) and ice water (2 x 50ml) and dried (calcium c h l o r i d e ) . F i l t r a t i o n and con-centration gave a syrup which was dissolved i n ether and cry-s t a l l i z e d on seeding. I t was r e c r y s t a l l i z e d from the same s o l -vent to give 17g of 12J (35$)J m.p. 98-100°? [ a ] D +139° (c 2, chloroform)? ( l i t . (13) m.p. 98-100°, [ o ] D +141° (c 3.2, chloroform)). A portion of the syrup (2g) c r y s t a l l i z e d spon-taneously a f t e r standing f o r 8 months i n ethyl acetate. This, however, was not 123 but probably the beta anomer, m.p. 130-186 1 3 2 ° ; [ a ] D +35° (c 1.7, chloroform); ( l i t . ( 1 5 8 ) m.p. I 3 6 - I 3 8 0 ) . 1» 3 ,4,6-Tetra-O-acetyl-a-D-galactopyranose 124 This compound was obtained s i m i l a r l y from galactose mono-hydrate. However, the solution (at 5°) of sodium acetate was added at 35 to 40°"(instead of 40 to 4 5 ° ) and a f t e r the addition was completed, the mixture was kept at 35 "to 40° (instead of 40 to 45°) f o r 25 min. From 26g of D-galactose monohydrate 15g of 124 were obtained, m.p. 148 - 1 5 0 ° ; ( l i t . (13) m.p. 151°).. 113»4,6-Tetra-0-ac etyl-p-D-mannopyranose 125 Compound 125 was obtained as described above f o r the ga-lactose derivative. The product was r e c r y s t a l l i z e d from ether or from chloroform-ether. D-Mannose (I3g) gave 3 .2 to 3 . 9 g of 125 (ca 2 7 $ ) , m.p. 1 6 4 - 1 6 5 ° ; ( l i t . (157) m.p. 1 6 4 - 1 6 5 ° ) . Rhamnose t r i a c e t a t e The procedure which follows was an attempt to prepare 1, 3»4-tri-0-acetyl-a-L-rhamnopyranose i n a s i m i l a r manner to the mannose compound. However, two c r y s t a l l i n e t r i a c e t a t e s were isol a t e d and one of them 127 (m.p. 1 0 0 - 1 1 0 ° ) , may be a mixture of the anomeric forms of 2 ,3» 4-tri - 0-acetyl-L-rhamnose (from p.m.r.). The other c r y s t a l l i n e form 126 (m.p. 1 6 7 - 1 7 0 ° ) has a low f i e l d anomeric hydrogen i n the p.m.r,, which suggests the presence of an acetate group at p o s i t i o n 1, The exact structures of these acetates are under further investigation. To a few mg L-rhamnose monohydrate i n acetic anhydride (50ml) was added perchloric acid (4 drops). L-Rhamnose mono-187 hydrate (13g) was added i n portions while keeping the i n t e r n a l temperature at 40 to 45° with an ice water hath. The mixture was kept at room temperature f o r l h , i t was cooled to 15°» and phosphorus tribromide (8.6ml) was added keeping the temperature at 20-25°. Water (4.6ml) was then added dropwise keeping the temperature at 20-25°. The mixture was allowed to stand at room temperature f o r lf-h and was divided into 3 equal portions a, b, and c. A solution (at 5°) of sodium acetate trihydrate (40g) i n water (50ml) was also divided into 3 equal portions (ca 24ml each) which was added to the reaction mixture (por-tions a,b,c) with s t i r r i n g and cooling as follows« Portion a- the sodium acetate solution (24ml) was added (4 min) keeping the temperature between 25 and 30°, and the r e s u l t i n g mixture was allowed to stand between 25 and 30° f o r 25; mint Portion b- the sodium acetate solu t i o n was added (4 min) keeping the temperature between 35 and 40° and the r e s u l t i n g mixture was allowed to stand at t h i s temperature f o r 15 minj Portion c- the sodium acetate solution was added (4 min) keeping the temperature between 35 and 40° and the r e s u l t i n g mixture was allowed to stand at t h i s temperature f o r 25 min (same as the mannose analog). Workup» the mixtures were separately poured into ice (200ml) and extracted with chloroform. The chloroform extracts were washed as before with cold water, cold saturated sodium hydrogen carbonate, and cold water , then dried (sodium s u l f a t e ) . 188 The syrups obtained were dissolved i n chloroform (15-25ml) and ether (100ml) was added, a f t e r which c r y s t a l l i z a t i o n began. Triacetate 126 ( c r y s t a l l i z e s f i r s t ) gave m.p. I 6 7 - I 7 0 0 , t r i a c e -tate 127 gave a m.p. 9 8 - 1 1 0 ° . Yields were as follows« 126 127 (m.p. 167-170°) (m.p. 98 - 1 1 0 ° ) portion a 510mg 2.1g portion b 560mg 2 . 1 g portion c 250mg 2 .1g R e c r y s t a l l i z a t i o n of 127 from ether did not change the m.p. substantially} [ a ] D of 126 +8° (c 4.9, chloroform), [ a ] D of 127 - 3 3 ° (c 2 . 1 , chloroform). Selective benzoylation of methyl 4 - 0-acetyl - a -L-rhamnopyranoside 128. Synthesis of a methyl 4 - 0-acetyl - 0-benzoyl - a -L-rhamnopyra-noside 129 Compound 125 was obtained by hydrolysis of methyl 4-0-acetyl - 2 , 3-O-isopropylidene - a -L-rhamnopyranoside (125, 128) (3g) i n a mixture of chloroform ( 1 3 5 m l ) , t r i f l u o r o a c e t i c acid (15ml) and water ( 0 . 3 m l ) at room temperature f o r 2h ( 1 2 5 ) . Evaporation of the bulk of the acid followed by n e u t r a l i z a t i o n (Duolite A-4 0H~ resin) and concentration gave c r y s t a l l i n e 128, which was r e c r y s t a l l i z e d from ethyl acetate, 2 g , m.p. 1 1 6 - 1 1 7 ° } ( l i t . (125) m.p. 1 1 7 ° , (128) m.p. 1 1 2 - 1 1 6 ° ) . Com-pound 128 ( l g , 4 .5 mmol) i n dry pyridine (10ml) was treated at 0° with benzoyl chloride ( 0 . 6 m l , 5 . 3 mmol). Af t e r l h at 0° t . l . c . (solvent A) gave R f values as follows t 128 0.14, a main 189 component (ca 85$) 0.55, and a minor component 0.73. A small amount of 128 remained unreacted. The mixture was kept an additional hour at 0° and the pyridine was then removed. Chloroform (75ml) was added and the solution was washed with water (3 x 20ml). Evaporation of the chloroform gave a syrup 129 which c r y s t a l l i z e d from absolute ethanol and was recry-s t a l l i z e d from the same solvent; y i e l d 600mg; [a3D=-27.7° (c 1.0, chloroform). The p.m.r. spectrum (60 MH CDC1,) i n d i -z j cated the presence of one benzoate group (1.9-2.8, $K)» acetate (8.07, 3H s i n g l e t ) , methoxy (6.57, 3H s i n g l e t ) , and methyl (8.74, 3H doublet, ^ = 6 ,HZ). The p.m.r. spectrum of the trimethyl-s i l y l ether of 129 indicated the presence of one trimethyl-s i l y l group, which confirms the presence of one free hydroxyl group i n 129. The p o s i t i o n of the free hydroxyl group i s under further investigation. 190 APPENDIX III Synthesis of 3-0-methyl-D-galactose Authentic samples of methylated sugars are invaluable as reference material i n s t r u c t u r a l investigations of polyssacha-ride structures. Many of the c l a s s i c studies i n these areas have been made i n connection with the structure of plant poly-saccharides, a large number of which contain 1-4 linkages. With our current interest i n b a c t e r i a l polysaccharides i n which both 1-3 and 1-2 linkages are more common, d i f f e r e n t standards are required. This and the following sections are concerned with the synthesis of methylated sugars necessary f o r the s t r u c t u r a l investigation of polysaccharides. The intermediates used, however, can also be employed f o r the synthesis of disaccharides. The synthesis of 3-0-methyl-D-galactose using a galactofura-nose intermediate i s f i r s t described. This i s followed (Ap-pendix 4) by a f a c i l e new synthesis of 3»4,6-tri-O-methyl-D-glucose, D-mannose, and D-galactose. 3-0-Methyl-D-galactose was prepared as shown i n Scheme 19 Diacetone glucose 130 was tosylated to give the corresponding 3-0-tosyl compound 131» which was then heated with base under vacuum and the corresponding 3-deoxy-l,2i5,6-di-0-isopropyli-d:ene-a-D-xylo-hex-3-enofuranose 132 was obtained i n low y i e l d (30$). Compound 132 was subjected to hydroboration-oxidation as described "by Paulsen and Behre (159) and t h i s gave l , 2 i 5 , 6 -di-O-isopropylidene-a-D-galactofuranose 133 i n 25$ y i e l d . Methylation of 133 by both the Hakomori and Purdie methods Scheme 19 192 gave syrupy 1 ,215,6-di-O-isopropylidene-3-0-methyl-a-D-galacto-furanose 13^ i n high y i e l d . Hydrolysis of the isopropylidene groups i n 2M t r i f l u o r o a c e t i c acid then afforded c r y s t a l l i n e 3-0-methyl-D-galactose 135. Compound 135 was l a t e r used i n the laboratory as part of a study on the c i r c u l a r dichroism of p a r t i a l l y methylated a l -d i t o l acetates ( 1 6 0 ) . Experimental l,2i5 , 6-Di - 0-isopropylidene-a-D-glucofuranose 130 (diacetone glucose Compound 130 was prepared by the two methods ( i . e . using s u l f u r i c acid or anhydrous CuSO^ as catalysts) described i n "Methods i n Carbohydrate Chemistry" 2, p.321 . The s u l f u r i c acid method was found to give better y i e l d s . When large amounts (over lOOg) were prepared, the a c i d i c solution (HgSO^ catalyst) containing 130 was cooled to about -25° with dry ice and i t was then neutralized as fast as possible with 50$ sodium hydroxide, but keeping the temperature from r i s i n g above 2 5 ° . Hydrolysis of the acetal groups (due to the presence of aqueous acid dur-ing p a r t i a l neutralization) i s minimized i n t h i s manner. 1,2i5 , 6-Di - 0 -isopropylidene-3 - 0-tosyl-a-D-glucofuranose 131 A 0.2 molar excess of p_-toluenesulfonyl chloride (23g, r e c r y s t a l l i z e d from benzene-petroleum ether) was added to a so l u t i o n cooled to 0° of diacetone glucose 130 (26g. 0 .1 mole) i n dry pyridine (70ml, d i s t i l l e d from KOH p e l l e t s ) . A f t e r standing at room temperature overnight the mixture was poured 193 into a s t i r r e d mixture of ice-water (300ml) and allowed to stand with occasional s t i r r i n g u n t i l c r y s t a l l i z a t i o n began (usually within 45 min; i f no c r y s t a l l i z a t i o n occurred, the aqueous mixture was extracted with ether and the ether extract washed with cold water. The pyridine-free syrup c r y s t a l l i z e d from ethanol). The c r 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 hot ethanol, giving pure 131, 34g (82$) m.p. 118 - 1 2 0 ° . 3-Deoxy-l, 21516-di -0-isopropylidene-a-D-xylo-hex-3-enofuranose 122 A mixture of ground 1 , 2 t 5 , 6-di - 0 -isopropylidene-3 - 0 -tosy-a-D-glueofuranose 131 ( 1 5 g ) » ground anhydrous sodium carbonate ( 1 5 g ) , and ground potassium hydroxide (lOg), was heated at 1 6 0 -1 7 0 ° , 0.05mm, i n a sublimation apparatus. The product 132 was c o l l e c t e d d i r e c t l y on the cold f i n g e r (water cooled), y i e l d 3g ( 3 0 $ ) , m.p. 48 - 5 0 ° , R f 0.77 (solvent A; ( l i t . (161) m.p. 5 0 ° ) . The product was stored at 0° i n a stoppered f l a s k . The following two conditions were t r i e d i n an attempt to improve the y i e l d of 132, but (a) gave only about 5$ product, (b) gave mainly diacetone glucose (due to removal of the t o s y l group) i (a) 5g of 131 and 15g of potassium t-butoxide, f i n e l y ground, were heated i n the sublimation apparatus at 140°, 0,1mm; (b) l g of 131 and 3g of potassium t-butoxide were refluxed i n 50ml t-butanol f o r 4 h . The reaction was followed by t . l . c . i n benzene-methanol ( 4 t l ) 194 1,2i5,6-Di-O-isopropylidene-a-D-galactofuranose 133 Compound 132 (3g, 12.4 mmol) and sodium borohydride (1.4g, 37 mmol) were s t i r r e d i n a 100ml 3-necked flask equip-ped with a thermometer and a drying tube (a piece of rubber tubing was connected to the drying tube and the other end of the tubing was placed i n the sink of the fume-hood, with running water). Boron t r i f l u o r i d e etherate (4 .5ml, 36 mmol) i n dry tetrahydrofuran (15ml) was added dropwise (2h) under anhydrous conditions keeping the temperature at 20-25°. The following solutions were added dropwise with s t i r r i n g and cool-ing ( i n t e r n a l temperature 25° or below)t (a) 1«1 mixture of tetrahydrofuran-water (3ml), (b) 2M sodium hydroxide (9ml), (c) 30$ hydrogen peroxide (4ml). The solution (containing 1 133) was decanted and the remaining gelatinous mass was washed with tetrahydrofuran (20ml) and the tetrahydrofuran wash was combined with the decanted solution. Concentration under re-duced pressure gave a syrup which was dissolved i n water (25ml) and extracted with ether (4 x 25ml). Evaporation of the com-bined ether extracts gave a syrup which c r y s t a l l i z e d on stan-ding. R e c r y s t a l l i z a t i o n from cyclohexane gave 133 (0.6-0.8g, 18-25$), m.p. 97-98$; (lit. (159) m.p. 97.5-98°) . 1,2« 5,6-Di -0-isopropylidene-3 -0-methyl-a-D-galactofuranose 134 (a) Compound 133 (50mg) i n dry methyl sulfoxide (2ml) was methylated with methylsulfinyl anion (lml, 1.5M) and methyl iodide (lml). The product was obtained by extraction of the reaction mixture with n-hexane. P u r i f i c a t i o n by preparative 195 t . l . c . gave 134 as a syrup; y i e l d , 44mg. (b) Compound 133 (57mg) and fres h l y prepared s i l v e r oxide (250mg) were refluxed i n methyl iodide (5ml) overnight under si anhydrous conditions. F i l t r a t i o n and evaporation gave a syrup 134 quantitatively, 60mg. The p.m.r. spectrum included (60 MHz CC1^)» 4.33 (1H doublet, J l j 2 - 4.0 H z, H-l of a-D form), 6.60 (3H si n g l e t , 0CH3), 8.50 and 8.69 (3H singlet and 9H sing-l e t respectively, isopropylidene CH^). 3-0-Methyl-D-galactose 135 1,2i5,6-Di-O-isopropylidene-3-0-methyl-a-D-galactofuranose 134 (60mg) i n 2M t r i f l u o r o a c e t i c acid (8ml) was hydrolyzed on a steam bath f o r 40 min. Concentration, removal of the bulk of the acid by co-evaporation with toluene and ne u t r a l i z a t i o n (Duolite A-4 OH" resin) gave c r y s t a l l i n e 135, 36mg, m.p. and mixed m.p. 144-146°; ( l i t . (162) m.p. 144-14?°). 196 APPENDIX IV Synthesis of 3,4,6-tri-O-methyl-D-glucose, D-mannose and D-galactose Many n a t u r a l l y occurring carbohydrates are p a r t i a l l y ace-ty l a t e d . In order to ascertain the pos i t i o n of the acetyl sub-stituents, procedures described i n the l i t e r a t u r e (163) often make use of methyl v i n y l ether as a blocking reagent f o r the free hydroxyl functions. The r e s u l t i n g per (O-(l-methoxyethyl)) sugar can then be methylated, the O-(l-methoxyethyl) groups hy-drolyzed and the methylated sugars i d e n t i f i e d , thus deter-mining the positions previously substituted by acetyl?:functions. With t h i s precedent i n the l i t e r a t u r e , i t was believed that treatment of 1,3,4,6 tetraacetates of glucose, mannose, and galactose with methyl v i n y l ether i n the presence of an acid c a t a l y s t , methylation (Hakomori) of the r e s u l t i n g blocked sug-ar, and hydrolysis, would be a convenient way of preparing the 3 ,4 ,6>trimethyl sugars. When t h i s was ca r r i e d out however (mainly with mannose), hydrolysis of the r e s u l t i n g methyl ethers gave e s s e n t i a l l y the 2,3,4,6 tetramethyl sugar. Some runs gave a small amount of the expected trimethyl sugar, but the tetramethyl compound was always predominant. This unexpected r e s u l t could be caused by incomplete re-action with methyl v i n y l ether, by removal of the r e s u l t i n g 0-(1-methoxyethyl) group during methylation, or by acetate mi-gration from the 1-hydroxyl to the 2-hydroxyl during the 197 blocking reaction. The l a s t p o s s i b i l i t y (migration) i s the more probable, because the v i n y l a t i o n reaction was checked by t . l . c , and methylation should not remove the 0-(l-methoxy-ethyl ether), since t h i s group i s stable under methylation conditions when used i n connection with polysaccharides. If. acetate migration i s responsible for the r e s u l t s , the question that a r i s e s i s whether t h i s can also occur i n poly-saccharides. The fact that the same re s u l t s were obtained using two other blocking groups suggests that migration here may be due to the highly l a b i l e 1-acetate group. A f t e r the O-(l-methoxyethyl) group f a i l e d to give the expected products, the use of other blocking groups, namely the t-butyl ether and the p_-toluenesulfonate group were inves-tigated. D e t a i l s of t h i s are given i n the experimental; i t s u f f i c e s to say that upon methylation and hydrolysis, mostly 2 ,3»^»6 tetramethyl sugar was obtained. F i n a l l y , the tetrahydropyranyl ether was used as a block-ing group f o r the 2-hydroxyl function and t h i s gave the expected 3 , 4 , 6-trimethyl sugars. This group has been used by Angyal and Gero (164) as a blocking group f o r the hydroxyl functions i n c y c l i t o l s . 3,4,6-Tri-O-methyl-D-glucose has been obtained previously during a study on sel e c t i v e methylation (I.65) and from l , 2 i 5 ,6-di-O-isopropylidene-D-glucose i n a multistep synthesis (166). The galactose isomer has been synthesized from D-galactal (167) and the mannose compound from the ortho-acetate (16B). The merits of the tetrahydropyranyl method described here are that 198 the same type of intermediate i s used i n each case; the re-quired tetra-acetates may be obtained i n 5b and the synthe-s i s can be completed i n three further steps (four i f deacetyla-t i o n i s effected separately). In a t y p i c a l procedure, l , 3 » 4 , 6-tetra - 0-acetyl-a-D-gluco-pyranose 123 (Scheme 20) was treated with dihydropyran and £-toluenesulfonic acid (or KC1) i n dichloromethane to give the corresponding 2 - 0 - ( 2-tetrahydropyranyl) ether 1 3 6 . Com-pound 136 was deacetylated p r i o r to methylation i n order to reduce the amount of methylsulfinyl anion required, thus f a c i -t a t i n g extraction of the product. When methyl iodide was added dropwise at about 1 0 ° to the sugar i n methylsulfinyl anion, much degradation occurred because of i n t e r n a l heating. This however, was lessened by freezing the reaction mixture i n dry ice-acetone p r i o r to the dropwise addition of methyl iodide. Hydrolysis of the methylated product 138 with 2M t r i -f l uoroacetic acid at 1 0 0 ° and subsequent p u r i f i c a t i o n by c e l l u -lose column chromatography or on a short column of s i l i c a gel (120) gave 3 , 4 , 6-tri - 0-methyl-D-glucose 139 i n 6 0 $ o v e r a l l y i e l d . l»3»4,6-Tetra-O-acetyl -0-D-mannopyranose was s i m i l a r l y converted to the tetrahydropyranyl derivative 140 i n high y i e l d . Deacetylation, methylation, hydrolysis and p u r i f i c a -t i o n afforded 3t4,6-tri -0-methyl-D-mannose 142 i n 28$ y i e l d . The lower y i e l d of 142 compared to the glucose analog was due mainly to lo s s of material due to degradation i n the me-Scheme 20 •OAc d ihydropyran/H NaOCH,-CH 0H J . 3 v-1. NaH/DMSO 2. CH 3I 200 t h y l a t i o n and hydrolysis steps. The same procedure was used f o r the corresponding galac-tose isomer, except that the 2-0-tetrahydropyranyl derivative was methylated f o r a shorter time (45 min) than used f o r glu-cose ( l h ) . Chromatographically pure 3»^»6-tri-0-methyl-D-galactose 145 was obtained i n 25$ o v e r a l l y i e l d . In addition, 3,5,6-tri-O-methyl-D-galactose 146 (8$) was also i s o l a t e d , making the t o t a l o v e r a l l y i e l d of galactose isomers 33$« Com-pound 146 r e s u l t s from methylation of the D-galactofuranose structure, which i s known to occur during the Haworth (73) and-Kuhn $98) methylations. The mass spectrum of the a l d i t o l acetate of 146 was consistent with i t s structure. 3,4,6-Tri-0-methyl-D-glucose, mannose, and galactose gave p.m.r. spectra which corresponded to t h e i r structure and the mass spectra of the corresponding a l d i t o l acetates were consistent with 3,k,6 s u b s t i t u t i o n (169,170). F i n a l l y , a l l three compounds c r y s t a l l i z e d on seeding with authentic samples and the expected melting points were obtained. Experimental Using the 0-(l-methoxyethyl)group (a) 1,3,4,6-Tetra-O-g-D-mannopyranose 12 5 (500mg) was d i s -solved i n dry methyl sulfoxide (5ml). Anhydrous £-toluene-sulfonic acid (lOmg) and excess methyl v i n y l ether (2ml, cooled to 0°) were added at 15°. After s t i r r i n g f o r 2h at t h i s tem-perature with the exclusion of moisture, excess methyl v i n y l ether was removed under vacuum. The r e s u l t i n g solution was 201 methylated (Hakomori) and hydrolyzed (0.5M s u l f u r i c acid, 8h at 100°). (b) As (a), but with absolute dichloromethane as solvent. The acid was neutralized with a few drops of pyridine and the mixture was concentrated to a syrup, i t was then methylated and hydrolyzed as i n (a). (c) as (a), but the v i n y l a t i o n reaction was conducted at -10° for 40 min (instead of 2h). The product was then methylated and hydrolyzed as (a). The v i n y l a t i o n reactions (before methylation) were monitored by t . l . c . i n chloroform-methanol (9*1) where 125 had R^ . 0 . 6 l , the product 140 0.75. Only a small amount of 125 remained un-reacted. The syrups from (b) and (c) were checked by p.m.r. before methylation. Paper chromatographic analysis of the hydrolysis products from the above three conditions indicated a large amount of 2,3»4,6-tetra-0-methyl-D-mannose and a small amount of the expected 3,4,6 sugar. Using r the t-butyl ether (a) Compound 125 (lOOmg) i n absolute dichloromethane (5ml) was treated with excess 2-methylpropene (0.8ml, at -10°) and concentrated s u l f u r i c acid (2 drops). After standing at -10° for 4h under anhydrous conditions the cooled solu t i o n was c a r e f u l l y washed with 2% ice cold sodium hydrogen carbonate and water, then dried (sodium s u l f a t e ) . This was concentrated to a syrup, methylated (Hakomori) and hydrolyzed (0.5M s u l f u r i c acid, 8h, 100°). n 202 (b) As (a), but the isobutene i n dichloromethane was treated with the acid f i r s t , the mixture was allowed to stand at -78° for 15 min, and the sugar 125 i n dichloromethane (2ml) was then added. The mixture was kept at -10° fo r 4h. The reactions were monitored using solvent A and two runs were p u r i f i e d by preparative t . l . c . and the p.m.r. obtained indicated , the presence of the t-butyl group. Paper chroma-tographic analysis of the hydrolysis products (solvent B) showed mainly 2,3,4,6-tetra-0-methyl-D-mannose. Using the p_-toluenesulfonate group Compound 125 (lOOmg) i n pyridine (5ml) was treated at 0° with p_-"toluenesulfonyl chloride (60mg) and was stored at -10° for 2 days. Workup gave two products, the major one c r y s t a l -l i z e d on standing (m.p. 153°) and contained a t o s y l group as s shown by the p.m.r. spectrum. This was deacetylated (0.1M sodium methoxide i n methanol), methylated 3 times (Purdie method), detosylated (using sodium amalgam) and hydrolyzed (2M t r i f l u o r o a c e t i c acid) to give mostly 2,3,4,6-tetra-0-methyl-D-mannose. Using the 2-0-(2-tetrahydropyranyl) ether (a) To 125 (lOOmg) i n absolute dichloromethane (5ml) were added absolute dihydropyran (0.5ml) and 2.5M hydrogen chloride i n dioxane (0.2ml) and the mixture was allowed to stand l h at room temperature. Neutralization (anhydrous KgCO^), methylation (Hakomori), and hydrolysis gave l a r g e l y (ca 90$ of products, 203 paper chromatography) the expected 3,4,6-tri-0-methyl-D-mannose ( d e t a i l s under ( c ) ) . (b) As above, but the reaction with dihydropyran was conduc-ted at 0°. Paper chromatography indicated a major spot cor-responding to 3,4,6-tri-0-methyl-D-mannose and a trace amount of tetramethyl sugar. (c) 1,3,4,6-Tetra-0-acetyl-2-0-(2-tetrahydropyranyl)-a-D-gluco-pyranose 136 l,3,4,6-Tetra-0-acetyl-a-D-glucopyranose 123 (2g) was dissolved i n absolute dichloromethane (45ml). Dihydropyran (0.65ml, dried with NagCO^, f r a c t i o n a l l y d i s t i l l e d and the f r a c t i o n b o i l i n g at 83.5-85° refluxed with sodium and d i s t i l -led) and anhydrous £-toluenesulfonic acid (hydrate heated at 95° f o r 2h at 10mm) were added and the mixture was s t i r r e d at 0°. Inspection by t . l . c . (solvent A) a f t e r l h showed a small amount of unreacted s t a r t i n g material (R^ 0.19), a major component (R^ . 0.50) corresponding to 136, and f a s t e r moving components (dihydropyran condensation products). The reaction mixture was neutralized with pyridine or with anhy-drous potassium carbonate. Evaporation of the solvent at 30° gave 136 as a syrup (2.5g). When p u r i f i e d by preparative t . l . c . * The f i n a l runs (preparative) of t h i s section were concluded i n c o l l a b o r a t i o n with Dr. Matthew Yuen-Min Choy who p a r t i c i -pated i n the methylation, hydrolysis and f i n a l p u r i f i c a t i o n of the sugars. 204 using solvent A, 136 had [ a ] D +76° (c 1.7, chloroform); p.m.r. (CDCl 3 ) t 7.8-8.0 (12H, OAc's), 8.3-8.7 (tetrahydropyranyl group). 3,4,6-Tri-O-methyl-D-glucose 139 The syrup 136 (2.5g) i n dry methanol (25ml) was treated with 0.2M sodium methoxide i n anhydrous methanol (10ml) f o r 30 min at room temperature gi v i n g a product of R f 0,34 ( s o l -vent B) on t . l . c . A l t e r n a t i v e l y , the dichloromethane solution of 136 (without n e u t r a l i z i n g the p_-toluenesulfonic acid) was treated d i r e c t l y with sodium methoxide. The syrup 2-0-(2-te-trahydropyranyl)-D-glucose 137 obtained on evaporation of the solvent (the sodium methoxide was not neutralized i n order to avoid hydrolysis of the tetrahydropyranyl ether) was dried 45 min on a vacuum pump and was then dissolved i n dry methyl sulfoxide (5ml). M e t h y l s u l f i n y l anion (2M, 25ml) was added under anhydrous conditions while keeping the temperature below 25°. The mixture was shaken f o r l h at room temperature, frozen i n a dry ice-acetone bath and excess methyl iodide (15ml) was added dropwise with shaking ( i f warming was detected the mix-ture was frozen again). The reaction mixture was shaken at room temperature overnight and extracted with petroleum ether (b.p. 65-70°, 10 x 100ml). Evaporation of the solvent gave a syrup 138, which on hydrolysis (2M t r i f l u o r o a c e t i c acid, 75ml, r e f l u x overnight) afforded a syrup containing 139 (R f O.58, paper, solvent B) and a small amount of tetramethyl sugar. This was dissolved i n water (100ml) and the solution was washed with chloroform (3 x 40ml„ removes methyl sulfoxide 205 degradation products, etc.) and the aqueous layer (containing 139) was concentrated. The syrup was p u r i f i e d using solvent B on either a c e l l u l o s e column (3.5cm x 4 5 c m , 8 - 1 0 m l f r a c t i o n s c o l l e c t e d every 25 min, f r a c t i o n s 120-170 contained 139) or on a short column of s i l i c a gel ( 1 2 0 ) ( I 5 0 g s i l i c a gel G, CHCl^-CH^OH ( 9 « 1 ) was found to be better than solvent B, 5 - 7 m l f r a c -tions c o l l e c t e d every 3 min, f r a c t i o n s 1 0 0 - 1 5 0 contained 139)• 3 , 4 , 6-Tri - 0-methyl-D-glucose 139 was obtained as a syrup ( 7 7 5 m g , 6 0 $ overall) which c r y s t a l l i z e d on seeding with the beta anomer. R e c r y s t a l l i z a t i o n from ether-petroleum ether (b.p. 3 0 - 6 0 ° ) ( 1 * 1 , lOOml/g) gave pure 3 i 4 , 6-tri - 0-methyl-£-D-glucose, m.p. 104 - 1 0 6 ° , mixed m.p. 104-106°; [<x]D + 4 l ° - » + 7 7 ° (2£h, c 1 . 5 . water): ( l i t . ( 1 6 6 ) m.p. 9 7 - 9 8 ° , [ a ] D + 4 1 0 - - * + 7 7 . 5 ° (c 1.6, water)); p.m.r. (DgO, i n t e r n a l DSS)« 4 . 8 5 (doublet, 2 • 3 . 4 H , H-l of a-D-form), 5.42 (doublet, J, 0 = 7 . 3 H . H-l of Z . I f c z e-D-form), 6 . 6 1 , 6 . 4 7 , 6 . 3 8 (3H s i n g l e t s , 0 C H 3 ) . Anal, calcd. f o r C^H^Ogi C, 48.64; H, 8 . 1 6 . Found* C, 48 . 5 8 ; H, 8 . 2 1 . A portion (lOmg) of 139 was reduced with sodium borohy-dride and the r e s u l t i n g a l d i t o l was acetylated i n pyridine ( 1 m l ) and acetic anhydride ( 1 m l ) . Injection of the a l d i t o l acetate onto column b.at ,185°(helium flow of 60ml/min) gave one peak of retention time 1 3 . 2 min. A sample was c o l l e c t e d and gave a mass spectrum which was consistent ( 1 6 9 , 1 7 0 ) with the a l -d i t o l acetate of 1 3 9 , the main peaks had m/e* 4 3 , 4 5 , 8 7 , 9 9 , 1 0 1 , 1 2 9 , 161, I 8 9 . 206 1 ,3,4,6-Tetra-0-acetyl-2-0-(2-tetrahydropyranyl)-0-D-mannopy-ranose 140 This compound was obtained as described f o r the glucose analog 136. 1,3,4,6-Tetra-O-acetyl-g-D-mannopyranose 125 (lg) gave 140 (1.2g) as a syrup: R f 0 .37 (solvent A); [ a ] D -41° (c 3.8, chloroform; p.m.r. (CDC13)« 7.8-8.0 (12H, OAc's), 8.1-8.6 (tetrahydropyranyl group). 3,4,6-Tri-O-methyl-D-mannose 142 1,3,4,6-Tetra-O-ac etyl-2-0-(2-tetrahydropyranyl) -g-D-mannopyranose 140 (1.2g) was deacetylated to the correspon-ding 2-0-(2-tetrahydropyranyl)-D-mannose 141 which had an R^ of 0 .35 ( t . l . c . solvent B). Methylation and hydrolysis as described f o r the glucose analog afforded 142 as a syrup with R^ 0.60 (paper, solvent B). P u r i f i c a t i o n by c e l l u l o s e column gave I80mg}(28$ ov e r a l l ) of product. C r y s t a l l i n e 3,4,6-tri-0-methyl-a-D-mannose was obtained on seeding and was r e c r y s t a l -l i z e d from ether-petroleum ether (1:1, lOOml/g), m.p. 104-106°, mixed m.p. 104-106°; [ a ] D +20°—> +8° (c 0.9, water l h ) ; ( l i t . (168) m.p. 104°, [ a ^ +21-^+8° (c 1.0, water)); p.m.r. (D 20, i n t e r n a l DSS): 4 . 8 5 (doublet, J± 2 - 4.0 H z , H-l of a-D form), 5.17 (doublet, J, 0 - 2.2 H . H-l of g-D form), 6 .51 , X , c z 6 . 5 5 , 6.60 (3H s i n g l e t s , 0CH 3). Anal. Calcd. f o r C ^ g P g i C, 48.64; H, 8.16. Found C, 48 . 3 5 ; H, 8.14. A portion of 142 (lOmg) was reduced with sodium borohy-dride (20mg) and the r e s u l t i n g a l d i t o l was acetylated (py-207 r i d i n e - a c e t i c anhydride). Injection of the r e s u l t i n g a l d i t o l acetate onto column b (same conditions as used f o r the glucose analog) gave one peak of retention time 13.2 min. The mass spectrum of a sample c o l l e c t e d gave main peaks with m/et 43, ^5$ 87, 99. 101, 129, 161, 189, consistent (169,170) with the a l d i t o l acetate of 142. 1 . 3 , 4 , 6-Tetra - 0-acetyl - 2 - 0 - ( 2-tetrahydropyranyl)-a-D-galacto-pyranose 143 This was obtained as a syrup from 1 , 3 , ^ » 6 - t e t r a - 0 - a c e t y l -a-D-galactopyranose 124 ( l g j j a s described f o r the glucose ana-log 136; y i e l d 1.2g, R f 0.50 (solvent A)j [ a ] D +72° (c 2.7, chloroform); p.m.r. (CDC1 3)1 7.8-8.1 (12H, OAc's), 8.3-8.6 (tetrahydropyranyl group). 3,4 ,6-Tri -0-methyl-D-galactose 145 1,3,4,6- Tetra-O-ac etyl - 2 - 0 - ( 2 -tetrahydropyranyl) -oc-D-ga-lactopyranose 143 (1.2g) was deacetylated to give 2-0-(2-te-trahydropyranyl)-D-galactose 144, R f 0.29 ( t . l . c . solvent B). Methylation (45 min i n methylsulfinyl anion) and hydrolysis gave two components on paper chromatography (solvent B); R^ O.37 corresponding to the 3,^.6 compound 145 and a fas t e r mov-ing component 146 with R f O.69. P u r i f i c a t i o n by c e l l u l o s e column (solvent B, 8ml f r a c t i o n s c o l l e c t e d every 25 min, fra c t i o n s 35-50 contained 146 and 9,0-1^0 contained 145) or by short column chromatography using chloroform-methanol (9tl) (5ml f r a c t i o n s c o l l e c t e d every 2.5 min, fract i o n s 90-125 con-208 tained IV?) gave 3,4,6-tri-0-methyl-D-galactose l4j> (l60mg, 25$ overall) which crystallized on seeding, m.p. and mixed m.p. 88-89°; M D + 1 5 2 ° - * + 1 1 0 ° (c 1.2, water); ( l i t . (167) m.p. 8 8 - 8 9 ° , M D +154° -^+110° (c 1 .0, water); p.m.r. (DgO, internal DSS)1 4.79 (doublet, J 1 2 - 3.7 H » H-l of a-D-form), 5.46 (doublet, J±^2 - 7.2 Hz, H-l of 0-D form), 6.51 and 6.61 (6H singlet and 3H singlet respectively, OCH^). Anal. Calcd. for C^gOgt C, 48.64; H, 8.16. Foundi C, 48.44; H, 8.00. Injection of the a l d i t o l acetate of 145 onto column (same conditions as used for the glucose analog) gave one peak of retentions time 14.2 min and the mass spectrum of a collected sample had m/et 43, 45, 87, 99, 101, 129, 161, I89. The column separation also gave 146 (54mg) - 1 5 ° (c 2 .0, water), a portion of which was converted to the a l d i t o l acetate. This had a retention time of 13.0 min and gave a mass spectrum consistent with the assignment of 146 as 3,5,6-tri-O-methyl-D-galactose (169,170), m/ei 43, 45, 129, 189, 273, 305. 209 BIBLIOGRAPHY 1. K. S t e l l n e r , 0. Luderitz, 0. Westphal, A. Staub, B. Leluc, C. Coynault and L. Minor. Ann. Inst. Pasteur, L 2 J , (1972). 2. D.A.R. Simmons. Immunology, 20, 17 (1971). 3. S. Stirm, A.M. Staub, B. Leluc, H. Mayer, 0. Luderitz and 0. Westphal. Biochem. Z e i t s c h r i f t , _3j*4, ^01 (1966). 4. M. T o r i i , K. Sakakibara and E. Kabate. J . 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