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Synthesis of a series of disaccharides of potential immunological significance Bebault, Gwendolyn Mary 1974

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cl SYNTHESIS OF A SERIES OF DISACCHARIDES OF POTENTIAL IMMUNOLOGICAL SIGNIFICANCE By GWENDOLYN MARY BEBAULT B.Sc, U n i v e r s i t y of B r i t i s h Columbia, 1969 A Thesis Submitted i n P a r t i a l Fulfilment of the Requirements f o r the Degree of Doctor of Philosophy i n the Department of Chemistry We accept t h i s thesis a a conforming to the required standard THE UNIVERSITY OF BRITISH"COLUMBIA A p r i l , 1974 EXTERNAL EXAMINER: P.J. GAREGG In s t i t u t e f o r Organic Chemistry, Stockholm U n i v e r s i t y , Stockholm Sweden In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT Disaccharides are us e f u l f o r immunological studies, the iden-t i f i c a t i o n of i s o l a t e d polymer fragments, and studies on model compounds but few have been synthesized. The syntheses of four new disaccharides of the 4 -0-glycopyranosyl-L-rhamnopyranose seri e s namely 4 - 0-g-D-glucopyranosyl--L-rhamnopyranose ( 1 5 ) , 4-0-a-L-rhamnopyranosyl-L-rhamnopyranose ( 2 6 ) , 4 - 0 --a-D-mannopyranosyl-L-rhamnopyranose ( 3 8 ), and 4-0-8-D-mannopyranosyl-L--rhamnopyranose (56) are reported. The s e l e c t i v e l y s u b s t i t u t e d aglycon used f o r a l l the members of thi s s e r i e s was methyl 2 , 3 - 0-isopropylidene-a-L-rhamnopyranoside (3) prepared by a c e t a l a t i o n of methyl a-L-rhamnopyranoside and p u r i f i e d through c r y s t a l -l i n e methyl 4 - 0-acetyl - 2 , 3 - 0-isopropylidene-a-L-rhamnopyranoside ( 2 ) . Compound 2_ was deacetalated to give methyl 4 -0-acetyl-a-L-rhamno--pyranoside which served as a key intermediate f o r the preparation of the standards 2,3-di-O-methyl-L-rhamnose and 4-deoxy-L-erythritol. Disaccharide J15_ was prepared i n an o v e r a l l y i e l d of 55% based on the aglycon by f i r s t condensing 2 , 3 , 4 , 6 - tetra-O-acetyl-a-D-glucopyranosyl bromide with 3^  i n the presence of mercuric cyanide i n a c e t o n i t r i l e to give c r y s t a l l i n e methyl 2 , 3 - C - i s o p r o p y l i d e n e - 4 - 0 - ( 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - g - D --glucopyranosyl)-a-L-rhamnopyranoside (10) i n 80% y i e l d based on 3_. Since the isopropylidene group of 1_0 could not be acetolyzed i t was removed with t r i f l u o r o a c e t i c a c i d to give methyl 4 - 0 - ( 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - B - D - g l u c o --pyranosyl)-a-L-rhamnopyranoside (11) . A c e t o l y s i s of n_ gave c r y s t a l l i n e 1 , 2 , 3 - t r i - 0 - a c e t y 1 - 4 - 0 - ( 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - i 3 - D - glucopyranosyl)-a-L--rhamnopyranose ( 1 4 ). Deacetylation of 1_1_ gave the methyl glycoside of the disaccharide which, as f o r the subsequent disaccharides, was subjected to methylation and periodate oxidation studies to confirm the 1,4 linkage. An improved method f o r the q u a n t i t a t i o n of periodate oxidation degradation products i s described. Deacetylation of 1_4_ gave L5_ which was reduced to give 4-0-B-D-glucopyranosyl-L-rhamnitol which upon a c e t y l a t i o n gave crys-t a l l i n e 1,2,3,5-tetra-C-acetyl-4-0-(2,3,4,6-tetra-O-acetyl-g-D-glucopyran--osyl)-L-rhamnitol. In an analogous manner disaccharides 26_ and 38 were prepared i n o v e r a l l y i e l d s of 60 and 55% based on the aglycon by condensing 2,3,4-tri--O-acetyl-a-L-rhamnopyranosyl bromide and 2,3,4,6-tetra-O-benzoyl-a-D--mannopyranosyl bromide r e s p e c t i v e l y with _3. The deblocking procedures p a r a l l e l e d those of the D-gluco analog. Disaccharide 26_ was characterized as c r y s t a l l i n e methyl 2,3-di-0-acetyl-4-0-(2 J3,4-tri-C-acetyl-a-L-rhamno--pyranosyl)-ct-L-rhamnopyranoside, 1,2,3-tri-O-acetyl-4-0-(2,3,4-tri-O-acetyl--a-L-rhamnopyranosyl)-a-L-rhamnopyranose, and 1,2,3,5-tetra-C-acetyl-4-0--(2,3,4-tri-0-acetyl-a-L-rhamnopyranosyl)-L-rhamnitol. Disaccharide 38^  was c r y s t a l l i n e and was also characterized as c r y s t a l l i n e 1,2,3-tri-c7-acetyl-4--0-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-a-L-rhamnopyranose and l,2,3,5-tetra-0-acetyl-4-0-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-L--rhamnitol. A combined reduction a c e t y l a t i o n procedure f o r the 2,3-di-O--methyl-L-rhamnose obtained from methylated 26 produced a boron-containing d e r i v a t i v e of 2,3-di-O-methyl-L-rhamnitol. Proton magnetic resonance spec-troscopy was used to substantiate the configuration of the linkage i n these disaccharides. A general method f o r the synthesis of 3-D-mannopyranosides was developed to synthesize disaccharide 56^ . This was done by preparing 4,6--di-0-acetyl-2,3-0-carbonyl-a-D-mannopyranosyl bromide (44) through sequential formation of the carbonate, a c e t o l y s i s , and bromination of methyl 4 ,.6-0-benzylidene-a-D-mannopyranoside. Bromide 44 was condensed with methanol to give c r y s t a l l i n e methyl 4,6-di-0-acetyl-2,3-0-carbonyl-g--D-mannopyranoside (45) i n 87% y i e l d . Deacylation of 45_ gave methyl 3-D--mannopyranoside which was characterized as i t s c r y s t a l l i n e isopropylate and peracetate. Disaccharide 5_6_ was prepared i n an o v e r a l l y i e l d of 50% based on the aglycon by condensing 44 with 3_ i n the presence of s i l v e r oxide i n chloroform. The deblocking procedures were s i m i l a r to those f o r the previous disaccharides. A c e t o l y s i s of the disaccharide intermediates i n the sequence was profoundly influenced by the presence of the c y c l i c carbonate group. Disaccharide 5_6_ was characterized as c r y s t a l l i n e methyl 4-0-8-D-mannopyranosyl-a-L-rhamnopyranoside isopropylate and 1,2,3-tri-O--acety1-4-0-(2,3,4,6-tetra-0-acetyl-B-D-mannopyranosyl)-a-L-rhamnppyranose i v ACKNOWLEDGEMENTS I remain s i n c e r e l y g r a t e f u l to Professor G.G.S. Dutton f o r his patience, guidance, and reassuring a t t i t u d e during the course of t h i s work. I deeply appreciate the opportunity f o r personal and p r o f e s s i o n a l develop-ment that he has given me. I would l i k e to thank the other members of the lab, p a r t i c u l a r l y J.M. Berry, N.A. Funnell, Y.M. Choy, M.T. Yang, and A.C. Wootton f o r many i n t e r e s t i n g and h e l p f u l discussions and C.K. Warfield f o r te c h n i c a l a s s i s -tance. I would also l i k e to thank Professors Rosenthal and H a l l and t h e i r research groups f o r several discussions r e l a t e d to t h i s work. I would l i k e to express my gratitude to the many s t a f f members of t h i s department who contributed time and energy to t h i s work, e s p e c i a l l y Mr. P. Borda, P h y l l i s Watson, Evert Koster, and George Gunn. I g r a t e f u l l y acknowledge the f i n a n c i a l support of the National Research Council (1969-1973) and an H.R. MacMillan Family Scholarship (1974).. F i n a l l y , I thank Beatrix Kriszan f o r assistance with the i l l u s t r a -tions and Jacque Houlden f o r typing the t h e s i s . V TABLE OF CONTENTS Page ABSTRACT i ACKNOWLEDGEMENTS i v TABLE OF CONTENTS v LIST OF FIGURES v i I INTRODUCTION 1. General 1 2. Rationale f o r Disaccharide Synthesis 3 3. H i s t o r i c a l Background 8 II RESULTS AND DISCUSSION 1. Aglycon 18 2. 4-0-g-D-Glucopyranosyl-L-rhamnopyranose 25 3. 4-0-a-L-Rhamnopyranosyl-L-rhamnopyranose 37 4. 4-0-a-D-Mannopyranosyl-L-rhamnopyranose 44 5. 4-0-g-D-Mannopyranosyl-L-rhamnopyranose 50 6. Conclusions 62 III EXPERIMENTAL 1. General Methods 65 2. Methyl 2,3-O-isopropylidene-a-L-rhamnopyranoside 67 3. 4-0-S-D-Glucopyranosy1-L-rhamnopyranose 74 4. 4-0-a-L-Rhamnopyranosyl-L-rhamnopyranose 81 5. 4-0-cx-D-Mannopyranosyl-L-rhamnopyranose 87 6. 4-0-B-D-Mannopyranosyl-L-rhamnopyranose 94 IV BIBLIOGRAPHY 106 APPENDIX 115 v i LIST OF FIGURES Figure Page 1 Chemically Synthesized Disaccharides of the Common 6 Neutral Sugars 2 Disaccharides Described i n t h i s Thesis 7 3 Side Reactions during Disaccharide Condensations 13 4 Synthetic Sequence f o r the Preparation of the Aglycon 20 5 Preparation of Methylation and Periodate Oxidation 23 Standards from Methyl 4-0-acetyl-a-L-rhamnopyranoside 6 4-0-3-D-Glucopyranosyl-L-rhamnopyranose 27 7 Hydrolysis Products of Methylated Disaccharide 30 8 Periodate Oxidation of Disaccharide 32 9 4-O-D-Glucopyranosyl-L-rhamnopyranose; Proton Magnetic 38 Resonance Spectra of the Anomeric Region 10 4-0-a-L-Rhamnopyranosyl-L-rhamnopyranose 39 11 4-0-a-D-Mannopyranosyl-L-rhamnopyranose 46 12 4-0-D-Mannopyranosyl-L-rhamnopyranose; Proton Magnetic 48 Resonance Spectra of the Anomeric Region 13 Synthetic Route f o r the Preparation of 51 4,6-Di-0-acetyl-2,3-C-carbonyl-a-D-mannopyranosyl bromide 14 4-0-3-D-Mannopyranosyl-L-rhamnopyranose 57 15 Competing Pathways i n A c e t o l y s i s 60 16 Physical Properties of Disaccharides and Derivatives 63 1 I INTRODUCTION 1. General Sugars along with amino acids, nucleotides, and l i p i d s comprise the basic b u i l d i n g blocks of nature. As do the other b u i l d i n g blocks, sugars occur i n nature l a r g e l y as polymers. These polymers are named according to the number of sugar u n i t s , i . e . monosaccharides, that they contain. For example polymers containing two, three, four, two to ten, and more than ten sugar units are c a l l e d d i - , t r i - , t e t r a - , o l i g o - , and polysaccharides r e s p e c t i v e l y . Many n a t u r a l l y occurring polymers are composed of more than one kind of b u i l d i n g block such as glycoproteins, g l y c o l i p i d s , and lipopolysaccharides. Polysaccharides and oligosaccharides are widely d i s t r i b u t e d i n a l l l i v i n g organisms and serve four main func-tions''': as food reserves, i l l u s t r a t e d by starch, glycogen, and galacto-mannans; as s t r u c t u r a l components, exemplified by c e l l u l o s e , hemicelluloses, and c h i t i n ; as p r o t e c t i v e coatings, i l l u s t r a t e d by plant gums and b a c t e r i a l capsules, and as determinants of s p e c i f i c i t y , demonstrated by blood group 2 3 4 S substances , microbial antigens ' and endotoxins, enzyme regulators , and gangliosides^. Most sugars can be described as polys u b s t i t u t e d tetrahydropyran rings where the substituents are hydroxyl and hydroxymethyl groups and the numbering i s as shown below. 2 Obviously many diastereoisomers are possible and t h i s leads to the s p e c i f i c sugars such as glucose, galactose, and mannose. Op t i c a l isomerism also gives r i s e to the enantiomeric D and L s e r i e s where C-6 i s above the plane of the r i n g i n the D-series and below i t i n the L-series i n most cases. The configuration at C - l (the anomeric carbon) i s designated a when the substituent i s on the opposite side of the r i n g to C-6 and 3 when i t i s on the same side of the r i n g as C-6. These m u l t i f u n c t i o n a l compounds contain hemi-acetal and secondary and primary hydroxyl groups. Other fun c t i o n a l v a r i a t i o n s such as 6-deoxy, 2-amino, and 6-carboxyl are common and lead to sugars such as rhamnose, glucosamine, and glucuronic a c i d . In polysaccharides a l l linkages between sugar units are formed from the loss of a molecule of water between C - l of one sugar and any other hydroxyl on the other sugar, giving r i s e to a s e r i e s of a c e t a l s . Although there are many combinations p o s s i b l e , natural polysaccharides tend to be r e p e t i t i v e rather than random i n both t h e i r constituent sugars and linkages. Thus c e l l u l o s e i s composed of g-D-glucose residues linked through C-4. Branching i s possible when more than one u n i t i s joined through C - l to the same sugar residue. Also several kinds of sugar r e s i -dues can combine to form heteropolymers. In both these cases there i s s t i l l a high degree of uniformity i n that the majority of the branches are attached to a s p e c i f i c p o s i t i o n of a c e r t a i n sugar and the sugars i n a heteropolymer are often arranged i n a d e f i n i t e order to form repeating u n i t s . Again each sugar has a s p e c i f i c configuration and point of attach-ment. Fragmentation of such polymers, therefore, gives d i s t i n c t o l i g o -saccharides. This holds true even though polysaccharides do not e x i s t as d i s c r e t e macromolecules a l l of the same molecular weight but rather as a homologous s e r i e s of polymers with a d i s t r i b u t i o n of molecular weights 3 about a mean value i . e . they are polydispersed. 2. Rationale for Disaccharide Synthesis Since t h e i r discovery man has been in t e r e s t e d i n determining the structure of these carbohydrate polymers. Synthetic oligosaccharides of unambiguous structure are invaluable i n the s t r u c t u r a l e l u c i d a t i o n of 7 i s o l a t e d polymer fragments. An e a r l y example of t h i s was the a c e t o l y s i s of c e l l u l o s e to give c e l l o b i o s e octaacetate which established the config-uration and the linkage between the glucose units i n c e l l u l o s e to be g-D-1,4. Because synthetic oligosaccharides are often a v a i l a b l e i n reasonable q u a n t i t i e s several d e r i v a t i v e s , each with d i s t i n c t i v e p h y s i c a l properties, may be prepared. This provides a v a r i e t y of authentic stan-dards from which the one or ones most appl i c a b l e to a small amount of a p a r t i c u l a r i s o l a t e d fragment can be chosen. Synthetic oligosaccharides not only provide important p h y s i c a l constants but also the defined model systems required f o r the development of new and/or improved methods of s t r u c t u r a l a n a l y s i s . Products or r e s u l t s from degradative techniques, such as methylation and periodate oxidation, that cannot be predicted or explained by known mechanisms or reactions can only be discovered and i n v e s t i g a t e d by subjecting compounds of known str u c t u r e to the same degradative procedures. The ever-increasing use of instrumental techni-ques, u t i l i z i n g p h y s i c a l measurements, such as nuclear magnetic resonance 8 > 9 « . « . 10 , . . . . . . . 11,12 . . , ^ , mass spectrometry , and c i r c u l a r tdichroism to elucidate s t r u c -tures requires a r e s e r v o i r of data on compounds of known structure i n order to e s t a b l i s h the d i s t i n g u i s h i n g c h a r a c t e r i s t i c s of each s t r u c t u r a l feature. 4 The other area of i n t e r e s t to man i s the r e l a t i o n s h i p between the chemical structure of these polymers and t h e i r b i o l o g i c a l function. Again, synthetic oligosaccharides of unambiguous structure play an impor-tant r o l e . Antigenic polysaccharides contain s p e c i f i c oligosaccharide units which combine with the antibody i n the immunological reaction and thus are c a l l e d immunodominant s i t e s , antigenic determinants, determinants of immunological s p e c i f i c i t y , or haptens. The s t r u c t u r a l composition of the determinant of immunological s p e c i f i c i t y can be determined by hapten i n h i b i t i o n ^ ' ' ^  or complement f i x a t i o n i n h i b i t i o n ^ studies using oligosaccharides of known s t r u c t u r e . The binding s i t e and the s i t e of action of enzymes on polysaccharides which are substrates can be deter- ' mined by i n h i b i t i o n studies or by varying the substrate using oligosacchar-17 ides of defined composition . S p e c i f i c a l l y l a b e l l e d r a d i o a c t i v e o l i g o -saccharides made a v a i l a b l e by s y n t h e s i s ^ ' ^ can be useful i n the el u c i d a t i o n of b i o s y n t h e t i c pathways when used i n feeding experiments. Since synthetic a n t i b i o t i c s with minor s t r u c t u r a l v a r i a t i o n s can be used to study the s t r u c t u r a l requirements f o r a c t i v i t y and since some a n t i -b i o t i c s contain sugars i t i s necessary to have a v a i l a b l e the methods f o r 20-22 coupling the sugar moieties i n a l l t h e i r s t r u c t u r a l v a r i a t i o n s 23 Toxins present an analogous . case . In many cases the bare attempt to synthesize a natural product sheds l i g h t on i t s function i n nature by revealing the properties, r e a c t i v i t y , s t a b i l i t y , and/or l a b i l i t y of cer-t a i n of i t s f u n c t i o n a l i t i e s . Synthesis provides an excellent t e s t i n g ground f o r the develop-ment of the fundamental p r i n c i p l e s of organic chemistry. Each new synthetic endeavour e i t h e r supports the e x i s t i n g p r i n c i p l e s or provides another exception which eventually leads to a new p r i n c i p l e being formulated. Synthetic studies of organic compounds enrich organic chemistry i n the areas of new reactions, r e a c t i o n mechanisms, and stereochemistry. For the foregoing reasons the synthesis of disaccharides was undertaken. As can be seen by Figure 1 the t o t a l number of synthetic disaccharides i s very l i m i t e d i n d i c a t i n g that disaccharide synthesis i s an area i n which much development i s needed. Although L-rhamnose i s widely d i s t r i b u t e d i n nature and i s commonly found as a constituent of 24 25 2627 plant gums , plant glycosides , and b a c t e r i a l polysaccharides ' , no 28 disaccharide having L-rhamnose as the aglycon has been synthesized with 29 the exception of 4-0-(3-<3-methyl-8-D-galactopyranosyl)-L-rhamnose Therefore four new disaccharides having L-rhamnose as the reducing end have been synthesized, namely 4-0-8-D-glucopyranosyl-L-rhamnopyranose ( s c i l l a b i o s e ) (15), 4-0-a-L-rhamnopyranosyl-L-rhamnopyranose (26), 4-0-a-D-mannopyranosyl-L-rhamnopyranose (38), and 4-0-B-D-mannopyranosyl--L-rhamnopyranose (56)- Their structures are shown i n Figure 2. 4-0-D-Mannopyranosyl-L-rhamnopyranose occurs with the g l y c o s i d i c linkage i n both the a-D and 3-D anomeric configurations i n the antigenic c e l l - w a l l lipopolysaccharides of d i f f e r e n t Salmonella species. Serogroups A, B, and contain an a-D linkage while D^ and E contain a 8-D linkage. The two c o n f i g u r a t i o n a l l y d i f f e r e n t disaccharides i s o l a t e d from natural sources were o r i g i n a l l y confused owing to the lack of authentic standards 30 The synthetic disaccharides would also be useful i n immunological 31 studies . 4-0-a-L-Rhamnopyranosyl-L-rhamnopyranose was thought to be a constituent of Shigella flexneri O-antigens by complement f i x a t i o n i n h i b i -16 32 t i o n studies but i t was l a t e r shown that i n these antigens the linkage 33 was not 1,4. A rhamnobiose has been i s o l a t e d from Viva conglobata and 34 preliminary studies have indicated that i t i s 4-0-a-L-rhamnopyranosyl-6 Figure 1. Chemically Synthesized Disaccharides of the Common Neutral Sugars V \ 0 ARABINOSE TO UJ CO O CO a CC 8 UJ cn .3 >-a X a 0 GALACTOSE TO GLUCOSE TO MANNOSE TO UJ CO o o D a u_ 8 . LU LO o z < X a CC 8 1-1 1*2 A R A B I N O ^ 1-4' 1*5 1-6 X X X X 1-1 1-2 R I B O S E ! 1-4 1-5 1-6 X X X 1-1 1-2 X Y L O S E ! 1-5 1-6 X X X X X X X X X X X X X X X X X X U l G A L A C T O S E 1-4 L-5 L-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 1-1 1-2 G L U C O S E 3 w> 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 M A N N O S E 1 1-4 1-5 1-6 X X X X X » X X X X X 1-1 1-2 F U C O S E 1-4 1-5 1./ X X X 1-1 1-2 R H A M N O S E 1-4 1-5 1-6 X X X X igure 2. Disaccharides Described i n t h i s Thesis 4-0-B-D-Glucopyranosyl-L-rhamnopyranose ( 1 5 ) O H 4-0-a-L-Rhamnopyranosy1-L-rhamnopyranose ( 2 6 ) 4-c9-a-D-Mannopyranosyl-L-rhamnopyranose ( 3 8 ) O H 4-0-3-D-Mannopyranosyl-L-rhamnopyranose ( 5 6 ) 8 -L-rhamnose but comparison of the p h y s i c a l constants with those of the synthetic disaccharide do not confirm t h i s . These s t r u c t u r a l studies would have been g r e a t l y s i m p l i f i e d had the authentic standard been a v a i l -able. 4-0-a-L-Rhamnopyranosyl-L-rhamnose i s l i k e l y to occur i n those Klebsiella capsular polysaccharides which contain a high percentage of L-rhamnose residues. S c i l l a b i o s e was f i r s t i s o l a t e d from the p a r t i a l acid hydrolysates of the glycosides s c i l l a r i n A and g l u c o s c i l l i p h a e o s i d e 35 36 obtained from S c i l l a maritima ' and i t s i d e n t i f i c a t i o n as 4-0-g-D-37 38 -glucopyranosyl-L-rhamnose was subsequently established ' . Since aldobiouronic acids may be e a s i l y characterized as the derived neutral disaccharides, 4-0-B-D-glucuronosyl-L-rhamnose from the p a r t i a l a c i d hydrolysates of the a c i d i c polysaccharides of the green seaweeds 39 40 Acrosiphoma centralis and Ulva lactuca has been characterized as 41 4-0-B-D-glucopyranosy1-L-rhamnose. Recently 4-0-B-D-glucuronosyl-L--rhamnose has been i d e n t i f i e d as a s t r u c t u r a l component of the capsular polysaccharide from Klebsiella type 9. These examples give some i n d i c a t i o n of where these four disaccharides could have been used had they been a v a i l a b l e , so t h e i r r e a l p o t e n t i a l l i e s i n f a c i l i t a t i n g the e l u c i d a t i o n of the structures and r e l a t e d functions of oligosaccharides yet to be d i s -covered. 3. H i s t o r i c a l Background Disaccharides can be synthesized e i t h e r enzymatically or chem-i c a l l y . Only chemical synthesis w i l l be discussed here. There are many chemical methods f o r disaccharide synthesis. Unsubstituted monosaccharides 42 43 44 ' or p a r t i a l l y blocked monosaccharides can be condensed under dehy-drating conditions such as s u l f u r i c acid, phosphorus pentoxide, or zinc c h l o r i d e . New disaccharides can also be prepared by the chemical a l t e r a -t i o n of e i t h e r the sugar r e s i d u e s ^ or the l i n k a g e ^ of a v a i l a b l e disacchar-ides. However the least ambiguous method of synthesizing disaccharides i s to couple two sugars, one of which has been a c t i v a t e d at C - l and blocked elsewhere and the other which has been s e l e c t i v e l y blocked to leave a s i n g l e hydroxyl group r e a c t i v e . The a d d i t i o n reactions of mono-saccharides having one free hydroxyl group to 1,2 anhydrides are examples 47 of t h i s as i n d i c a t e d by Lemieux and Huber's synthesis of sucrose heptaacetate from B r i g l ' s anhydride (3,4,6-tri-O-acetyl-l,2-anhydro-cx--D-glucose) and 1,3,4,6-tetra-O-acetyl-D-fructofuranose. Also i n t h i s class are the reactions of acetylated monosaccharide l , 2 - ( a l k y l ortho-acetates) with other s u i t a b l y blocked monosaccharides having a free hydroxyl group i n the presence of c a t a l y t i c amounts of mercuric bromide to give good y i e l d s of the 1,2-trans glycosides. This r e a c t i o n has been 48 49 31 perfected by Kochetkov ' and used s u c c e s s f u l l y by others . Another type involves the e l i m i n a t i o n of an a l k a l i s a l t such as sodium bromide from the sodium s a l t of a monosaccharide having a free hydroxyl group and an acylated g l y c o s y l bromide as exemplified by the r e a c t i o n of the sodium s a l t of 1,2,3,4-tetra-O-acetyl-g-D-glucopyranose with 2,3,4,6-tetra-0--acetyl-a-D-glucopyranosyl bromide to form gentiobiose o c t a a c e t a t e ^ . However the most important and v e r s a t i l e method i s the Koenigs-Knorr reaction which involves the e l i m i n a t i o n of a hydrogen hal i d e between a substituted g l y c o s y l h a l i d e and an unsubstituted hydroxyl group of a second monosaccharide i n the presence of an acid acceptor such as a heavy metal s a l t or organic base. The h a l i d e most commonly used i s bromide since i t i s at the same time s u f f i c i e n t l y stable to be e a s i l y prepared and s u f f i c -10 ently r e a c t i v e towards free hydroxyl groups. The chlorides are less reac-t i v e but are more e a s i l y prepared i n the less common 6-D configuration which i s sometimes u s e f u l . The f l u o r i d e s do not normally r e a c t . The iodides are too r e a c t i v e to be prepared but may be prepared in situ. The most commonly used condensing agents are s i l v e r carbonate, s i l v e r oxide, quinoline, and c o l l i d i n e . S i l v e r perchlorate i s used with t r i t y l deriva-t i v e s (Bredereck r e a c t i o n ) . Cadmium c a r b o n a t e ^ has been used with s t e r o i d a l aglycons. Since the r e a c t i o n of the hydrogen hal i d e with s i l v e r oxide or s i l v e r carbonate l i b e r a t e s water which competes with the glycoside formation an i n t e r n a l desiccant such as anhydrous calcium s u l f a t e , sodium s u l f a t e , copper s u l f a t e , or calcium c h l o r i d e i s added. Iodine i s also com-monly added. The Koenigs-Knorr reaction i s u s u a l l y c a r r i e d out i n an i n e r t solvent such as chloroform or benzene. The other necessary component i s a s u i t a b l y substituted monosaccharide which w i l l react at a s i n g l e p o s i -t i o n . The development of the chemistry of hydroxyl blocking groups was necessary i n order to provide these. The Koenigs-Knorr r e a c t i o n has been 52-57 extensively reviewed and a t y p i c a l example i s the r e a c t i o n of 2,3,4,6-tetra-c3-acetyl-a-D-glucopyranosyl bromide with l,2,3,4-tetra-0-58 -acetyl-3-D-glucopyranose to give gentiobiose octaacetate 11 The anomeric e f f e c t generally d i r e c t s the bromine at C-l to the a x i a l p o s i t i o n . Bromides having an acyloxy group at C-2 ois to the bromine at C - l normally react with inv e r s i o n whereas those with an acyloxy group at C-2 trans to the bromine at C-l react with predominant retention of configuration. In both cases the 1,2 trans glycoside i s formed. The f i r s t case can be explained e i t h e r by a S^2 type (ion-pair) mechanism or a S ^ l mechanism followed by p a r t i c i p a t i o n of the acyloxy group on C-2 to form a c y c l i c acetoxonium ion which forces the incoming nucleophile to attack from a trans p o s i t i o n at C - l . Case 1: The second case can be explained by a S^2 a s s i s t e d displacement of the halogen by the p a r t i c i p a t i n g acyloxy group on C-2 to form the c y c l i c acetoxonium ion followed by replacement by the aglycon from the opposite side. o 12 Anomeric and s t e r i c e f f e c t s are probably also operative but are not predominant when a p a r t i c i p a t i n g group i s present on C-2. E i t h e r may become dominant i n other circumstances. Thus the Koenigs-Knorr r e a c t i o n provides a 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 glycosides almost e x c l u s i v e l y . Later the Koenigs-Knorr r e a c t i o n was modified by using mercury s a l t s such as mercuric acetate i n benzene (Zemplen reaction) or mercuric cyanide and mercuric bromide which were soluble i n the newer polar s o l -vents such as a c e t o n i t r i l e and nitromethane ( H e l f e r i c h reaction) . Some cases have been reported where the mercury s a l t s give some 1,2 cis glycosides The y i e l d s of Koenigs-Knorr reactions are l i m i t e d by two f a c t o r s , the r e a c t i v i t y of the a l c o h o l i c f u n c t i o n a l i t y and the side reactions to which the g l y c o s y l bromide i s prone. The monosaccharides being large and c y c l i c are not as r e a c t i v e as the small a l i p h a t i c alcohols such as methanol, ethanol, etc. Usually a c y c l i c sugars are more r e a c t i v e than c y c l i c ones, primary hydroxyls are more r e a c t i v e than secondary ones, and equatorial hydroxyls are more r e a c t i v e than a x i a l ones. S t e r i c a l l y hindered hydroxyls are also less r e a c t i v e . Several side reactions as well as the desired glycoside formation may a r i s e from the intermediate c y c l i c acetoxonium ion as shown i n Figure 3. Attack of the incoming nucleophile 6 2 at B rather than A leads to the formation of two isomeric orthoesters Attack of cyanide ion at A gives the 1-cyano compound whereas attack at B 6 3 gives two isomeric products . Water can hydrolyze the bromide to give the 1-hydroxy sugar or attack the c y c l i c acetoxonium ion at A or B to give 64 the 1- or 2-hydroxy sugars . These are then a v a i l a b l e to condense and form unwanted disaccharides. Goldschmidt and P e r l i n ^ have shown that 14 s i l v e r oxide i t s e l f decomposes the g l y c o s y l bromide leading to products such as 1-hydroxy sugars, dimeric orthoesters, and even a t r i s a c c h a r i d e 66 orthoester . They suggest that iodine acts as an i n h i b i t o r of these reactions. These side reactions explain why condensation reactions give multicomponent mixtures. The previously mentioned developments paved the way f o r the pre-paration, described i n t h i s t h e s i s , of 4-0-g-D-glucopyranosyl-L-rhamno-pyranose from 2, 3,4,6-tetra-c9-acetyl-a-D-glucopyranosyl bromide and the s u i t a b l y blocked aglycon methyl 2,3-0-isopropylidene-a-L-rhaninopyranoside (3) i n the presence of mercuric cyanide i n a c e t o n i t r i l e . This provides a t y p i c a l example of Case 1 s t e r i c r e l a t i o n s h i p s . In an analogous manner 4-0-a-L-rhamnopyranosyl-L-rhamnopyranose and 4-0-ct-D-mannopyranosyl-L- ' -rhamnopyranose were prepared by re a c t i n g 2,3,4-tri-O-acetyl-a-L-rhamnopy-ranosyl bromide and 2,3,4,6-tetra-O-benzoyl-a-D-mannopyranosyl bromide r e s p e c t i v e l y with the aglycon 3 i n the presence of mercuric cyanide i n a c e t o n i t r i l e . These are t y p i c a l examples of Case 2 s t e r i c r e l a t i o n s h i p s . The synthesis of 4-0-g-D-mannopyranosyl-L-rhamnopyranose required the formation of a 1,2 ais glycoside. The 1,2 cis glycosides have presented much more of a challenge to chemists of organic synthesis, but are i n t e r e s t i n g because they are often the n a t u r a l l y occurring forms. a-D-Glucosides occur i n glycogen, amylose, amylopectin, sucrose, a n t i b i o t i c s , and fungal and 28 b a c t e r i a l polysaccharides. B-D-Mannosides occur as plant mannans , as 28 67 68 glucomannans i n hemicelluloses , as galactomannans i n seeds ' , and i n 69 Klebs%ella capsular polysaccharides . Most of the synthetic work to date on 1,2 cis glycosides has been done on glucose. Once the d i r e c t i n g e f f e c t of the p a r t i c i p a t i n g group on C-2 i n the formation of 1,2 trans glycosides was understood the obvious choice f o r the synthesis of 1 ,2 cis glycosides was to put a n o n p a r t i c i p a t i n g group on C - 2 . Several nonparticipating groups 70 71 72 73 74 75 were t r i e d such as free hydroxyl ' , methyl , n i t r o ' , t o s y l , 76 77 78 t r i c h l o r o a c e t y 1 and benzyl ' . In most cases the configuration of the o r i g i n a l h a l i d e was now inverted by an incoming nucleophile. This was 79 s a t i s f a c t o r y for the synthesis of B-D-arabinofuranosides but required a _ . i • j * , ™ i * j T 7 0 , 7 1 , 7 3 , 7 5 , 7 6 , B-halide i n order to get a-D-glucosides. In the cases where B-D-glucopyranosy1 chlorides were employed a-D-glucosides were produced. 80 However B-chlorides are not r e a d i l y prepared i n high y i e l d s . Noting that halides could be s t a b i l i z e d by electron withdrawing groups Ishikawa and 81 Fletcher now synthesized 2 - 0 - b e n z y l - 3 , 4 , 6 - t r i - 0 - p - n i t r o b e n z o y l - B - D --glucopyranosyl bromide, which combines a nonparticipating group at C-2 with a s t a b i l i z e d g-bromide and demonstrated i t s s t e r e o s e l e c t i v i t y for the production of a-D-glucosides. This h a l i d e has proven quite useful i n the preparation of a -D-gluco-. 8 2 , 8 3 sides 84 Frechet and Schuerch have extended t h i s idea by using 2 , 3 , 4 --tri-C-benzyl-D-glucopyranosyl bromides with various electron donating and e l e c t r o n withdrawing groups on C-6 i n order to get s t e r e o s e l e c t i v e reactions. 85 86 Zorbach and coworkers (and references therein) and Flowers and coworkers have each studied the e f f e c t s of substituents at the various positions i n the r i n g on reactions at C - l . 87 Lemieux and coworkers have developed an a l t e r n a t i v e method using n i t r o s y l chlorides for the synthesis of a-D-glucosides and a-D--galactosides. It i s p a r t i c u l a r l y useful f o r disaccharides containing keto oo and amino sugars. Another method developed by Lemieux and Hendriks involves 16 s u b s t i t u t i o n with no n p a r t i c i p a t i n g benzyl groups and the rapid e q u i l i b r a -t i o n of the a- and B-bromides i n the presence of a quaternary ammonium bro-mide while doing the condensation with a s u f f i c i e n t l y sluggish condensing agent such as diisopropylamine or c o l l i d i n e so that e s s e n t i a l l y only the more re a c t i v e g-bromide reacts. This has been used s u c c e s s f u l l y to make a-L-fucosides. Some attempts have now been made to replace the halogen at C - l with a good leaving group that prefers the equatorial p o s i t i o n and there-89 fore i n v e r s i o n would lead to a-D-glucosides. On t h i s theme F e r r i e r has 90 t r i e d phenyl 1-thio-g-D-glucopyranoside and James and Angyal have used 91 the nitrobenzene-p-sulphonyloxy group i n another context. F e r r i e r et al. have also used acylated 2-hydroxyglycals with boron t r i f l u o r i d e f o r the production of 1,2 cis glycosides. This i s p a r t i c u l a r l y u s e f u l f o r the synthesis of disaccharides having 3-deoxy sugars as t h e i r non-reducing 92 93 moieties . West and Schuerch have reviewed the attempts at making 1,2 cis glycosides and have introduced e l e c t r o p o s i t i v e equatorial substituents at C - l with n o n p a r t i c i p a t i n g groups elsewhere i n order to get stereoselec-t i v e i n v e r s i o n . The 3-D-mannopyranos.ides f i r s t reportedly synthesized were l a t e r 94 shown to be i n c o r r e c t l y assigned and were a c t u a l l y a-D-mannopyranosides Considering the foregoing developments the synthesis of B-D-mannosides should have been straightforward since the commonly a v a i l a b l e a-bromide should on inversion produce the 1,2 cis glycoside. The only requirement should have been a nonparticipating group on C-2. Nonparticipating groups such as 95 methyl, benzyl, and hydroxyl did not lead to B-D-mannosides s t e r e o s e l e c t i v e l y It should be noted that often i n the attempts to produce 1,2 cis glycosides 17 s t e r e o s e l e c t i v e i n v e r s i o n was achieved with small r e l a t i v e l y strong nucleophiles such as methanol but not with s e l e c t i v e l y s ubstituted mono-95 saccharides. However, when Gorin and P e r l i n used a 2,3 c y c l i c carbonate as the non p a r t i c i p a t i n g group s t e r e o s e l e c t i v e i n v e r s i o n was achieved. 96 97 C y c l i c carbonates had previously been used ' to prepare 1,2 cis gly-98 cosides i n the furanose s e r i e s . Zorbach et at. have condensed a c y c l i c 72 carbonate of rhamnopyranose with s t e r o i d a l aglycons. It has been suggested that the trans a x i a l substituent at C-2 of mannose impedes an incoming nucleophile from approaching from that side and hence prevents S^2 type su b s t i t u t i o n s of a-D-mannoses. Perhaps the fused 5-membered carbonate r i n g modifies the conformation of the pyranose r i n g so as to allow t h i s to occur. 99 It has also been proposed that the modified conformation r e s i s t s the formation of a planar carbonium ion about C - l . L i n d b e r g ^ ^ has recently developed an a l t e r n a t i v e method f o r the synthesis of B-D-mannosides which involves the synthesis of g-D-glucosides and subsequent s e l e c t i v e epimerization. 95 Following up the idea of Gorin and P e r l i n 4,6-di-C-acetyl-2,3--a-carbonyl-D-mannopyranosyl bromide (44) was used to synthesize 4-0-B-D--mannopyranosyl-L-rhamnopyranose. An improved synthetic route f o r the preparation of t h i s valuable b i c y c l i c bromide and condensation conditions modified to make t h i s a p r a c t i c a l method f o r the preparation of B-D-manno--pyranosides are described i n t h i s t h e s i s . 18 II RESULTS AND DISCUSSION The discussion w i l l f a l l i nto f i v e main areas; the aglycon which also served as a key intermediate f or the preparation of necessary stan-dards; 4-0-3-D-glucopyranosyl-L-rhamnopyranose which functioned as a model f o r the proof of s t r u c t u r e , deblocking, and c h a r a c t e r i z a t i o n procedures of the s e r i e s ; 4-0-a-L-rhamnopyranosyl-L-rharnnopyranose which served as a t e s t i n g ground f o r an a l t e r n a t i v e reduction a c e t y l a t i o n procedure which led to an anomalous boron-containing product; 4-0-a-D-mannopyranosyl-L--rhamnopyranose which demonstrates the u t i l i t y of proton magnetic resonance spectroscopy i n assigning configurations; and 4<-0-g-D-mannopyranosyl-L--rhamnopyranose f o r which i t was necessary to develop a general method f o r the synthesis of g-D-mannopyranosides. Many of the reactions employed i n this work are standard methods which w i l l not be discussed i n d e t a i l but the r e s u l t s w i l l be i l l u s t r a t e d by structures. Only the i n t r i g u i n g and p o t e n t i a l l y important facets of t h i s work w i l l be discussed. 1. Aglycon As previously mentioned a requirement f o r discaccharide synthesis i s a monosaccharide s e l e c t i v e l y s ubstituted so that a s i n g l e hydroxyl group i s r e a c t i v e . Methyl 2,3-0-isopropylidene-a-L-rhamnopyranoside (3) f i l l s these requirements. 19 It also happens that the hydroxyl group on C-4 of _3 i s r e l a t i v e l y r e a c t i v e and hence leads to good y i e l d s of disaccharides. L-Rhamnose has other advantages for the synthesis of disaccharides such as the f a c t that under the v i s u a l i z a t i o n procedures used f o r t h i n - l a y e r chromatography 6-deoxy sugars such as L-rhamnose appear yellow whereas a l l the ordinary hexoses appear black and hence compounds containing only L-rhamnose (yellow), h a l f L-rhamnose ( o l i v e green), and no L-rhamnose (black) can be d i s t i n g u i s h e d . The proton magnetic resonance spectrum of compounds containing 6-deoxy sugars such as 'L-rhamnose i s p a r t i c u l a r l y informative because the quanti-tation of the high f i e l d doublet a r i s i n g from the C-6 protons provides an i n t e r n a l standard for determining the amount of any other s i g n a l r e l a t i v e to L-rhamnose. This provides an easy method f o r determining the number of the various substituents per L-rhamnose moiety i n any molecule. The disaccharides synthesized i n t h i s t hesis a l l have a common aglycon, namely _3 so therefore form a s e r i e s of disaccharides a l l linked through p o s i t i o n 4 of L-rhamnose, that i s a s e r i e s of 4-0-glycopyranosyl-L-rhamnopyranoses. The synthetic scheme f o r the preparation of _3 i s shown i n Figure 4. Methyl a-L-rhamnopyranoside (1) was prepared e s s e n t i a l l y as described by Levene and Muskat^*. Compound 1_ c r y s t a l l i z e s r e a d i l y when seeded, but only when seeded," and thus provides a s t a r t i n g material f o r the aglycon which i s pure i n anomeric configuration and locked i n the pyranose r i n g structure. This pure methyl a-L-anomer assures a permanent configuration around C - l for a l l subsequent d e r i v a t i v e s i n c l u d i n g derived disaccharides. This greatly f a c i l i t a t e s t h e i r c r y s t a l l i z a t i o n . This s t a b i l i z e d linkage i s also b e n e f i c i a l f or immunochemical studies where the methyl glycoside of a disaccharide can be used to study the s p e c i f i c i t y of two linkages i n 31 sequence . The methyl a-L-glycosides of 38 and 5_6 are p a r t i c u l a r l y r e l e -vant as i t has been shown that L-rhamnose i s a-linked i n the Salmonella lipopolysaccharides . Insoluble acids i n the form of c a t i o n exchange resins can be used to catalyze acetal formation'''^ and were found to be the most conven-ient method for preparing _3 from 1_. The condition of the r e s i n i s the most i n f l u e n t i a l f a c t o r . It performs best when i t i s f r e s h l y regenerated and 103 dehydrated as much as p o s s i b l e with anhydrous methanol. As recommended i t was found advantageous to use 2,2-dimethoxypropane as the a c e t a l a t i n g reagent but since the s t a r t i n g material d i d not d i s s o l v e i n 2,2-dimethoxy--propane, acetone was also employed. The f a c t that i n t e r n a l desiccants d i d not improve the r e a c t i o n may be explained by the greater r e a c t i v i t y of 2,2-dimethoxypropane and hence the production of methanol rather than water. The formation of acetone polymers can be c o n t r o l l e d by carrying out the reaction at 0°. Under these conditions resins are s u f f i c i e n t c a t a l y s t s for the acetal formation yet weak enough acids that they do not d i s t u r b the methyl glycoside appreciably during the time required f o r the r e a c t i o n . It was found necessary to f i l t e r the product through calcium oxide to remove the traces of a c i d r e s u l t i n g from bleeding of the r e s i n . Compound 3 was 107 reported c r y s t a l l i n e once i n the l i t e r a t u r e and also c r y s t a l l i z e d neat once i n t h i s work but could not be persuaded to c r y s t a l l i z e from solvent. Hence compound 3^  was p u r i f i e d through c r y s t a l l i n e methyl 4-<9-acetyl-2,3--O-isopropylidene-a-L-rhamnopyranoside (2). Because a c e t a l a t i o n never goes 100% to completion, some acetone polymers are always formed, and since deacetylation i s a clean q u a n t i t a t i v e procedure t h i s c r y s t a l l i n e intermed-i a t e .2 provides an easy method f o r obtaining the pure 3_ required for disaccharide synthesis. Care must be taken during the deacetylation when cleionizing the sodium methoxide with cation exchange r e s i n to keep the temperature of the s o l u t i o n around 5° and the time of contact with the r e s i n minimal i n order to prevent hydrolysis of the isopropylidene group. Subsequent passage of the s o l u t i o n through an anion exchange r e s i n removes the traces of a c i d r e s u l t i n g from bleeding of the c a t i o n i c r e s i n and pre-vents deacetalation. 108 Shapiro et al. have reported a very mild method f o r the removal of isopropylidene groups i n the presence of acetates. It involves the use of t r i f l u o r o a c e t i c a c i d i n wet chloroform which i s conveniently removed by evaporation. This procedure s e l e c t i v e l y removes isopropylidene groups while leaving methyl glycosides and acetates i n t a c t . Subjecting compound 2_ to t h i s treatment r e s u l t s i n the preparation of c r y s t a l l i n e methyl 4-0-acetyl- a-L-rhamnopyranoside (4). Since compound 4_ has a substituent on C-4 i t serves as an analog of the methyl glycosides of the disaccharides and thus can be used to prepare the necessary standards f o r the methylation and periodate oxidation products r e s u l t i n g from the L-rhamnose moiety as shown i n Figure 5. Compound 4_ should also be a u s e f u l intermediate f o r the preparation of L-rhamnose residues s e l e c t i v e l y substituted to leave p o s i t i o n 3 r e a c t i v e as i t has been demonstrated that v i c i n a l hydroxyls react with ethyl orthoacetate to 23 Figure 5. Preparation of Methylation and Periodate Oxidation Standards from Methyl 4-<9-acetyl-a-L-rhamnopyranoside OMe CH 2N 2/BF 3 OMe HO 1 HO— Ac 9 0/pyr CHs Ac0\/^CHO (VleOH/HCl CH2OH HO 1 HO— CH, 8 M e ( \ yOMe c \:hLpH + CHjOAc —OMe —OMe Act) AcO Ac20/NaOAc p-N0 2C 6H 4C0Cl/pyr CHOAC AcO-AcO-CH 3 7 CW-p-NBz p-NBz-O-p-NBz-O-CH 3 CH3 24 give monoacetylation of the a x i a l hydroxyl. This would then lead to the i n t e r e s t i n g s e r i e s of disaccharides linked through p o s i t i o n 3 on L-rhamnose but w i l l not be discussed f u r t h e r here. Methylation of compound 4 leads to the standards required f or comparison with the product obtained from the methylation of the L-rhamnose moiety of the disaccharides. Since u n t i l recently a l l methylation proced-ures involved the u s e - o f strong bases, compounds containing base l a b i l e 109 groups such as acetates could not be methylated. However Gros et al. have developed a procedure f o r methylating compounds containing base l a b i l e groups by using boron t r i f l u o r i d e and diazomethane. Using t h i s procedure compound 4_ was methylated to give methyl 4-0-acetyl-2,3-di-<9-methyl-a-L--rhamnopyranoside. It was not found advantageous to cool the re a c t i o n mix-ture to -78° as has been s u g g e s t e d ^ but the suggestion to repeat the pro-cedure i f i t i s not complete the f i r s t time has proven p r a c t i c a l . Care should be taken to obtain pure s t a r t i n g material and to maintain anhydrous conditions. Subsequent deacetylation gave methyl 2,3-di-O-methyl-a-L--rhamnopyranoside (5). Compound 5 was characterized as c r y s t a l l i n e methyl 2,3-di-O-methyl-4-0-toluene-p-sulphonyl-a-L-rhamnopyranoside. Now the standard methyl 2,3-di-O-methyl-a-L-rhamnopyranoside (5) can be hydrolyzed r e a d i l y to give standard 2,3-di-C-methyl-L-rhamnose (6_) which can be reduced to give standard 2,3-di-c9-methyl-L-rhamnitol which i n turn can be acetylated to give standard l,4,5-tri-0-acetyl-2,3-di-0-methyl-L-rhamnitol (7). Periodate oxidation of 4_ leads to the standards required f o r com-parison with the product obtained from the periodate oxidation of the L-rhamnose moiety of the disaccharides. Therefore periodate oxidation of £ followed by deacetylation, reduction and methanolysis gave 4-deoxy-L-25 - e r y t h r i t o l (1-deoxy-D-erythritol) (8). Methanolysis rather than hydrolysis was employed in order to convert the reducing portion of the molecule to v o l a t i l e glycolaldehyde dimethyl acetal which could be conveniently removed by evaporation. Methanolysis also prevents the unwanted formation of acetals between glycolaldehyde and 8. This aspect w i l l be discussed more f u l l y i n the periodate oxidation of the disaccharides. Compound 8_ was characterized as i t s c r y s t a l l i n e t ri-p-nitrobenzoate. Standard 4-deoxy-L-- e r y t h r i t o l (8) was acetylated with sodium acetate and a c e t i c anhydride to give standard 4-deoxy-L-erythritol t r i a c e t a t e . Sodium acetate was used as a c a t a l y s t instead of the usual p y r i d i n e because the evaporation condi-tions used f o r the removal of p y r i d i n e may lead to the loss of some of t h i s f a i r l y v o l a t i l e product. 2. 4-0-$-D-Glucopyranosyl-L-rhamnopyranose Since 4-0-8-D-glucopyranosyl-L-rhamnopyranose (15) r e s u l t s from the condensation of 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide (acetobromoglucose) (9) with the aglycon 3_ and since acetobromoglucose i s the cheapest, most commonly used, best understood, and most e a s i l y prepared g l y c o s y l h a l i d e a v a i l a b l e t h i s condensation was the one used to determine the optimal condensation conditions f o r t h i s s e r i e s of disaccharides. Many v a r i a t i o n s of the standard Koenigs-Knorr conditions were compared with the H e l f e r i c h conditions since Kamiya et al.^® have recommended the H e l f e r i c h r e a c t i o n f o r the preparation of L-rhamnopyranosides. However i t was noted that the isopropylidene group of 3 was not stable to the H e l f e r i c h reaction conditions. By doing a ser i e s of control experiments where only 3 was sub-jected to condensation conditions each varying in one component i t was deter-26 mined that the mercuric bromide was causing hydrolysis of the isopropylidene group. Neither the a c e t o n i t r i l e alone nor i n combination with mercuric cyanide caused cleavage but when mercuric bromide was added considerable deacetalation was observed. Kamiya et al . d i d not report any deaceta-l a t i o n of t h e i r aglycons i n the presence of mercuric bromide which perhaps indicates the l a b i l i t y of the isopropylidene group of 3_. Consequently the H e l f e r i c h r e a c t i o n conditions were modified i n that the mercuric bromide was omitted. P a r a l l e l microscale condensations were performed with s i l v e r oxide and anhydrous calcium s u l f a t e i n chloroform; s i l v e r oxide, iodine, and anhydrous calcium s u l f a t e i n chloroform; s i l v e r oxide, s i l v e r perchlorate, and anhydrous calcium s u l f a t e i n chloroform; and mercuric cyanide i n aceto-n i t r i l e . C onsistently the modified H e l f e r i c h r e a c t i o n conditions gave r i s e to a more complete re a c t i o n ; that i s , gave a higher y i e l d of product and less of fewer degradation products. The major degradation product observed i n a l l the condensation reactions described i n t h i s thesis was the so c a l l e d "hydrolysis product" of the bromide, that i s the product with a free hydroxyl at C - l r e s u l t i n g from h y d r o l y s i s of the g l y c o s y l bromide. This by-product was i d e n t i f i e d by t h i n - l a y e r chromatographic comparison with the product obtained by d e l i b e r a t e h y d r o l y s i s of the bromide with aqueous s i l v e r n i t r a t e . The major d i f f e r e n c e between the s i l v e r oxide condensations and the mercuric cyanide condensation i s that the l a t t e r i s a homogeneous system whereas the others are heterogeneous. Therefore acetobromoglucose 9^  was condensed with 3^  i n the presence of mercuric cyanide i n a c e t o n i t r i l e , as shown i n Figure 6, to give c r y s t a l l i n e methyl 2,3-0-isopropylidene-4--0-(2,3,4,6-tetra-0-acetyl-3-D-glucopyranosyl)-a-L-rhamnopyranoside (10) i n 80% y i e l d . 27 Figure 6. 4-0-8-D-Glucopyranosyl-L-rhamnopyranose R 2 R 3 R 4 R 5 R 6 R 7 Me Ac Ac Ac Ac 11 MQ H H Ac Ac Ac Ac 12 MQ H H H H H H 13 MQ MG Me MG Me Me Me 14 Ac Ac Ac Ac Ac Ac Ac 15 H H H H H H H 28 Since 4-(9-8-D-glucopyranosyl-L-rhamnopyranose i n the form of 10 was the most e a s i l y obtained member of the 4-0-glycopyranosyl-L-rhamno--pyranose s e r i e s i t was the most appropriate one to use to work out the proof of structure procedures and the deblocking and d e r i v a t i z a t i o n reactions f o r the s e r i e s . Although the mode of synthesis gives a good i n d i c a t i o n of the structure of a synthetic disaccharide i t cannot be used to assign the structure unambiguously without confirmation from the usual proof of structure procedures such as methylation and periodate oxidation. By prac-t i s i n g these procedures on a system of predicted structure the procedures can be improved to give optimum r e s u l t s i n general and/or f o r the s p e c i f i c s t r u c t u r a l feature under i n v e s t i g a t i o n and then these improved procedures can be extended to systems of unknown or suspected s t r u c t u r e . The methyl glycoside of the disaccharide i s the most convenient d e r i v a t i v e to use f o r methylation and periodate-oxidation studies because i t has the r i n g s i z e and configuration of the reducing sugar locked, thus only one degradation product r e s u l t s from the reducing moiety of the disaccharide. The methyl glycoside also prevents base degradation from the reducing end during methylation and prevents the formation and slow hydrol-y s i s of a formate ester during periodate oxidation. Consequently 10_ was 108 deacetalated by the previously discussed method of Shapiro et al. to give methyl 4-0-(2,3,4,6-tetra-0-acetyl-8-D-glucopyranosy1)-a-L-rhamnopyranoside (11) which was deacetylated to give methyl 4-0-8-D-glucopyranosyl-a-L--rhamnopyranoside (12). The methyl glycoside 1_2 was methylated by the Hakomori method^* 112 as d e t a i l e d by Sandford and Conrad . Since the disaccharide could not be dialyzed the methyl sulphoxide extract was d i l u t e d with a small amount of 29 water and extracted with large amounts of petroleum ether (b.p. 65-70°). This procedure i s s a t i s f a c t o r y f o r separating the permethylated disaccharide 13 from the methyl sulf o x i d e and provides a clean permethylated product as nothing else i s extracted by the petroleum ether. The permethylated d i -saccharide 13 was then cleaved into monomeric u n i t s since the determination of the methyl s u b s t i t u t i o n pattern of the derived monomeric units indicates the p o s i t i o n of linkage. The methyl s u b s t i t u t i o n pattern i s r o u t i n e l y established by g a s - l i q u i d and paper chromotographic comparison with authen-t i c standards and by mass spectrometric fragmentation a n a l y s i s . A v a r i e t y of d e r i v a t i v e s of the p a r t i a l l y methylated monomeric units can be used to determine the s u b s t i t u t i o n pattern as i l l u s t r a t e d by the various ones used f o r the monomeric fragments of 1^ shown i n Figure 7. Methanolysis of 13 gave the monomeric methyl glycosides but they were not separable by gas-l i q u i d chromatography. However since the L-rhamnose d e r i v a t i v e s had a free hydroxyl group but the D-glucose d e r i v a t i v e s d i d not, i t was p o s s i b l e to make the t r i m e t h y l s i l y 1 d e r i v a t i v e thus decreasing s u b s t a n t i a l l y the re t e n t i o n time of the L-rhamnosides while leaving the D-glucosides unchanged. This method has been used with p a r t i c u l a r success i n the D-mannose se r i e s 113,114^ that the. fragments were separated they were shown to cochromatograph with authentic standards 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 of the methyl 2,3-di-O-methyl-L-rhamnopyranosides and with the authentic standards of the methyl 2,3,4,6-tetra-0-methyl-D-glucopyranosides. Methyl 2,3,4,6-tetra-O-methyl-B-D-glucopyranoside was c o l l e c t e d and characterized as a c r y s t a l l i n e d e r i v a t i v e . Hydrolysis of 1_3 gave 2,3,-di-O-methyl-L-rhamnose and 2,3,4,6-tetra-O-methyl-D-glucose as i n d i c a t e d by paper chroma-tographic comparison with authentic standards. They could also be separated 30 igure 7. Hydrolysis Products of Methylated Disaccharide 31 and i d e n t i f i e d by g a s - l i q u i d chromatographic analysis of t h e i r acetate d e r i v a t i v e s ^ ^ . Hydrolysis of 1_3 followed by reduction and subsequent a c e t y l a t i o n gave the a l d i t o l acetates. These were not separable by gas-l i q u i d chromatography using butanediol succinate**^ as a l i q u i d phase but 117 when OS138 was used as suggested by Lindberg and coworkers separation was achieved on an ordinary packed column. The two equimolar peaks then obtained were i d e n t i f i e d as l,4,5-tri-0-acetyl-2,3-di-O-methyl-L-rhamnitol and l,5-di-<9-acetyl-2,3,4,6-tetra-0-methyl-D-glucitol by g a s - l i q u i d chroma-tographic and mass spectrometric comparison with authentic standards. Thus 2,3-di-O-methyl-L-rhamnose and 2,3,4,6-tetra-O-methyl-D-glucose can be separated and i d e n t i f e d by paper chromatography and by g a s - l i q u i d chroma-tography as e i t h e r t h e i r methyl glycosides, t h e i r acetates, or t h e i r a l d i t o l acetates. In a l l cases authentic standards are required which emphasizes the importance of compounds such as S_, 6^  and 7. The methyl glycoside 12 was subjected to periodate oxidation degradation as shown i n Figure 8. S t r u c t u r a l information i s obtained from both the consumption of periodate and the products obtained a f t e r degrada-t i o n . A consumption of three moles per mole of 1_2 i n d i c a t e d that the l i n k -age was e i t h e r 1,2 or 1,4 but could not be 1,3. The products obtained from the subsequent degradation procedures dist i n g u i s h e d 1,2 and 1,4 linkages. In both cases the non-reducing p o r t i o n of the molecule would produce g l y c e r o l and i f the linkage was 1,2 the reducing portion would produce 3-deoxy-L-- g l y c e r o l but i f the linkage was 1,4 the reducing p o r t i o n would produce 4-deoxy-L-erythritol. The 1,4 linkage was confirmed by the production of g l y c e r o l and 4-deoxy-L-erythritol as i d e n t i f i e d by paper chromatography as well as the g a s - l i q u i d chromatography and mass spectrometry of t h e i r Figure 8 . Periodate Oxidation of Disaccharide 32 3 NalO, CHO - f - HCOOH NaBH^ , CHjOH u CHJIOH A A C H J C'H 20H a\OH CH 2 0H CHjOH OH - L . ^ C CHjOH L y / NCHJPH MeOH/HCl CHaOH - f HC -f-Ac^O/NaAc H,OH CHjOAc CHjtjAc AcO-AcO CH$ 33 peracetylated d e r i v a t i v e s with the use of authentic standards such as _8. Glycerol and 4-deoxy-L-erythritol should be produced q u a n t i t a t i v e l y and i n an equimolar r a t i o . Although t h i s i s not c r i t i c a l f o r this s e r i e s of disaccharides i t should hold true i f the method i s to be generally useful f o r undefined systems where more components are present and t h e i r r a t i o s are informative. As mentioned previously (page 25) h y d r o l y s i s of the poly-alcohol intermediate led to the formation of acetals between g l y c o l aldehyde and g l y c e r o l and 4-deoxy-L-erythritol. Ga s- li qu id chromatographic analy-s i s of the acetylated mixture i n d i c a t e d one large peak and several smaller ones i n a d d i t i o n to the peaks expected f o r g l y c e r o l and 4-deoxy-L-erythritol. Therefore h y d r o l y s i s conditions are not s a t i s f a c t o r y i f q u a n t i t a t i v e pro-118 cedures are required. This and r e l a t e d problems have been reviewed Methanolysis produced a much more q u a n t i t a t i v e r e a c t i o n . The dimethyl ac e t a l of glycolaldehyde i s formed during methanolysis thus rendering i t i n e r t toward the formation of acetals with the higher alcohols. G l y c o l -aldehyde i s also conveniently removed by evaporation as i t s dimethyl a c e t a l . G a s - l i q u i d chromatographic analysis of the acetylated product obtained from methanolysis showed two major equimolar peaks corresponding to g l y c e r o l and 4-deoxy-L-erythritol with a few other smaller components. Methanolysis gave s a t i s f a c t o r y r e s u l t s f o r a l l the disaccharides i n t h i s thesis, however 119 on the suggestion of P.E. Reid the polyalcohol was also hydrolyzed with cation exchange r e s i n i n the presence of an anion exchange r e s i n . The anion exchange r e s i n i r r e v e r s i b l y adsorbs the glycolaldehyde (or any aldehyde) as soon as i t i s released thus e f f e c t i v e l y removing i t from the reaction mix-ture. G a s - l i q u i d chromatographic analysis of the acetylated product obtained from mixed r e s i n h y d r o l y s i s showed only the two peaks expected corresponding 34 to g l y c e r o l .and 4-deoxy-L-erythritol plus one other very small peak. This method c e r t a i n l y looks promising f o r the q u a n t i t a t i o n of more complicated mixtures. To be of use i n the i d e n t i f i c a t i o n of i s o l a t e d polymer fragments synthetic disaccharides of unambiguous structure have to be characterized as d e r i v a t i v e s , preferably c r y s t a l l i n e , that can be r e a d i l y prepared from the i s o l a t e d fragments. The peracetate of the disaccharide was an obvious candidate. Since a c e t o l y s i s has been used s u c c e s s f u l l y to replace methyl ., 81,120,121 , ^ . 121,122 , . ... glycosides , benzylidene acetals , and isopropylidene 120 123 ketals ' with acetates i t was assumed that t h i s would provide a convenient one-step procedure f o r converting 10_ into 1,2,3-tri-O-acetyl--4-0-(2,3,4,6-tetra-0-acetyl-8-D-glucopyranosyl)-a-L-rhamnopyranose ( s c i l l a b i o s e heptaacetate) (14). However, a l l of the several sets of a c e t o l y s i s conditions t r i e d only gave the product i n a very low y i e l d (up to 15%). Once c r y s t a l l i n e l-0-acetyl-2,3-O-isopyropylidene-4-C">- (2,3,4,6--tetra-0-acetyl-8-D-glucopyranosyl)-a-L-rhamnopyranose as i d e n t i f i e d by i t s proton magnetic resonance spectrum was i s o l a t e d . A c l o s e r i n v e s t i g a -t i o n of the l i t e r a t u r e revealed that a c e t o l y s i s had only been used success-f u l l y f o r isopropylidene groups spanning a primary and a secondary hydroxyl i . e . 1,2-; 5,6-; 6,7-0-isopropylidene groups and not for isopropylidene groups spanning two secondary hydroxyl groups such as the 2,3-<9-isopropyli-120 -dene group i n 10_. It has also been noted that secondary-secondary methylene acetals are considerably less susceptible to a c e t o l y s i s than are primary-secondary methylene a c e t a l s . A c e t o l y s i s of isopropylidene ketals 124 formed between two secondary hydroxyl groups has been studied by Sowa 125 i n extending an e a r l i e r observation made by Jerkeman . They give examples of cis 2,3-0-isopropylidene ketals of furanoses that are epimerized at C-2 35 when acetolyzed i n high concentrations of a c e t i c a c i d . It seemed probable that the isopropylidene group of I0_ could not be acetolyzed to any extent but may have been hydrolyzed during the work-up thus making the product water soluble and therefore l o s t during the washing of the organic solvent layer. This would account f o r the observed 15% y i e l d of uncontaminated pre-dicted product. Once i t was r e a l i z e d that the isopropylidene group could not be acetolyzed i t was e a s i l y removed by the mild method of Shapiro 108 et al. to give methyl 4-0-(2,3,4,6-tetra-0-acetyl-6-D-glucopyranosyl)--a-L-rhamnopyranoside (11) . Now the a c e t o l y s i s of 1_1 e s s e n t i a l l y as des-121 cribed by A s p i n a l l and coworkers gave 14 i n good y i e l d (70%). Since 126 a c e t o l y s i s p r e f e r e n t i a l l y cleaves small aglycons i t i s a convenient method f o r removing the methyl glycoside while leaving the disaccharide linkage i n t a c t . Since 1,4 linkages are least susceptible to cleavage by a c e t o l y s i s i t i s most appropriate f o r t h i s s e r i e s of disaccharides. S c i l l a b i o s e heptaacetate 14_ thus obtained had m.p. 139-140° and the proton magnetic resonance spectrum showed c l e a r l y the presence of seven acetate groups per L-rhamnose moiety. Neither the melting point nor the o p t i c a l 35 36 r o t a t i o n agree with that given i n the l i t e r a t u r e ' f o r " s c i l l a b i o s e hexaacetate". Although s a t i s f a c t o r y a n a l y t i c a l data are given f o r a hexa-acetate there i s no i n d i c a t i o n why such an acetate should be formed and 28 Bailey has recognized t h i s ambiguity by l i s t i n g only s c i l l a b i o s e acetate. Straightforward deacetylation of l_4gave the free disaccharide, 4-0-8-D-glucopyranosyl-L-rhamnopyranose (15) . Two other d e r i v a t i v e s , the free a l d i t o l , 4-0-6-D-glucopyranosyl-L-rhamnitol (16) and i t s c r y s t a l l i n e peracetate, 1,2,3,5-tetra-O-acetyl-4-0-(2,3,4,6-tetra-O-acetyl-B-D--glucopyranosyl)-L-rhamnitol (17) were prepared by standard procedures. 36 CHjOR OR RO -OR 16 R = H C H 3 1 7 R = A c In order to extend the data of P e r c i v a l 127 the g a s - l i q u i d chromatographic retention times of the per-O- ( 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 of 1_5 and 1_6 r e l a t i v e to p e r - O - ( t r i m e t h y l s i l y l ) sucrose were recorded. The configuration of the disaccharide linkage can be determined by Hudson's rules or by enzymatic hydrolysis studies but most conveniently by 128 proton magnetic resonance spectroscopy as demonstrated by van der Veen , 129 pursuing the e a r l i e r observations of Lemieux et al. . Because C-l i s bonded to two oxygen atoms the resonance of the proton on C - l (the anomeric proton) i s to low f i e l d of the other r i n g protons and hence can be d i s t i n g -uished. a-Linked disaccharides have an equatorial H-l which resonates at lower f i e l d (4.5 - 5.0 x) than the a x i a l H-l of B-linked disaccharides which resonates at 5.0 - 5.5 x. Coupling constants are also informative f o r d i s -t inguishing a-D- and B-D - linked glucosides since an a-D-linkage leads to a small (^  3 Hz) a x i a l - e q u a t o r i a l coupling constant whereas a g-D-linkage leads to a large ('v 7 Hz) a x i a l - a x i a l coupling constant. As has been repor-130 ted the proton magnetic resonance spectra of permethylated disaccharides 131 also d i s t i n g u i s h a- and B-anomers. Whyte has pointed out the advantage of using disaccharide a l d i t o l s f o r determining the g l y c o s i d i c configuration 37 as they only e x h i b i t the one anomeric proton of i n t e r e s t and eliminate complications a r i s i n g from the anomeric protons of the reducing moiety. Proton magnetic resonance spectroscopy was used to confirm the 8-D-linkage of 4-0-B-D-glucopyranosyl-L-rhamnopyranose. The signals from the anomeric protons of the free disaccharide, the permethylated disaccharide, and the free a l d i t o l provide p a r t i c u l a r l y c l e a r evidence of a 8-D-linked glucoside 83 as shown-in Figure 9 which compares the a-D- and B-D-linked glucosides. The synthesis of 4-0-8-D-glucopyranosyl-L-rhamnopyranose described 132 here has now been published 3. 4-Q-a-L-Rhamnopyranosyl-L-rhamnopyvanose 4-0-a-L-Rhamnopyranosyl-L-rhamnopyranose (26) was prepared i n a manner analogous to 1_5_ by condensing 2,3,4-tri-C-acetyl-a-L-rhamnopyranosyl bromide (19) with _3 i n the presence of mercuric cyanide i n a c e t o n i t r i l e as suggested by Kamiya et al. to give methyl 2,3-C-isopropylidene-4-C--(2,3,4-tri-C-acetyl-a-L-rhamnopyranosyl)-a-L-rhamnopyranoside (20) as shown i n Figure 10. In t r i a l condensations of 19_ Kamiya et al found that whereas the Koenigs-Knorr conditions gave mainly orthoesters the H e l f e r i c h conditions produced the desired disaccharides. Since 4-0-8-D-glucopyranosyl-L-rhamnopyranose was the only d i -saccharide of t h i s s e r i e s where the d i r e c t product of the condensation c r y s t a l l i z e d and since i t was desired to prepare the methyl glycoside, the peracetate, the free disaccharide, the free a l d i t o l , and the a l d i t o l acetate for each disaccharide of the s e r i e s , i t was necessary to use c r y s t a l l i n e intermediates for p u r i f y i n g the condensation mixture. As mentioned e a r l i e r 3.8 a — l i n k e d J Glc 1—^Rha 1.5 P- linked T 4 . 8 8 i 1 — 5.12 5 . 2 6 3.7 G!c 1-^Rha OMe-7.7 1.5 fJ ' 1 1 4 . 9 5 5 . 2 5 5 . 3 7 3.8 GIc^RhamnitoI 7 2 I J —I — , — 4 . 7 0 5 . 3 7 gure 9. 4-0-D-Glucopyranosyl-L-rhamnopyranose; Proton Magnetic Resonance Spectra of the Anomeric Region 40 condensations are never very clean reactions and thus produce multicom-ponent mixtures. In almost a l l cases there i s a small amount of the other anomer formed which i s s i m i l a r to, and therefore d i f f i c u l t to separate from, the major product.. Chromatography can be used for p u r i f i c a t i o n but i t i s tiresome f o r large q u a n t i t i e s and not as absolute as c r y s t a l l i n e intermed-i a t e s . Thus c r y s t a l l i n e intermediates are u s e f u l stepping stones between the d i r e c t condensation product and the desired d e r i v a t i v e s . The c r y s t a l -l i n e intermediates are obtained on a t r i a l and error basis by preparing small quantities of the various intermediates, p o s s i b l e , p u r i f y i n g them chromatographically, and u t i l i z i n g the ones that c r y s t a l l i z e . Since each disaccharide has d i f f e r e n t c r y s t a l l i n e intermediates the exact sequence of deblocking and d e r i v a t i z a t i o n procedures w i l l vary from disaccharide to disaccharide although the same general operations are employed i n each case. For example, i n the 4-0-a-L-rhamnopyranosyl-L-rhamnopyranose case removal of the isopropylidene group and subsequent a c e t y l a t i o n gave c r y s t a l l i n e methyl 2,3-di-0-acetyl-4-0-(2,3,4-tri-t9-acetyl-a-L-rhamno--pyranosyl)-ct-L-rhamnopyranoside (22) which served as a c r y s t a l l i n e i n t e r -mediate f o r the preparation of methyl 4-0-a-L-rhamnopyranosyl-a-L-rhamno--pyranoside (23). In f a c t 22^  i s also a c r y s t a l l i n e d e r i v a t i v e since i t could be prepared from the i s o l a t e d disaccharide although i t i s not a commonly used d e r i v a t i v e . The f i r s t c r y s t a l l i n e intermediate i s also used to c a l c u l a t e the y i e l d of the condensation as no meaningful numbers can be arr i v e d at u n t i l a pure product i s obtained. In t h i s case 22_ was obtained i n 65% y i e l d based on _3. This y i e l d could probably be increased by using benzobromorhamnose rather than acetobromorhamnose (19) as Kamiya et al-^^ obtained higher y i e l d s when the benzoyl analog was employed. Benzobromo-41 mannose has also been reported to give b e t t e r y i e l d s and higher stereo-s e l e c t i v i t y than acetobromomannose so was chosen f o r the synthesis of 4-0-a-D-mannopyranosy1-L-rhamnopyranose (38). Although a higher y i e l d (76%) was obtained, benzoates introduce t h e i r own d i f f i c u l t i e s as w i l l be mentioned l a t e r . So although acetates may not produce as high a y i e l d as benzoates they o f f e r a more a t t r a c t i v e synthetic sequence. The methyl glycoside 23 obtained by deacetylation of c r y s t a l l i n e 22 was subjected to methylation and periodate oxidation studies to substan-t i a t e the 1,4 linkage. As f o r subsequent disaccharides i t was decided to characterize the fragments of the permethylated disaccharide 24_ only as t h e i r a l d i t o l acetates. The a l d i t o l acetates were chosen so that each sugar would r e s u l t i n only one compound and hence give only one peak when subjected to g a s - l i q u i d chromatographic a n a l y s i s . They also lend themselves to mass spectrometric analysis and the authentic standards i . e . 7 were a v a i l a b l e . 133 Albersheim et at- have devised a procedure f o r the combined reduction and a c e t y l a t i o n of aldoses which circumvents the i s o l a t i o n of the free a l d i t o l . It involves destroying the excess borohydride with a c e t i c a c i d and removing the borate as methyl borate thus leaving the sodium ace-tate to catalyze the subsequent a c e t y l a t i o n with a c e t i c anhydride. It was assumed that t h i s would provide a convenient method f o r the preparation of the methylated a l d i t o l acetates. However, when th i s method was employed three peaks designated Peak 1, Peak 2, and Peak 3 i n a 4:1:3 r a t i o respec-t i v e l y rather than the expected two peaks were observed on g a s - l i q u i d chroma-tographic a n a l y s i s . Peak 1 and Peak 3 were i d e n t i f e d as 1,5-di-0-acetyl--2,3,4-tri-O-methyl-L-rhamnitol and 1,4,5-tri-C~>-acetyl-2,3-di-O-methyl-L--rhamnitol (7) r e s p e c t i v e l y by g a s - l i q u i d chromatographic and mass spectro-42 metric comparison with authentic standards. Peak 2 has been assigned the structure shown below on the basis of the mass spectrum, the proton magnetic resonance spectrum, and the product obtained a f t e r methanolysis and subsequent a c e t y l a t i o n . The mass spectrum obtained showed peaks at m/e 161, 131, 117, 101, and 71 i n d i c a t i n g Fragment 1. It also contained m/e 87, p o s s i b l y i n d i c a t i n g Fragment 2. The peak at m/e 143 which arises from Fragment 3 i n 7_ could also a r i s e from the proposed s t r u c t u r e . + H Cj O M Q + H C r f -H,pAc >Me O M Q H- -OAc H-Fragment 1 JUS Fragment 2 —OAc Fragment 3 The proton magnetic resonance spectrum showed the presence of two acetate substituents and four methyl substituents per rhamnose moiety. The fa c t that Peak 2 can be transformed into 1_ by methanolysis and subsequent acety-l a t i o n also supports t h i s structure. Consequently i t was concluded that 1,5-di-O-acetyl-2,3,4-tri-O-methyl-L-rhamnito1 and 1,4,5-tri-O-acetyl-2,3--di-O-methyl-L-rhamnitol (7) were obtained i n an equimolar r a t i o thus sub-43 s t a n t i a t i n g the 1,4 linkage, but that t h i s combined reduction a c e t y l a t i o n method when applied to the preparation of methylated a l d i t o l acetates does not produce q u a n t i t a t i v e r e s u l t s . The hydrolyzates of the permethyl-ated d e r i v a t i v e s of the other disaccharides i n t h i s s e r i e s were also sub-jected to t h i s procedure and i n each case a p o r t i o n of the 1,4,5-tri-C--acetyl-2,3-di-0-methyl-L-rhamnitol appeared as t h i s (l,5-di-0-acetyJ-2,3--di-O-methyl-L-rhamnityl)-4-dimethyl borate, although the r a t i o s v a r i e d s l i g h t l y from disaccharide to disaccharide. Therefore, the phenomenon i s not associated with the other compounds present but only with 2,3-di-O-133 -methyl-L-rhamnose i t s e l f . Albersheim et al• noted that the borate present formed a complex with the a l d i t o l s and suggested the a d d i t i o n and evaporation of f i v e portions of methanol to remove i t as methyl borate. However, t h i s borate compound i s not decomposed by successive additions and evaporations of methanol but requires methanolysis at r e f l u x overnight f o r complete removal. This type of compound has since been observed i n t h i s laboratory with other methylated aldoses when subjected to t h i s procedure. Whenever the free a l d i t o l s were i s o l a t e d (as was done i n the s c i l l a b i o s e case), that i s the sodium ions were removed with cation exchange r e s i n p r i o r to removal of the borate as methyl borate, the borate was removed completely by successive additions and evaporations of methanol and t h i s anomalous compound di d not a r i s e . This was the procedure adopted f o r the subsequent disaccharides since i t appears that t h i s combined reduction a c e t y l a t i o n procedure i s not s a t i s f a c t o r y f or methylated aldoses. This i s a prime example of the u t i l i t y of defined systems f o r checking degradative proced-ures before they are applied to undefined systems as should t h i s extraneous peak appear i n a g a s - l i q u i d chromatographic analysis of a permethylated 44 polysaccharide hydrolyzate i t would severely hamper meaningful i n t e r p r e -t a t i o n . The d e r i v a t i z a t i o n procedures were c a r r i e d out i n a manner analogous to those for 4-0-8-D-glucopyranosyl-L-rhamnopyranose and proceeded without i n c i d e n t . 4-0-a-L-Rhamnopyranosyl-L-rhamnopyranose (26_) was characterized as c r y s t a l l i n e l ,2,3-tri-0-acetyl-4-0-(2,3,4-tri-O-acetyl--a-L-rhamnopyranosyl)-a-L-rhamnopyranose (25) and as c r y s t a l l i n e 1,2,3,5--tetra-0-acetyl-4-0-(2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl)-L-rhamnitol 128 (28). The a-L-linkage was confirmed by the chemical s h i f t of the non-reducing anomeric proton i n the proton magnetic resonance spectra of a l l the d e r i v a t i v e s prepared but p a r t i c u l a r l y i n the free disaccharide where the anomeric proton of the non-reducing sugar i s c l e a r l y to low f i e l d and therefore a x i a l . Although i t was not p o s s i b l e to assign the s i g n a l s from the C-6 protons to the reducing and non-reducing sugars, comparison with the C-6 protons from the reducing sugar of the other members of the ser i e s suggests that the high f i e l d s i g n a l comes from the reducing sugar i n most cases. A report of the synthesis and properties of 4-0-a-L-rhamnopyran-134 -osyl-L-rhamnopyranose described here i s i n press 4. 4-0-a-D-Mannopyranosyl-L-vhamnopyranose 4-0-a-D-Mannopyranosyl-L-rhamnopyranose (38) was prepared i n a manner p a r a l l e l to 1_5 and 26_ by f i r s t condensing 2,3,4,5-tetra - o-benzoyl--a-D-mannopyranosyl bromide (30) with 3 i n the presence of mercuric cyanide i n a c e t o n i t r i l e to give methyl 2,3-0-isopropylidene-4 - o -(2,3,4,6-tetra-O--benzoyl-a-D-mannopyranosy1)-a-L-rhamnopyranoside (31) as shown i n Figure 45 11. Benzobromomannose 3_0_ was used rather than the more common acetate 95 analog since Gorin and P e r l i n have reported that i t gives higher y i e l d s and greater s t e r e o s e l e c t i v i t y than acetobromomannose. However, benzoates also present some complications since deacylation with sodium methoxide produces methyl benzoate which has to be removed by steam d i s t i l l a t i o n . They also tend to retard the separation by t h i n - l a y e r chromatography of c l o s e l y r e l a t e d compounds. When several benzoates are present on one molecule the product forms a hard glass and becomes too ins o l u b l e to c r y s t a l l i z e r e a d i l y as was the case with 31 which was debenzoylated and acetylated to give c r y s t a l l i n e methyl 2,3-0-isopropylidene-4-0-(2,3,4,6--tetra-O-acetyl-a-D-mannopyranosyl)-a-L-rhamnopyranoside (32) i n 76% y i e l d based on _3. The deblocking and d e r i v a t i z a t i o n sequence was e s s e n t i a l l y the same as f o r 1_5_. In t h i s case the free disaccharide 38 c r y s t a l l i z e d and was also characterized as c r y s t a l l i n e l , 2 , 3 - t r i - 0 - a c e t y l - 4 - 0 - (2,3,4,6-tetra--0-acetyl-a-D-mannopyranosyl)-a-L-rhamnopyranose (37) and c r y s t a l l i n e 1,2,3,5-tetra-0-acetyl-4-0-(2,3,4,6-tetra-0-acetyl-a-D-mannopyranosy1)-L--rhamnitol (4_0). The a l d i t o l acetates of disaccharides frequently c r y s t a l -l i z e since they only e x i s t i n one anomeric form. Again the 1,4 linkage was substantiated by methylation and periodate oxidation studies. Proton magnetic resonance spectroscopy has been used extensively i n t h i s work f o r confirming the number and type of substituents present i n 135 a molecule. It has also been used to determine the number of free hy-droxyl groups i n a molecule. The r i n g s i z e can often be confirmed by proton 136 137 magnetic resonance spectroscopy ' as w i l l be i l l u s t r a t e d l a t e r . As previously mentioned the other area where proton magnetic resonance spectros-Figure 11. 4-0-a-D-Mannopyranosyl-L-rhamnopyranose 46 47 copy provides valuable information i s i n assigning the configuration of the disaccharide linkage. Proton magnetic resonance spectroscopy does not d i f f e r e n t i a t e a-D- and 8-D-mannopyranosides as well as i t does a-D- and 8-D-gluco or galactopyranosides since the coupling constant f o r e q u a t o r i a l -equatorial hydrogens does not d i f f e r s u b s t a n t i a l l y from that of e q u a t o r i a l -a x i a l hydrogens. Also the chemical s h i f t d i f f e r e n c e between the a-D- and B-D-anomers of mannopyranosides i s not as great as f o r glucopyranosides or galactopyranosides. However, the p a i r of anomeric mannopyranosides syn-thesized i n t h i s t hesis demonstrates that there i s c o n s i s t e n t l y a s i g n i f i -cant small d i f f e r e n c e i n the chemical s h i f t of the anomeric proton of a-D-and B-D-mannopyranosides as well as i n t h e i r coupling constants as shown 138-143 i n Figure 12. Lindberg et al. when determining the structure of the Salmonella c e l l - w a l l lipopolysaccharides containing these disaccharides 58 and 56^  used the proton magnetic resonance spectra of the per-<9- (trimethyl-- s i l y l ) oligosaccharide a l d i t o l s to aid the assignment of the configuration of the glucose and galactose residues but could not assign the configuration " of the mannose residues by t h i s technique, although t h e i r data are consis-tent with the f a c t that the B-D-linkages, assigned p o l a r i m e t r i c a l l y , reson-ate at l e a s t 0.2 parts per m i l l i o n higher than the corresponding a-D-linkages. 144 Ballou et al. assigned the configuration of mannose and 3-0-methylmannose units i n a c y c l i c polysaccharide on the basis of the chemical s h i f t of t h e i r anomeric protons. Gorin et al used the d i f f e r e n c e between the chemical s h i f t s of the anomeric protons to d i s t i n g u i s h a-D- and B-D-mannose residues i n yeast mannans. Gahan, Sandford, and Conrad*^ assigned the con-f i g u r a t i o n of D-mannose i n an oligosaccharide i s o l a t e d from the capsular polysaccharide of Klebsiella type 2 on the basis of the chemical s h i f t of 1.8 a-l inked u 4.90 5.03 5.16 Man1-^-Rha 1.6 4.93 5.27 Man 1 —Rha 0Me 7 1.9 4.89 Man 1 — 1 Rhamnitol Figure 12. /3-linked 0.9 0.8 0.9 4.97 5.20 5.23 4.93 5.25 5.37 4.89 526 4-0-D-Mannopyranosyl-L-rhamnopyranose; Proton Magnetic Resonance Spectra of the Anomeric Region 00 49 69 147 the anomeric proton. Dutton et al • ' have used chemical s h i f t s to determine the configuration of D-mannose residues i n the i n t a c t capsular polysaccharides of Klebsiella types 5 and 24. It seems apparent that a-D-and B-D-mannopyranosides can be distinguished by t h e i r proton magnetic reson-ance chemical s h i f t s p a r t i c u l a r l y when both anomers are a v a i l a b l e . In most cases the B-D-anomer resonates about 0.2 to 0.3 parts per m i l l i o n higher than the a-D-anomer. The actual p o s i t i o n of the resonance depends on the d e r i v a t i v e being examined i.e. p e r - 0 - ( t r i m e t h y l s i l y l ) d e r i v a t i v e , permethyl-ated d e r i v a t i v e , or free oligosaccharide and on the p o s i t i o n of the linkage i . e . 1,2; 1,3; 1,4; or 1,6. Therefore, i n order to assign configuration from a s i n g l e resonance, appropriate standards are required such as the disaccharides and t h e i r d e r i v a t i v e s described i n t h i s t h e s i s . As noted by Gorin et al. 1 4 5 the B-D-glycosides of mannose c o n s i s t e n t l y e x h i b i t a smaller coup-l i n g constant than the cx-D-glycosides. Although the d i f f e r e n c e i s not large, f o r small oligosaccharides i t can e a s i l y be observed with the present technology. Thus proton magnetic resonance spectroscopy complements polar-imetry and enzyme studies i n the assignment of the configuration of o l i g o -saccharide linkages as i l l u s t r a t e d by 4-O-a-D-mannopyranosyl-L-rhamnopyranose and the proton magnetic resonance data presented here w i l l extend the u s e f u l -ness of proton magnetic resonance spectroscopy f o r the assignment of 1,4-linked a-D- and B-D-mannopyranosides. The synthesis of 4-O-a-D-mannopyranosyl-L-rhamnopyranose described 148 here has now been published 50 5. 4-0-$-D~Mannopyranosyl-L-rhamnopyranose In order to synthesize the complementary anomer of 38, namely 4-0-8-D-mannopyranosyl-L-rhamnopyranose i t was necessary to develop a gen-e r a l method f o r the synthesis of 8-D-mannopyranosides. It was decided to 95 pursue the 2,3 c y c l i c carbonate procedure of Gorin and P e r l i n . Consider-ing the low y i e l d s obtained i n many of t h e i r steps an a l t e r n a t i v e synthetic route f o r the preparation of 4,6-di-0-acetyl-2,3-0-carbonyl-a-D-mannopyran--osyl bromide (44) was devised as shown i n Figure 13. In view of the pre-vious success i n ac e t o l y z i n g methyl glycosides i t was decided to use methyl 4,6-0-benzylidene-a-D-mannopyranoside (41) rather than benzyl 6-0-triphenyl--methyl-a-D-mannopyranoside f o r preparing the 2,3 carbonate. Compound 4jL_ was e a s i l y prepared although not i n high y i e l d by the method described by 149 Gibney . Methyl 4,6-<?-benzylidene-2,3-0-carbonyl-a-D-mannopyranoside (42) was prepared by modifying the procedure of Doane et a l f o r the D-gluco analog which r e a d i l y produced c y c l i c carbonates i n high y i e l d s by employing the convenient reagent ethyl chloroformate i n the presence of triethylamine. 150 The molar q u a n t i t i e s recommended by Doane et al. f o r the D-gluco analog produced i n addi t i o n to the desired product s u b s t a n t i a l q u a n t i t i e s of the a c y c l i c 0-ethyloxycarbonyl d e r i v a t i v e s i n the D-manno s e r i e s . However, i f instead of adding the amount of triethylamine suggested by Doane et al. j u s t enough triethylamine to make the rea c t i o n mixture basic was added the de-s i r e d compound was e s s e n t i a l l y the only product. The a d d i t i o n of any more triethylamine only removes any c y c l i c carbonate already formed as c y c l i c carbonates are very base l a b i l e . This procedure worked well f o r small reac-tions (under one gram) but was less s a t i s f a c t o r y f o r larger q u a n t i t i e s . 51 Figure 13. Synthetic Route f o r the Preparation of 4,6-Di-(9-acetyl-2,3-0-carbonyl-a-D-mannopyranosyl bromide 52 Since i t was noted (by t h i n - l a y e r chromatography) that very l i t t l e r e action took place u n t i l the r e a c t i o n mixture became basic and since i t was believed that the triethylamine should aid the removal of the hydroxylic proton, the triethylamine was combined with the s t a r t i n g material and the ethyl chloroformate was added dropwise instead of the reverse procedure employed by Doane et al . The molar r a t i o s of the previous r e a c t i o n were not changed, that i s the amount of triethylamine to just make a c e r t a i n quan-t i t y of ethyl chloroformate b a s i c was added to the r e a c t i o n mixture and that c e r t a i n quantity of ethyl chloroformate was added dropwise. Doing the reaction at 0° prevents decomposition of the c y c l i c carbonate once i t i s formed. I t i s imperative f o r the r e a c t i o n to proceed that the mixture remain ba s i c . Furthermore, i f i t i s neutral the excess ethyl chloroformate w i l l slowly hydrolyze during the various stages of the work-up thus produc-ing ethanol and hydrogen c h l o r i d e which w i l l hydrolyze the benzylidene group and basic n e u t r a l i z a t i o n procedures w i l l remove the c y c l i c carbonate. Using t h i s modified procedure a very good y i e l d of a s u b s t a n t i a l quantity of c y c l i c carbonate 42_ was e a s i l y obtained. The large d i f f e r e n c e between the o p t i c a l r o t a t i o n s of 41 (+64.3°) and 42^  (-19.3°) i s a t t r i b u t e d 1 5 1 to a modification of the conformation of the pyranose chair towards a boat when a five-membered carbonate r i n g i s superimposed. A c e t o l y s i s of 4_2_ provides l , 4 , 6 - t r i - 0 - a c e t y l - 2 , 3 - O-carbonyl-a--D-mannopyranose (43). The mild a c e t o l y s i s conditions used (0.75% (v/v) concentrated s u l f u r i c acid i n a c e t i c anhydride f o r two hours) replace the methyl glycoside and the benzylidene group with acetates while leaving the c y c l i c carbonate i n t a c t . A c e t o l y s i s furnishes a method by which 4_2 can be 136 137 coverted to 4_3 without a change i n r i n g form. It has been shown ' that 53 2,3-0-carbonyl-D-mannose e x i s t s i n the furanose form since a five-membered r i n g fused to a five-membered r i n g i s more stable than a five-membered r i n g fused to a six-membered r i n g . Therefore, i f at any time C - l and C-4 were simultaneously free the mannopyranose r i n g with the fused five-membered carbonate r i n g would convert to a mannofuranose system. The mechanism of a c e t o l y s i s i s such that a p o s i t i o n i s blocked with an acetate as i t i s r e l i e v e d of i t s previous substituent so that no p o s i t i o n ever had a free hydroxyl substituent during a c e t o l y s i s . This permits replacement of the benzylidene group and the methyl glycoside with acetates while preserving the pyranose r i n g form. The pyranose r i n g structure i s substantiated by 152 the proton magnetic resonance spectrum . In the furanose form H-5 w i l l be geminal to an acetoxy group and hence downfield of H-4 but i n the pyranose form H-4 w i l l be geminal to an acetoxy group and hence downfield r e l a t i v e to H-5 as i s observed f o r 43^ . The discrepancy between the o p t i c a l r o t a t i o n 95 presented here and that of Gorin and P e r l i n i s probably due to the f a c t that they d i d not have a pure anomer. 4,6-Di-0-acetyl-2,3-0-carbonyl-a-D-mannopyranosyl bromide (44) was 95 prepared from 43 e s s e n t i a l l y as described by Gorin and P e r l i n . The reaction time was extended somewhat since reactions worked up a f t e r two hours often contained a small amount of s t a r t i n g material which was d i f f i c u l t to remove as i t c o c r y s t a l l i z e d with the product. It was found advantageous to f i l t e r the chloroform extract of 44 through calcium oxide and s i l i c a gel to remove remaining traces of acid which lead to decomposition. The synthe-t i c sequence presented here permits the preparation of the valuable b i c y c l i c bromide 4_4 i n four easy steps. Condensation conditions which engender a S M2 type mechanism for the 54 replacement of the bromine of 44 by an incoming nucleophile thus producing net in v e r s i o n are required f o r the formation of 3-D-mannopyranosides from th i s a-D-bromide with a nonparticipating group on C-2 and C-3, I f a free carbonium ion i s produced at C - l the thermodynamically more stable a-D-mannopyranosides w i l l be formed since both the anomeric e f f e c t and the s t e r i c r e l a t i o n s h i p s favor a-D-mannopyranosides. The use of tremendous excesses of nucleophile f a c i l i t a t e s the operation of the S^2 type mechanism. Con-densation of the bromide 44 i n the presence of mercuric cyanide i n aceto-n i t r i l e produces B-D-mannopyranosides s t e r e o s e l e c t i v e l y only when a r e l a t i v e l y strong nucleophile such as methanol i s used i n excess as was done f o r the preparation of c r y s t a l l i n e methyl 4,6-di-c>acetyl-2,3-(9-carbonyl-8-D--mannopyranoside (45) i n 87% y i e l d . Again the discrepancy between the o p t i c a l 95 r o t a t i o n obtained here and that of Gorin and P e r l i n i s probably due to the fa c t that they d i d not obtain a c r y s t a l l i n e product and therefore a pure anomer. Compound 45_ was deacylated to give methyl B-D-wannopyranoside (46) which was characterized as i t s c r y s t a l l i n e isopropylate and as i t s c r y s t a l -l i n e peracetate 47. When a monosaccharide nucleophile such as 3^ even i n t e n - f o l d excess, was condensed with the bromide 44 i n the presence of mercuric cya-nide i n a c e t o n i t r i l e equal amounts of a-D- and B-D-mannopyranosides were produced and when equimolar r a t i o s were condensed the a-D-anomer was pro-duced almost e x c l u s i v e l y . The r a t i o s of anomers presented here were those observed by t h i n - l a y e r chromatography of the re a c t i o n product. The g-D-anomer has a s i g n i f i c a n t l y smaller R^ . value than the a-D-anomer. Thin-layer 153 chromatography was also used f o r monitoring the progress of the re a c t i o n Also the f a c t that the condensation of 44 with methanol i n equimolar qu a n t i t i e s 55 i n the presence of mercuric cyanide i n a c e t o n i t r i l e gave l a r g e l y the a-D-anomer ind i c a t e s the inappropriateness of these condensation conditions f o r the synthesis of B-D-mannopyranosides. The use of other solvents such as nitromethane and benzene instead of a c e t o n i t r i l e increases the percentage of the 8-D-anomer (up to 70% when equimolar r a t i o s were employed) but does not produce a s t e r e o s e l e c t i v e r e a c t i o n . C o n s i s t e n t l y the ad d i t i o n of ethyl acetate to any condensation.conditions t r i e d increased^the percentage of the a-D-anomer. 95 Gorin and P e r l i n used s i l v e r oxide and iodine i n chloroform and a t e n - f o l d excess of aglycon to obtain a s t e r e o s e l e c t i v e r e a c t i o n with 44. These conditions also produce the desired product when 3 i s used as the aglycon but the use of a t e n - f o l d excess of aglycon i s most impractical f o r the synthesis of disaccharides. The use of large excesses of aglycon i s acceptable only when the aglycon i s e a s i l y obtained and when the excess i s r e a d i l y separated from the product such as i s the case with methanol. When the aglycon i s a s p e c i f i c a l l y s u b s t i t u t e d monosaccharide t h i s i s not the case' so the necessity of using large excesses of aglycon makes the method unman-ageable except f o r the preparation of very small q u a n t i t i t e s of product. When the aglycon i s not present i n excess these condensation conditions ( s i l v e r oxide and iodine i n chloroform) produce ^ 75% B-D-mannopyranosides; however, i f the iodine i s omitted the condensation proceeds with stereoselec-t i v e i n v e r s i o n . The use of mercuric acetate i n benzene (Zemplen reaction) also provides condensation conditions which generate s t e r e o s e l e c t i v e inver-sion and hence B-D-mannopyranosides from 44_when the aglycon i s present i n an equimolar r a t i o , but more bromide degradation products are formed than when s i l v e r oxide i n chloroform i s employed. 56 Preliminary condensation experiments of 44 with other aglycons such as 1,2,3,4-tetra-0-acetyl-B-D-glucopyranose and l } 2;3,4-di-O-isopropylidene--a-D-galactopyranoside i n the presence of s i l v e r oxide i n chloroform i n d i r cated that again the product was the B-D-anomer thus confirming the gener-a l i t y of the method f o r the preparation of B-D-mannopyranosides. Consequently 44 was condensed with 3^  i n the presence of s i l v e r oxide i n chloroform with anhydrous calcium s u l f a t e as an i n t e r n a l desiccant to give the desired methyl 4-0-(4,6-di-0-acetyl-2,3-0-carbonyl-8-D-manno--pyranosyl)-2,3-0-isopropylidene-a-L-rhamnopyranoside (48) as shown i n Figure 14. Alcohol-free chloroform i s , r e q u i r e d f o r these condensations since any primary alcohol of low molecular weight such as ethanol would condense p r e f e r e n t i a l l y with the bromide thus producing unwanted glycosides. The a-D-anomer was formed i n less than 5% y i e l d as i t was j u s t detectable on t h i n - l a y e r chromatography but not by proton magnetic resonance spectros-copy . As before, the synthetic sequence was determined by the c r y s t a l -l i n e intermediates and d e r i v a t i v e s . In t h i s case removal of the i s o p r o p y l i -dene group to give methyl 4-0-(4,6-di-0-acetyl-2,3-0-carbonyl-8-D-manno--pyranosyl)-a-L-rhamnopyranoside (49) and subsequent a c e t y l a t i o n gave the c r y s t a l l i n e intermediate methyl 2 , 3 - d i - 0 - a c e t y l - 4 - 0 - ( 4 , 6 - d i - 0 - a c e t y l - 2 , 3 --O-carbonyl-8-D-mannopyranosyl)-a-L-rhamnopyranoside (50) i n 73% y i e l d based on 3. Deacylation of 50 gave methyl 4-0-B-D-mannopyranosyl-a-L-rhamnopyrano-side (51) which c r y s t a l l i z e d as an isopropylate containing two moles of 5J_ per mole of 2-propanol. Methylation and periodate oxidation studies of 51 confirmed the 1,4 linkage. A c e t y l a t i o n of 51 gave sirupy methyl 2,3-0--acetyl-4-0-(2,3,4,6-tetra-0-acetyl-3-D-mannopyranosyl)-a-L-rhamnopyranoside ( 5 3 ) . 58 For t h i s disaccharide the most d i r e c t route to the peracetate 55 (1,2,3-tri-0-acetyl-4-0-(2,3,4,6-tetra-0-acetyl-$-D-mannopyranosyl)-a--L-rhamnopyranose) appeared to be the a c e t o l y s i s of the methyl glycoside 51. On the basis of the previous success of the a c e t o l y s i s of methyl 4-0- (2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-a-L-rhamnopyranoside (33) to give the corresponding peracetate 37 i t was assumed that the a c e t o l y s i s of 5JL_ would conveniently provide 55_. However, the a c e t o l y s i s of e i t h e r 5_1_ or i t s peracetate 53 gave two compounds i n approximately equal amounts as indicated by t h i n - l a y e r chromatography. Proton magnetic resonance spec-troscopy i d e n t i f i e d the slower moving compound as the desired peracetate 55. The r a t i o of the two components could be varied by varying the concen-t r a t i o n of the s u l f u r i c a c i d and the amount of acid r e l a t i v e to the amount of s t a r t i n g material but at no time could the desired product be obtained i n more than 65% y i e l d . The f a s t e r moving component on t h i n - l a y e r chromatography was i d e n t i f i e d as 1,2,3-tri-O-acety1-4-0-(1,2,Z,4,5,6-hexa-O-acetyl-aldehydo-D--mannosyl) -ct-L-rhamnopyranose on the basis of the proton magnetic resonance spectrum, the mass spectrum, and the products obtained on deacylation. The structure i s shown below. 59 The proton magnetic resonance spectrum indicated the presence of nine acetate groups per rhamnose moiety and also that both anomeric protons were very low f i e l d (T 4.05) and therefore probably both geminal to acetoxy groups. The mass spectrum contained peaks at m/e 43, 85, 115, 145, 157, 229, 289, and 331 i n d i c a t i v e of the peracetylated a c y c l i c mannose portion of the molecule''' 5 4' 1 5 5 and a peak at m/e 273 corresponding to the rhamnose portion of the molecule. The f a c t that t h i s compound exhibits one spot on t h i n - l a y e r chromatography (appearing o l i v e green i n d i c a t i n g a hybrid of rhamnose and mannose) but when deacetylated gives monomeric rhamnose and mannose supports t h i s hemiacetal structure since hemiacetals hydrolyze i n base whereas f u l l acetals (glycosides) do not. This compound presumably arises from attack of the acetylium ion 53 120 on the r i n g oxygen rather than on the C - l oxygen ' as shown i n Figure 15. Attack at the C-l oxygen would lead to cleavage of the disaccharide linkage and concurrent a c e t y l a t i o n which i s the phenomenon normally obser-ved i n the a c e t o l y s i s of p o l y s a c c h a r i d e s 1 5 ^ . A c e t o l y s i s at the reducing anomeric carbon proceeded as i n the previous disaccharides by r e p l a c i n g the methyl glycoside with an acetate group. The three cis substituents on C - l , C-2, and C-3 of B-D-mannopyran--osides create a s t e r i c a l l y s t r a i n e d system which i s r e l i e v e d when the r i n g opens to form the a c y c l i c compound. Another example of r i n g opening to r e l i e v e s t e r i c s t r a i n during a c e t o l y s i s , observed i n t h i s laboratory, was the a c e t o l y s i s of methyl 4,6-0-benzylidene-2,3-O-carbonyl-tx-D-glucopyranoside 157 to give l-0-methyl-l,4,6-tri-0-acetyl-2,3-0-carbonyl-a7cie^z/cio-D-glucose A c y c l i c products have also been observed i n boron t r i f l u o r i d e catalyzed acetolyses igure 15. Competing Pathways i n A c e t o l y s i s 60 61 Conversely, when 50 was subjected to a c e t o l y s i s only one product resulted and i t was i d e n t i f i e d as the expected 1,2,3-tri-O-acetyl-4-0-- (4,6-di-0-acetyl-2,3-0-carbonyl-g-D-mannopyranosyl)-L-rhamnopyranose (54). The 2,3 c y c l i c carbonate appears to d i s t o r t the conformation of the r i n g so as to r e l i e v e the s t e r i c crowding r e s u l t i n g from the three cis s u b s t i t -uents and therefore prevents r i n g opening during a c e t o l y s i s . This i s another example of the 2,3 c y c l i c carbonate a l t e r i n g the s t e r i c r e l a t i o n s h i p s i n i t s environment and consequently the course of reactions t h e r e i n . To obtain the peracetate 55 i t was now necessary to deacylate 54_ and acetylate i t . The l a t t e r step was achieved with p y r i d i n e and a c e t i c anhydride but the r e s u l t i n g peracetate contained a s u b s t a n t i a l quantity of the sirupy g-L-anomer so was anomerized with zinc c h l o r i d e * ^ ' ^ ® to give c r y s t a l l i n e 55. The a c e t y l a t i o n could have been done d i r e c t l y with zinc chloride i n a c e t i c anhydride but the free disaccharide did not d i s s o l v e s u f f i c i e n t l y i n zinc c h l o r i d e and a c e t i c anhydride at room temperature and ° when heated considerable degradation was observed. The anomerization pro-ceeded smoothly at room temperature. No doubt t h i s anomerization procedure could have been used f o r other compounds i n t h i s t h e s i s to improve the y i e l d of c r y s t a l l i n e product such as 14 or 43. The free disaccharide 4-O-g-D--mannopyranosyl-L-rhamnopyranose (56) could be obtained d i r e c t l y from 54_ but was preferably obtained from c r y s t a l l i n e 55_ by deacetylation. The derived a l d i t o l 4-<9-g-D-mannopyranosyl-L-rhamnitol (57) and i t s peracetate 5_8_ were prepared by the usual procedures. 95 Thus the method of Gorin and P e r l i n f o r the synthesis of g-D--mannopyranosides has been r e f i n e d and i s being extended to other aglycons. 62 6. Conclusions The uniqueness of t h i s work l i e s not only i n the syntheses of four new disaccharides of p o t e n t i a l immunological s i g n i f i c a n c e but also i n the thoroughness with which these syntheses have been inv e s t i g a t e d . Many disaccharides are synthesized with the sole objective of acquiring a small quantity of a c e r t a i n disaccharide f o r a s p e c i f i e d purpose with no exploration into determining the best general method f o r the synthetic sequence employed. In t h i s work an attempt was made to improve the methods used for the synthesis and c h a r a c t e r i z a t i o n of these disaccharides as well as to obtain new products. This involved comparing various reaction con-d i t i o n s i n order to determine the ones which produced the maximum y i e l d of the desired product most conveniently. These r e f i n e d procedures can then be extended to other systems. Another requirement f o r an optimal y i e l d i s an adequate method of i s o l a t i o n which i s f u l f i l l e d by chromatography or more a t t r a c t i v e l y by c r y s t a l l i z a t i o n . Thus much time and e f f o r t has been spent i n obtaining the many c r y s t a l l i n e intermediates and de r i v a t i v e s pre-sented here. The improved r e a c t i o n conditions combined with the ease and e f f i c i e n c y of i s o l a t i o n provided by c r y s t a l l i n e intermediates allows the synthesis of these disaccharides i n s u f f i c i e n t quantity to fur n i s h defined systems f o r examining the r e l i a b i l i t y of, and s e l e c t i n g the optimal procedures for, the methods of s t r u c t u r a l e l u c i d a t i o n both instrumental and chemical as well as to prepare and characterize several d e r i v a t i v e s to serve as stan-dards f o r the i d e n t i f i c a t i o n of i s o l a t e d polymer fragments as shown i n Figure 16. In the course of t h i s d e t a i l e d synthetic study many noteworthy points have been brought to l i g h t . For example, the homogeneous system, 63 Figure 16. Physical Properties of Disaccharides and Derivatives 1 4 G l c Rha 3 Rha — Rha a Man — - Rha a 1 4 Man Rha B -113-114.5° m.p . M e t h y l g l y c o s i d e -58° -109° 13° -74.8° [ a ] D 2 3 ( H 2 0 ) S . 3 0 , 7 . 7 4 . 8 1 , 1 . 8 5 . 0 1 , 1 . 8 " 5 . 1 6 , 0 . 9 T H - l ' , J V 2 , (D 2 0 ) 2 . 9 4 . 0 2 . 9 1 .44 R g l u c o s e < s o l v e n t C > 182-183° m.p . M e t h y l g l y c o s i d e -51.7° 4° -57° [ a ] D 2 3 (CHC1 3 ) p e r a c e t a t e — — — — 5 . 0 2 , 1 . 4 5 . 0 1 , 1 . 6 5 . 2 5 , 0 . 8 x H - l ' , J L L J 2 , (CDC1 3 ) 139-140° 162-163° 1 4 9 . 5 - l ' 5 0 . 5 o 164-165° m.p . P e r a c e t a t e -62.3° -63.6° -S .5 ° -67.8° , [ a ] D 2 3 (CHC1 3 ) - - - - 4 . 9 9 , 1 . 9 5 . 0 0 , 1 . 8 5 . 2 7 , 1 . 0 T H - l ' , J 1 1 2 , (CDC1 3 ) 143-145° m.p . F r e e d i s a c c h a r i d e -24° -68° 60.3° -46° [ a ] D 2 3 ( H 2 0 ) 5 . 2 6 , 7 . 5 4 . 8 2 , 1 . 7 5 . 0 3 , 1 . 8 5 . 2 0 , 0 . 9 T H - l ' , J V > 2 , (D 2 0 ) 1 .2 2 . 2 1 .0 0 . 6 R g l u c o s e ( s o l v e n t C> m . p . F r e e a l d i t o l -50° 57° -36° [ a ] D 2 3 ( H 2 0 ) 5 . 3 7 , 7 . 2 5 . 0 2 , 1 . 7 4 . 8 9 , 1 . 9 5 . 2 6 , 0 . 9 T H - l ' , J 1 I J 2 , (D 2 0 ) 0 . 8 1 .3 0 . 5 0 . 5 R g l u c o s e ^ S 0 l v e n t C> 132-133° 138.5-139.5° 84-85° m.p . A l d i t o l p e r a c e t a t e -78.6° -67.2° 5 . 1 3 , 1 . 8 - 4 .4 ° 4 . 9 8 , 1 . 6 -67° 5 . 2 1 , 0 . 9 [ a ] D 2 3 (CHC1 3 ) T H - l ' , J 2 , (CDC1 3 ) 64 mercuric cyanide i n a c e t o n i t r i l e , i s a superior condensing agent f o r the synthesis of 1,2 trans glycosides aided by a p a r t i c i p a t i n g group on C-2. Some isopropylidene groups are not stable i n the presence of mercuric bromide i n a c e t o n i t r i l e . The f a c t that secondary-secondary i s o p r o p y l i -dene groups cannot be acetolyzed and that s t e r i c a l l y s t r a i n e d systems can r i n g open during a c e t o l y s i s to r e l i e v e that s t r a i n contributes to the pool of information about a c e t o l y s i s which w i l l eventually lead to an encom-passing mechanism. An e x c e l l e n t method f o r the q u a n t i t a t i o n of periodate oxidation degradation products has been elaborated and boron containing compounds were discovered when the borate was not adequately removed a f t e r reduction with sodium borohydride.* This work demonstrates the exceptional a b i l i t y of the c y c l i c carbonate group to act not only as a n o n p a r t i c i p a t i n g blocking group but more importantly as an implement f o r a l t e r i n g the s t e r i c r e l a t i o n s h i p s around i t so as to allow otherwise i n a c c e s s i b l e reactions to take place. The evidence f o r t h i s i s provided by the preparation of g-D-mannopyranosides from 44_ whereas they could not be prepared from 3,4,6-tri-c9-acetyl-2-C--benzyl-a-D-mannopyranosyl bromide and by the a c e t o l y s i s of 50_ to give the desired product whereas the a c e t o l y s i s of 51_ or 5_3 led to a d i f f e r e n t pro-duct. An amended procedure f o r the preparation of c y c l i c carbonates has a l s o been devised. The most s i g n i f i c a n t development i n t h i s work has been the f a c i l e entry into the 8-D-mannopyranoside s e r i e s presented here. The 95 method of Gorin and P e r l i n has been promoted to a p r a c t i c a b l e procedure. *Since w r i t i n g t h i s thesis i t has been noted that carbohydrate borate esters have been prepared and examined [Ya. Ya. Makarov-Zembyanskii and V.V. Gertsev, Zh. Obshch. Khim. , 35 (1965) 272; Chem. Abstr. , 62 (1965) 13216 g]. 65 III EXPERIMENTAL General Methods Melting points were obtained for samples between glass s l i d e s on a Fisher-Johns apparatus and are uncorrected. O p t i c a l rotations were meas-ured with a Perkin-Elmer model 141 polarimeter at 23 ± 1°. Proton magnetic resonance (p.m.r.) spectra were recorded on a Varian XL-100 instrument, with tetramethylsilane as the i n t e r n a l standard, except as noted. The integrated area, m u l t i p l i c i t y , and type of proton are i n d i c a t e d i n paren-theses. G a s - l i q u i d p a r t i t i o n chromatography (g.l.c.) was conducted with an F and M 720 instrument equipped with dual, thermal-conductivity detectors using helium as a c a r r i e r gas at a flow-rate of 60 ml/min, with the follow-ing columns: (a) 2 f t x 0.25 i n . of 20% of SE-30 (F and M D i v i s i o n , Hewlett Packard, Avondale, Pennsylvania), (b) 4 f t x 0.25 i n . of 5% of butanediol succinate on Diatoport S (80 - 100 mesh), and (c) 6 f t x 0.25 i n . of 15% of OS-138 on Gas Chrom Q (100 - 120 mesh). Peak areas were determined with an Infotronics CRS-100 e l e c t r o n i c i n t e g r a t o r . Mass spectra were recorded e i t h e r with a Micromass 12 g a s - l i q u i d chromatography-mass spectrometer, or on an AEI MS 9 instrument. Thin-layer chromatography ( t . l . c . ) was performed with solvent systems A and B on s i l i c a gel G (from EM Reagents, type 60); solvent A, e t h y l ether-toluene (2:1); solvent B, butanone-water azeotrope. The plates were d r i e d and components were detected by spraying with 35% ethanolic s u l f u r i c a c i d and heating f o r 3 - 5 min at ^ 150°. Paper-chromatographic separations were conducted on Whatman No. 1 paper with the upper layer of solvent systems C and D; solvent C, ethyl acetate-pyridine-66 -water (4:1:1); solvent D, 1-butanol-ethanol-water (4:1:5). Zones were made v i s i b l e by using s i l v e r n i t r a t e i n ac e t o n e 1 ^ 1 f o r reducing and non-162 reducing underivatized compounds and p - a n i s i d i n e i n t r i c h l o r o a c e t i c a c i d f o r methylated reducing sugars. indicates with respect to 2,3,4,6--tetra-C-methyl-D-glucose. Microanalyses were performed by Mr. P. Borda, M i c r o a n a l y t i c a l Laboratory, U n i v e r s i t y of B r i t i s h Columbia, Vancouver. Solutions were evaporated below 50° under diminished pressure. 163 Dry solvents where in d i c a t e d were prepared as follows : -methanol - d r i e d over sodium metal and then d i s t i l l e d , -acetone - d r i e d with anhydrous calcium s u l f a t e and then d i s t i l l e d , -pyridine - drie d by r e f l u x i n g with s o l i d potassium hydroxide, d i s t i l l e d , and stored over potassium hydroxide p e l l e t s , - a c e t i c anhydride- f r a c t i o n a l l y d i s t i l l e d or used as received from Mallinclcrodt ( a n a l y t i c a l reagent), - a c e t o n i t r i l e - s t i r r e d with calcium hydride and then d i s t i l l e d , -methyl s u l f o x i d e - s t i r r e d with calcium hydride overnight, d i s t i l l e d under' diminished pressure, and stored over Linde type 4A molecular sieves, -ethyl ether (anhydrous) - used as received from Ma l l i n c k r o d t , -petroleum ether (b.p. 65-70°) - dri e d over sodium metal and d i s t i l l e d , -benzene - refluxed over sodium metal and then d i s t i l l e d , -chloroform (alcohol-free) 58 - prepared according to Reynolds and Evans 67 Descriptions of t y p i c a l r e c u r r i n g operations are described i n d e t a i l on the pages indicated below and thereafter are merely c i t e d . The actual amounts of reagents were adjusted to the quantity of substrate to preserve the same molar r a t i o s . - a c e t y l a t i o n with p y r i d i n e , compound 2, page 68 -deacetylation (deacylation), compound 3_, page 69 -deacetalation, compound 4_, page 69 -reduction, compound 6, page 72 - a c e t y l a t i o n with sodium acetate, compound 8, page 73 - H e l f e r i c h condensation (work-up), compound 10_, page 74 -methylation, compound 12, page 75 -periodate oxidation, page 77 - a c e t o l y s i s (work-up), compound 14, page 78 - t r i m e t h y l s i l y l a t i o n , compound 15, page 79 Methyl a-L-rhamnopyranoside (1) Compound (1) was prepared e s s e n t i a l l y as described by Levene and Muskat 1^ 1. L-Rhamnose monohydrate (10.0 g, commercial preparation, from Eastman Kodak) was dissolved i n hydrogen chloride i n methanol (0.4 M, 1.6%, 100 ml, prepared by adding 2.6 ml of acetyl c h l o r i d e to 100 ml of methanol) and refluxed f o r 2 h. The cooled s o l u t i o n was rendered neutral to pH i n d i c a t o r paper with lead carbonate f i l t e r e d , and evaporated to a thick sirup. The remaining lead chloride was removed by d i s s o l v i n g the sirup i n ethyl acetate (13 ml) and f i l t e r i n g . The product c r y s t a l l i z e d from the ethyl acetate immediately upon being seeded; y i e l d 7.80 g (80%). Recrystal-l i z a t i o n from et h y l acetate gave pure 1, m.p. 108.5 - 109°; [a]p - 60.4° (c 9.6, water); l i t . 1 6 4 m.p. 109 - 110°, [ a ] ^ 0 - 62.48° (c 10.2, water), 68 l i t . 1 6 5 m.p. 108 - 109°, [a] D 2° - 62.5° ( a 9.1, water); R f 0.39 (solvent B); p.m.r. (^2®' e x t e r n a l t et ra methylsilane): x 5.36 (1 H doublet, ^ 1.5 Hz, H - l ) , 6.64 (3 H s i n g l e t , OMe), 8.75 (3 H doublet, J g 6 6 Hz, CH^). Methyl 4-Q-aoetyl-2i3-0-isopropylidene-a-L-rhcvmopyranoside (2) A s o l u t i o n of 1_ (5.00 g, f i n e l y powdered) i n dry acetone (150 ml) was magnetically s t i r r e d f o r 5 h i n an ice-water bath with 2,2-dimethoxy--propane (50 ml) and Amberlite IR-120 (H +) r e s i n (10 ml, f r e s h l y regenerated, soaked i n dry methanol overnight). The r e s i n was removed by f i l t r a t i o n through a layer of calcium oxide to remove remaining traces of acid and the f i l t r a t e was evaporated to a si r u p . T . l . c . (solvent B) showed a major component with R^ . 0.76 and a small amount of s t a r t i n g m a t e r i a l . The sirupy product was acetylated with dry pyridine (30 ml) and a c e t i c anhydride (30 ml) overnight at room temper-ature. The excess a c e t i c anhydride was removed as ethyl acetate by succes-sive additions and evaporations of ethanol. The excess p y r i d i n e was removed as an azeotrope by successive additions and evaporations of water. Insoluble impurities r e s u l t i n g from p y r i d i n e decomposition were removed by d i s s o l v i n g the dr i e d s i r u p i n ethyl acetate and f i l t e r i n g . The r e s u l t i n g acetate 2_ c r y s t a l l i z e d from ethanol (25 ml); y i e l d 4.90 g (67%). R e c r y s t a l l i z a t i o n from ethanol gave pure 2^, m.p..66 - 67°; [ a ] Q - 16.5° (<? 2.4, chloroform); l i t . 1 6 6 m.p. 61 - 63°, [a] - 14.5° (c 2.8, chloroform); R f 0.69 (solvent A); p.m.r. (CDClj): x 5.10 (1 H s i n g l e t , H - l ) , 6.62 (3 H s i n g l e t , OMe), 7.92 (3 H s i n g l e t , OAc), 8.44, 8.66 (3 H s i n g l e t s , CMe 2), 8.83 (3 H doublet, J5,6 6 H Z> CH3>" 69 Anal. Calcd. for C 1 2 H 2 0 ° 6 : C ' 5 5 ' 3 7 ; H ' 7' 7 5> 0 M e ' n - 9 2 -Found: C, 55.73; H, 7.73; OMe, 12.11. Methyl 2,3-0-isopropylidene-a-L-rhcwmopyranoside (Z) Compound 2 (5.90 g) was deacetylated with sodium methoxide i n dry methanol (0.2 J4, 30 ml, prepared by reacting sodium metal (0.5 g) with methanol (100 ml) f o r 1 h at room temperature. Sodium ions were quickly removed from the c h i l l e d s o l u t i o n with Amberlite IR-120 (H +) r e s i n (60 ml), rinsed with methanol and the remaining traces of ac i d were removed with Duolite A-4 (0H~) r e s i n (rinsed with methanol). The sirup obtained on f i l t r a t i o n and evaporation showed one spot on t . l . c , 0.54 (solvent A); y i e l d 4.90 g (99%); [ a ] Q - 29.5° ( G 5.0, chloroform), [ a ] D - 16.4° (c 3.1, acetone); l i t . 1 0 1 [ a ] D 2 4 - 14.1° { Q 1.5, water), l i t . 1 0 7 m.p. 36°, [ c O n 2 ° - 15.9° (c 1.6, acetone); p.m.r. (CDC1 3): T 5.14 (1 H s i n g l e t , H - l ) , 6.62 (3 H s i n g l e t , OMe), 8.48, 8.65 (3 H s i n g l e t s , endo-, exo- CMe2 ), 8.70 (3 H doublet, J c , 6 Hz, CH_). Methyl 4-0-acetyl-a-L-rharimopyranoside (4) A s o l u t i o n of compound 2 (3.00 g) i n chloroform (135 ml) was deacetalated with t r i f l u o r o a c e t i c a c i d containing 1% (v/v) water (15 ml) f o r 1 h at room temperature. The reac t i o n mixture was then concentrated, and remaining t r i f l u o r o a c e t i c a c i d was removed by addition and evaporation of toluene. The r e s u l t i n g sirup c r y s t a l l i z e d neat; y i e l d 2.50 g (98%). It was further p u r i f i e d by r e c r y s t a l l i z a t i o n from ethyl acetate (50 ml) or by 70 sublimation, m.p. 117°; [ a ] D - 104.1° (c 2.2, chloroform), [ a ] D - 56.0° (o 2.8, water); l i t . m.p. 112 - 116°, [ a ] D - 55° (a 1.8, water); R f 0.66 (solvent B); p.m.r. (CDClj): T 5.30 (1 H doublet, 2 1.5 Hz, H - l ) , 6.64 (3 H s i n g l e t , OMe), 7.90 (3 H s i n g l e t , OAc), 8.80 (3 H doublet, J 5 6 Hz, CH 3). Anal. Calcd. f o r C„H n^: C, 49.09, H, 7.32. Found: C, 49.22; H, 7.53. Methyl 2,3-di-O-methyl-a-L-rhamnopyranoside (5) Compound 4 (0.50 g) i n dichloromethane (5 ml) was cooled i n an i c e - s a l t water bath (-5°). Boron t r i f l u o r i d e etherate s o l u t i o n (1 ml, prepared by adding 0.4 ml of boron t r i f l u o r i d e etherate to 10 ml of d i c h l o r o -methane) was added and then diazomethane i n dichloromethane (at -78°, 168 prepared according to Vogel except that dichloromethane was substituted fo r ether) was added dropwise while s w i r l i n g the f l a s k u n t i l a f a i n t yellow c o l o r p e r s i s t e d . The r e a c t i o n mixture was kept at -5° f o r 30 min and then allowed to warm up. The s o l i d polymethylene was removed by f i l t r a t i o n and a l l v o l a t i l e components were removed from the f i l t r a t e by evaporation. T . l . c . (solvent A) showed a major spot at R^ . 0.47 with a minor monomethyl compon-ent at R^ 0.29. The product was p u r i f i e d by a preparative t . l . c . separa-169 t i o n (solvent A); y i e l d 0.45 g (80%); p.m.r. (CDC1 3): x 5.25 (1 H doub-l e t , J x 2 1.5 Hz, H - l ) , 6.49, 6.58, 6.61 (3 H s i n g l e t s , 3 OMe), 7.93 (3 H s i n g l e t , OAc), 8.80 (3 H doublet, J"5 6 6 Hz, CH 3) . The methyl 4-0-acetyl--2,3-di-0-methyl-a-L-rhamnopyranoside (0.45 g) was deacetylated i n the usual way (see page 69) to give 5 as a s i r u p ; y i e l d 0.36 g (96%), [a]^ - 6° 1 70 ( a 2.0, water), [ a ] D - 2 8 ° ( a 2.0, chloroform); l i t . [ a l D - 6 ° ( c 2.0, water), l i t . 1 7 1 [ a ] D - 14° ( e 1 . 9 7 ) , l i t . 1 7 2 [a] - 26.2° ( c 0 . 3 , chloro-71 form); R f 0.24 (solvent A) ; p.m.r. (CDClj): T 5.19 (1 H doublet, J j 2 1.5 Hz, H - l ) , 6.46, 6.48, 6.57 (3 H s i n g l e t s , 3 OMe), 8.65 (3 H doublet, J 5 6 6 Hz, CH 3). A po r t i o n of 5_ (0.20 g) was dissol v e d i n dry pyridine (0.2 ml) and treated with p-toluenesulfonyl ch l o r i d e (0.3 g, r e c r y s t a l l i z e d from benzene-petroleum ether) at 55° f o r 24 h. T . l . c . (solvent A) showed the reac t i o n to be complete with one spot of 0.57. A few drops of water were added to the rea c t i o n mixture which was l e f t standing f o r 1 h and then d i l u t e d with chloroform (100 ml). The chloroform extract was successively washed with water (2 x 75 ml), saturated sodium hydrogen carbonate s o l u t i o n (2 x 75 ml), hydroch l o r i c a c i d (0.5 M, 2 x 75 ml) and water (3 x 75 ml). The siru p obtained on evaporation c r y s t a l l i z e d from 2-propanol (3 ml); y i e l d 0.30 g (86%). R e c r y s t a l l i z a t i o n from 2-propanol gave pure methyl 2,3-di-O-methy1-4-0-toluene-p-sulphonyl-a-L-rhamnopyranoside, m.p. 114°; [ a ] D - 36.7° (c 2.6, chloroform); l i t . m.p. 111°, [ a ] D - 33° (c 2.0, chloroform); p.m.r. (CDC1 3): T 2.12 - 2.74 (4 H, 0H); 5.30 (1 H doublet, J l 2 1.5 Hz, H - l ) , 6.56, 6.68, 7.06 (3 H s i n g l e t s , 3 OMe), 7.58 (3 H si n g -l e t , 0Me), 8.67 (3 H doublet, J" 5 g 6 Hz, CHj) . Anal. Calcd. f o r C 1 6 H 2 4 0 ? S : C, 53.32; H, 6.71. Found: C, 53.11; H, 6.72. 2, 3-lK-O-methyl-L-rhamnose (6) The methyl glycoside _5 (0.30 g) was hydrolyzed with t r i f l u o r o a c e t i c a c i d (2 M, 30 ml) by r e f l u x i n g f o r 5 h. The sirup (0.26 g, 93%) obtained on evaporation showed one spot on t . l . c . R- 0.41 (solvent B) and on paper 72 chromatography gave a reddish-brown spot with R„ 0.85 (solvent D) when sprayed with a n i l i n e t r i c h l o r o a c e t a t e spray; [ a ] n 42° (c 1.5, water); l i t . 1 7 0 [ct] D 40° {a 0.7, water), l i t . 1 7 1 RQ 0.83. Compound (5 (0.20 g) was reduced with sodium borohydride (0.1 g) i n water (3 ml) overnight. Passage through Amberlite IR-120 (H +) r e s i n to remove the sodium ions, concentration, and a d d i t i o n and evaporation of methanol (5 x 50 ml) to remove the borate as methyl borate gave 2,3-di-(9--methyl-L-rhamnitol which was acetylated with p y r i d i n e and a c e t i c anhydride (see page 68) to give l,4,5-tri-0-acetyl-2,3-di-0-methyl-L-rhamnitol (7) (0.30 g, 90%); p.m.r. (CDClj): T 6.55, 6.63 (3 H s i n g l e t s , 2 OMe), 7.87, 7.90, 7.94 (3 H s i n g l e t s , 3 OAc), 8.75 (3 H doublet, J 5 6 6 Hz, CH^). I n j e c t i o n of 1_ i - n ethyl acetate onto column b programmed from 150 to 200° at 2°/min gave a major peak at 24.4 min and on column c at 225° gave a major peak at 38.6 min. A sample was c o l l e c t e d and the mass spectrum obtain-173 ed showed fragments at m/e 43, 44, 45, 58, 59, 69, 71, 74, 85, 87, 101, 113, 117, 143, 161, and 2 0 3 1 1 7 . 4-Deoxy-L-evythritol (1-deoxy-D-erythvitol) (8) Reaction of 4_ (2.40 g) with sodium metaperiodate (0.05 M, 400 ml) at 5° i n the dark reached a constant uptake of 1 mole per mole of sugar (monitored by t i t r a t i o n with sodium t h i o s u l f a t e 1 7 4 , 1 7 5 ) i n 20 h. The iodate and excess periodate were p r e c i p i t a t e d with barium acetate s o l u t i o n and removed by c e n t r i f u g a t i o n . The r e s i d u a l polyaldehyde was deacetylated by standard procedures (see page 69) and reduced with sodium borohydride (2 g) overnight at 5°. The p o l y a l c o h o l , obtained a f t e r the usual work-up (see 73 page 72), was subjected to methanolysis with hydrogen ch l o r i d e i n methanol (3%, 30 ml, prepared by adding 5 ml of a c e t y l c h l o r i d e to 100 ml of meth-anol), r e f l u x i n g f o r 6.5 h and then n e u t r a l i z e d with Duolite A-4 (OH ) r e s i n Glycolaldehyde dimethyl acetal and solvent were evaporated and the r e s u l t i n g crude si r u p (1.10 g, 95%) gave a small p o s i t i v e r o t a t i o n (e 1.6, methanol); l i t . 1 7 6 [ a ] D 7.4° ( e 2, methanol); R f 0.57 (solvent D); l i t . 1 7 7 R f 0.53 (solvent D). A p o r t i o n (0.05 g) was acetylated with sodium acetate (0.1 g) and a c e t i c anhydride (1 ml) f o r 30 min on the steam bath. The excess a c e t i c anhydride was removed as ethyl acetate by successive additions and evaporations of ethanol. The a d d i t i o n of e t h y l acetate (15 ml) and sub-sequent f i l t r a t i o n removed the sodium acetate. Evaporation of most of the ethyl acetate and i n j e c t i o n onto column b at 140° gave a major peak at 12 min. The mass spectrum was obtained and showed fragments at m/e 43, 44, 70, 71, 86, 87, 103, 115, 117, 130, 145, 159, and 189. Another portion of 8^  (1.00 g) i n dry pyridine (60 ml) was treated with p-nitrobenzoyl c h l o r i d e (5.6 g) f o r 24 h at room temperature ( i n i t i a l l y warmed to a i d s o l u t i o n ) . The reaction mixture was d i l u t e d with a few drops of water and l e f t stand f o r 2 h. The p y r i d i n e was evaporated under dimin-ished pressure and the r e s u l t i n g sirup t r i t u r a t e d with saturated sodium hydrogen carbonate s o l u t i o n . The aqeuous phase was decanted and the c r y s t a l -l i n e residue d i s s o l v e d i n chloroform (100 ml) and washed with saturated sodium hydrogen carbonate s o l u t i o n (2 x 100 ml) and then with water (2 x 200 ml). The amorphous residue obtained on evaporation c r y s t a l l i z e d from acetone (10 ml); y i e l d 2.70 g (52%). R e c r y s t a l l i z a t i o n from acetone gave pure 4-deoxy-L-erythritol t r i - p - n i t r o b e n z o a t e , m.p. 161 - 162°; [a]^ 81.9° (c 2.2, chloroform); l i t . 1 7 7 m.p. 159 - 160°, [ a ] D 81.07° [o 2.05, chloroform). Anal. Calcd. f o r c 2 5 H i 9 ° i 2 N 3 : C ' 5 4 - 2 6 5 H> 2.7,6; N, 7.59. 74 Found: C, 54.08; H, 3.31; N, 7.81. 2J3i4J6-Tetra-0-acetyl-a-D-glucopyranosyl bromide (9_) Compound 9_ was prepared from anhydrous D-glucose e i t h e r as 178 reported by Lemieux i n 68% y i e l d or more conveniently but i n lower 179 y i e l d (55%) by the method of Dale . In both cases r e c r y s t a l l i z a t i o n from ether gave pure 9_, m.p. 90.5 - 91°; [ a ] Q 199.9° (c 2.2, chloroform); 178 l i t . m.p. 88 - 89°, [ a ] D 197.8° (e 2, chloroform). Methyl 2, 3-0-isopropylidene-4-0-(2, 3, 4,6-tetra-0-acetyl-&-D-glucopyranosyl--a-L-rhamnopyranoside)(10) To a s o l u t i o n of 3 (4.90 g, 22.5 mmoles) and" dry mercuric cyanide (5.0 g) i n dry a c e t o n i t r i l e (50 ml) was added the bromide 9_ (15.0 g) with s t i r r i n g over 2 h and the s t i r r i n g was continued f o r 3 h. The a c e t o n i t r i l e was evaporated under diminished pressure and the r e s u l t i n g residue was d i s -solved i n chloroform (150 ml). The chloroform extract was washed succes-s i v e l y with potassium bromide (1 M, 2 x 75 ml), water (75 ml), saturated sodium hydrogen carbonate s o l u t i o n (75 ml), and water (2 x 75 ml). The sirup obtained on evaporation was dissolved i n warm ethanol (50 ml) and c r y s t a l l i z e d upon cooling; y i e l d 9.90 g, 18.0 mmoles (80% based on 3). R e c r y s t a l l i z a t i o n from ethanol gave pure 1TJ, m.p. 158.5 - 159°; [ a ] Q - 30.6° ( c 1.7, chloroform); R f 0.56 (solvent A); p.m.r. (CDC1 3): x 6.66 (3 H s i n g l e t , OMe), 7.96 - 8.03 (12 H, 4 OAc), 8.49, 8.67 (3 H s i n g l e t s , CMe 2), 8.75 (3 H doublet, J" 5 fi 6 Hz, CHj) . Anal. Calcd. f o r C 2 4 H 3 6 ° 1 4 : C ' 5 2 - 5 5 > H » 6-62. Found: C, 52.63; H, 6.74. 75 Methyl 4-0- (23 Z3 43 6-tetra-0-acetyl-^-D-glucopyranosyl)-a-L-vhajrrnopyranoside (11) Compound 1_0_ (2.00 g) was deacetalated with t r i f l u o r o a c e t i c a c i d i n chloroform by a previously described procedure (see page 69). The r e s u l t i n g s i r u p showed one spot on t . l . c . with R^ . 0.09 (solvent A) and 0.66 (solvent B); y i e l d 1.83 g (99%); p.m.r. ( C D C l ^ : T 6.65 (3 H s i n g l e t , OMe), 7.93 - 8.01 (12 H, 4 OAc), 8.70 (3 H doublet, J g fi 6 Hz, CHj). Methyl 4-0-$-D-glucopyranosyl-a-L-rhamnopyranoside (12) Compound 1_1_ (1.83 g) was deacetylated i n the usual way (see page 69). T . l . c . (solvent B) showed a major component with R^ 0.04 and a small amount of a f a s t e r running compound; y i e l d 1.10 g (90%). A portion 169 of the sirupy product was p u r i f i e d by preparative t . l . c . f o r a n a l y t i c a l purposes, [ a ] Q - 58° (e 1.5, methanol); R g i u c o s e 2.9 (solvent C); p.m.r. (D 20, external t e t r a m e t h y l s i l a n e ) : T 5.30 (1 H doublet, J - ^ t 7 - 7 H z > H ~ 1 ' ) > 5.32 (1 H doublet, 2 1.5 Hz, H - l ) , 6.61 (3 H s i n g l e t , OMe), 8.65 (3 H doublet, J 5 6 6 Hz, CH^. The methyl glycoside 12_ was methylated by the Hakomori method1'''1. Sirupy 12_ (1.10 g) was d i s s o l v e d i n dry methyl s u l f o x i d e (20 ml) and shaken with methyl s u l f i n y l anion (1.5 M, 20 ml, prepared according to Sandford 112 and Conrad ) f o r 4 h i n an atmosphere o f nitrogen supplied v i a a hypoder-mic needle through a serum cap. A small portion was withdrawn with a syringe and tested for excess anion by reaction with triphenylmethane. To the reaction mixture, maintained i n a nitrogen atmosphere and cooled to 0°, was added methyl iodide (6 ml) with shaking and the shaking was continued 76 overnight. The excess methyl iodide was evaporated under diminished pres-sure and the r e s u l t i n g s o l u t i o n was d i l u t e d with water (20 ml). The s o l u t i o n was extracted with petroleum ether (b.p. 65 - 70°, 5 x 100 ml). The sirup 13. obtained on evaporation showed one spot on t . l . c . (solvent A) with R f 0.28; y i e l d 1.00 g (73%); [a] - 51° (c 2.6, methanol); l i t . [ct] D 7° (e 2.0, methanol); p.m.r. (CDClj) : T 5.26 (1 H doublet, J ] [ 2 1.5 Hz, H- l ) , 5.37 (1 H doublet, v 7.7 Hz, H - l 1 ) , 6.36-6.63 (21 H, 7 OMe), 8.68 (3 H doublet, J 5 6 6 Hz, CH^ . Methanolysis of 13 (0.30 g) with hydrogen chloride i n methanol (3%, 30 ml, prepared by adding 5 ml of a c e t y l chloride to 100 ml of methanol) r e f l u x i n g f o r 8 h, n e u t r a l i z a t i o n with Duolite A-4 (OH ) r e s i n , and concentration gave the corresponding monomeric methyl glycosides which were examined by g.I.e. as 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 (prepared as described on page 79). I n j e c t i o n i n e t h y l acetate onto column b pro-grammed from 110 to 205° at 2°/min gave peaks corresponding to the methyl 2,3-di-O-methyl-L-rhamnopyranosides (6.8, 9.8 min) and the methyl 2,3,4,6--tetra-O-methyl-D-glucopyranosides (15.4, 20.7 min), i d e n t i f i e d by cochroma-tography with authentic standards (i.e. compound 5). The peak corresponding to methyl 2,3,4,6-tetra-0-methyl-B-D-glucopyranoside was c o l l e c t e d , m.p. 36°; 180 l i t . m.p. 39 - 41°. The mixed melting point with authentic standard was undepressed. Hydrolysis of 13_ (0.30 g) i n methanol (5 ml) with t r i f l u o r o -a c e t i c a c i d (2 M, 25 ml) r e f l u x i n g f o r 16 h followed by evaporation gave a mixture of 2,3-di-O-methyl-L-rhamnose and 2,3,4,6-tetra-O-methyl-D-glucose as shown by paper chromatography (solvent D) using authentic standards ( i . e . compound 6). The mixture was acetylated i n the usual way (see page 68) and then could be separated***' by g . l . c . on column b programmed from 110 to 210° at 2°/min. The glucose d e r i v a t i v e s gave peaks at 27.9 min (29%) and 30.2 min 77 (21%) while the rhamnose d e r i v a t i v e s gave peaks at 37.0 min (38%) and 40.7 min (4%). Two u n i d e n t i f i e d peaks at 16.1 min (5%) and 18.5 min (3%) may be rhamnofuranose d e r i v a t i v e s . The peaks were c o l l e c t e d and subjected to t . l . c . i n order to assign the parent sugars, as L-rhamnose and d e r i v a t i v e s appear yellow under the v i s u a l i z a t i o n conditions. A portion of the hydro-lysate was reduced by standard procedure (see page 72) and subsequently acetylated (see page 68) to give the corresponding a l d i t o l acetates. Injection i n ethyl acetate, onto column b at any temperature gave one peak 117 while on column c at 225° gave an equimolar r a t i o of two peaks which cochromatographed with authentic standards of l , 4 , 5 - t r i - 0 - a c e t y l - 2 , 3 - d i --C-methyl-L-rhamnitol (7) (38.6 min) and l,5-di-c9-acetyl-2,3,4,6-tetra--c9-methyl-D-glucitol (46.3 min). Samples were c o l l e c t e d and the mass spec-t r a obtained corresponded to those of the authentic standards. Reaction of the methyl glycoside of the disaccharide 1_2_ (0.625 g) with sodium metaperiodate (0.05 M, 40 ml) at 5° i n the dark showed a r a p i d .. uptake of 1 mole per mole of sugar with the consumption of periodate becoming constant (3.0 moles per mole of 1_2_ , monitored by t i t r a t i o n with sodium t h i o s u l f a t e ^ 7 ^ ' * 7 ^ ) i n 48 h. The iodate and excess periodate were p r e c i p i t a t e d with barium acetate s o l u t i o n and removed by c e n t r i f u g a t i o n . The r e s i d u a l polyaldehyde i n the supernatant (500 ml) was reduced with sodium borohydride (0.6 g) overnight at 5°. Passage through Amberlite IR-120 (H +) r e s i n to remove the cations, concentration, and ad d i t i o n and evaporation of methanol (5 x 50 ml) to remove the borate as methyl borate and the excess a c e t i c ac i d as methyl acetate gave the polyalcohol which was subsequently subjected to methanolysis with hydrogen chl o r i d e i n methanol (3%, .10 ml, prepared by adding 5 ml of a c e t y l c h l o r i d e to 100 ml of methanol) by r e f l u x i n g f or 6.5 h. 78 The chloride ions were removed with Duolite A-4 (OH") r e s i n and the glycolaldehyde dimethyl acetal and solvents were evaporated. The r e s u l t -ing sirup showed two major spots on paper chromatography (solvent D) corresponding to standard 4-deoxy-L-erythritol (8) and g l y c e r o l . A por-t i o n was acetylated with sodium acetate and a c e t i c anhydride as on page 73. Injection i n e t h y l acetate, onto column b at 140° gave a 1:1 r a t i o of peaks i d e n t i c a l to authentic standards of the peracetylated d e r i v a t i v e s of 4-deoxy-L-erythritol (8) (12 min) and g l y c e r o l (18 min). Mass spectra were obtained and corresponded to those of the authentic standards. A l t e r n a t i v e hydrolysis of the poly alcohol The polyalcohol (0.01 g) i n water (10 ml) was refluxed overnight i n the presence of Amberlite IR-120 (H +) r e s i n (4 ml) and Duolite A-4 (0H~) r e s i n (4 ml). The resins were removed by f i l t r a t i o n and the solvent evapor-ated. The r e s u l t i n g sirup was acetylated with sodium acetate and a c e t i c anhydride as on page 73. Injection i n ethyl acetate, onto column b at 140° gave two major peaks i n a 1:1 r a t i o i d e n t i c a l to authentic standards of the peracetylated d e r i v a t i v e s of 4-deoxy-L-erythritol (8) and g l y c e r o l plus one small u n i d e n t i f i e d peak. I, 2,3-Tri-0-acetyl-4-0-(2, 3,4,6-tetra-0-acetyl-$-D-glucopyranosyl)-a-L--rhamnopyranose (scillabiose heptaacetate) (14) Compound 1_1 (1.00 g) i n a c e t i c anhydride (5 ml) was shaken with 2% (v/v) concentrated s u l f u r i c a c i d i n a c e t i c anhydride (10 ml) f o r 5 h at room temperature. The r e a c t i o n mixture was d i l u t e d with chloroform (100 ml) and successively washed with i c e - c o l d water (2 x 100 ml), saturated sodium 79 hydrogen carbonate s o l u t i o n (2 x 100 ml), and water (2 x 100 ml). The chloroform was evaporated under diminished pressure and remaining a c e t i c anhydride, and/or a c e t i c acid was removed as eth y l acetate by addition and evaporation of ethanol. The r e s u l t i n g s i r u p , containing only a trace of cleavage products as indic a t e d by t . l . c , was dissol v e d i n 2-propanol (20 ml) and c r y s t a l l i z e d immediately when nucleated with a c r y s t a l obtained 169 from a t . l . c . separation of a small p o r t i o n ; y i e l d 0.85 g (70%). R e c r y s t a l l i z a t i o n from 2-propanol gave pure 14, m.p. 139 - 140°; [ a ] Q -62.3° [c 2.6, chloroform); R f 0.34 (solvent A); p.m.r. (CDC1 3): T 4.04 (1 H doublet, J : 2 1.5 Hz, H - l ) , 7.86 - 8.04 (21 H, 7 OAc), 8.70 (3 H doublet, J 5 6 6 Hz, CH 3). Anal. Calcd. f o r C^H.^0^: C, 50.32; H, 5.85. Found: C, 50.07; H, 5.84. 4-0-$-D-Glucopyranosyl-L-rharmopyranose (15) S c i l l a b i o s e heptaacetate 1_4 (0.50 g) was deacetylated by the customary method (see page 69). The r e s u l t i n g s i r u p (0.25 g, 95%) showed one spot on a paper chromatogram (solvent C) with R g ^ u c o s e 1-2; [ a ] n ~ 24° 35 (a 1.1, water); l i t . [ a ] D - 24.8° (c 3.3, water); p.m.r. (D 20, external tetramethylsilane): T 4.88 (0.63 H doublet, J 9 1.5 Hz, H-l, a-L-form), 5.12 (0.37 H doublet, 2 0.8 Hz, H-l, 8-L form), 5.26 (1 H doublet, 2 , 7.5 Hz, H - l ' ) , 8.65 (3 H doublet, J 5 6 Hz, CH 3). 127 The p e r - 0 - ( t r i m e t h y l s i l y l ) d e r i v a t i v e of 1_5_ was prepared by d i s s o l v i n g 1_5_ (2-6 mg) i n pyridine (0.4 ml) and rea c t i o n with hexamethyl-d i s i l a z a n e (0.2 ml) and t r i m e t h y l c h l o r o s i l a n e (0.1 ml) f o r 2 h at 40°. The 80 excess reagents and solvent were evaporated i n anhydrous conditions. G.l.c. (column a at 250°) of the p e r - O - ( t r i m e t h y l s i l y l ) disaccharide gave one peak (74%) at 10.1 min and a second peak which has the same re t e n t i o n time as p e r - O - ( t r i m e t h y l s i l y l ) sucrose at 12.6 min. 4-0-$-D-Glucopyranosyl-L-rharrrnitol (16) The free disaccharide 15 (0.20 g) was reduced with sodium boro-hydride (0.05 g) i n water (10 ml) overnight. The usual work-up (see page 72) gave 16 as a s i r u p ; y i e l d 0.20 g (99%); R g l u c o s e °- 8 (solvent C); p.m.r. (D~0, external tetramethylsilane): T 5.37 (1 H doublet, J 1 ( 7.2 Hz ^ 1,2 H - l 1 ) , 8.70 (3 H doublet, J G fi 6 Hz, CH 3). G.l.c. (column a at 250°) of the per-0- ( t r i m e t h y l s i l y l ) a l d i t o l (prepared by the procedure described on page 79) gave one peak at 13.3 min [ p e r - O - ( t r i m e t h y l s i l y l ) sucrose, 16.6 m i n ] 1 2 7 . The a l d i t o l 16_ (0.20 g) was acetylated i n the usual way (see page 68) to give c r y s t a l l i n e 4-0-8-D-glucopyranosyl-L-rhamnitol octaacetate ( s c i l l a b i i t o l octaacetate) (17) (0.38 g, 94%, c r y s t a l l i z e d from ethanol (20 ml)). R e c r y s t a l l i z a t i o n from ethanol gave pure _17, m.p. 132 - 133°; [ a ] D - 78.6° (c 2.0, chloroform); R f 0.33 (solvent A); p.m.r. ( C D C l ^ : T 7.84 - 7.99 (24 H, 8 OAc), 8.66 (3 H doublet, J 5 6 6 Hz, CH 3). Anal. Calcd. f o r C ^ H ^ O ^ : C, 50.60; H, 6.07. Found: Zo 4U io C, 50.63; H, 6.06. 81 I, 2, Z, 4-Tetra-O-aoetyl-a-L-rhamnopyranose (18) L-Rhamnose monohydrate (1.00 g> commercial preparation, from Eastman Kodak) was acetylated with p y r i d i n e (25 ml) and a c e t i c anhydride (25 ml) for 5 h at room temperature. This i s e s s e n t i a l l y the procedure 181 of Fischer et al- , except that the excess reagents were removed by successive evaporations with ethanol, and then with water (see page 68), y i e l d i n g the sirupy product (1.80 g, 98%); [ a ] Q - 63° (e 2.3, chloroform); 182 l i t . [ a ] D - 61.7° (o 2.7, chloroform); R f 0.58 (solvent A); p.m.r. (CDC1 3): T 3.98 (1 H doublet, J± 2 1.4 Hz, H - l ) , 7.85, 7.86, 7.95, 8.01 (3 H s i n g l e t s , 4 OAc), 8.77 (3 H doublet, J $ g 6 Hz, CH 3). 2i3J4-Tri-0-acetyl-a-L-rhamnopyranosyl bromide (19) Compound 18 (6.00 g) was s t i r r e d with g l a c i a l a c e t i c a c i d (6 ml), chloroform (6 ml), and hydrogen bromide i n a c e t i c a c i d (30 - 32%, 12 ml, commercial preparation, from Eastman Kodak) f o r 3 h at 0°, e s s e n t i a l l y as 181 described by Fischer et al. . The mixture was d i l u t e d with chloroform (100 ml), qu i c k l y washed with ice-water (2 x 75 ml), saturated sodium hydro-gen carbonate s o l u t i o n (2 x 75 ml), and then ice-water (2 x 75 ml), d r i e d over sodium s u l f a t e and calcium s u l f a t e at 5°, f i l t e r e d through a layer of calcium oxide and s i l i c a g e l , and evaporated to a sirup that c r y s t a l l i z e d from ethyl ether (anhydrous, 6 ml) upon the gradual addition of petroleum ether (b.p. 65 - 70°, 6 ml) at -10°; y i e l d 5.10 g (80%). R e c r y s t a l l i z a t i o n from ethyl ether gave pure 19, m.p. 64.5 - 65.6°, [ a ] D - 173.5° (c 2.4, chloro-181 form); l i t . m.p. 71 - 72°, [ a ] D - 169.0° (c 12.3, tetrachloroethane); R f 0.65 (solvent A); p.m.r. (CDC1 3): T 3.70 (1 H doublet, 2 1.6 Hz, H - l ) , 7.86, 82 7.94, 8.02 (3 H s i n g l e t s , 3 OAc), 8.73 (3 H doublet, J & & 6 Hz, CHj). Methyl 2, 3-0~isopropylidene-4-Q-(2, 3,4-tri-Q-acetyl-a-L-rhamnopyranosyl--a-L-rhamnopyranoside (20) To a s t i r r e d s o l u t i o n of compound _3 (1.00 g; 4.6 mmoles) and dry mercuric cyanide (1.0 g) i n dry a c e t o n i t r i l e (2 ml) was added the bromide 19 (3.50 g), i n portions during 3 h. At 4 h, the standard work-up (see page 74) gave an impure, amorphous material (3.00 g) which showed a major component (y 80%) on t . l . c . with 0.62 (solvent A). For a n a l y t i c a l 169 purposes a small amount was p u r i f i e d by preparative t . l . c . ; [alp - 74° (a 2.0, chlorform); p.m.r. (CDClj) : T 4.66 (1 H doublet, J-l,2, 1.9 Hz, H - l 1 ) , 5.12 (1 H s i n g l e t , H - l ) , 6.60 (3 H s i n g l e t , OMe), 7.83, 7.94, 8.01 (3 H s i n g l e t s , 3 OAc), 8.45, 8.65 (3 H s i n g l e t s , CMep, 8.66 (3 H doublet, J $ 6 6 Hz, CH 3), 8.75 (3 H doublet, J $ fi 6 Hz, CH 3). Methyl 4-0-(2} 3>4-tri-O-aaetyl-a-L-rhamnopyranosyl)-a-L-rhamnopyranoside (21) A s o l u t i o n of impure, amorphous compound 21_ (3.00 g) i n chloro-form (135 ml) was deacetalated by a previously described procedure (see page 69). The r e s u l t i n g sirup (2.70 g) showed mainly one spot on t . l . c , R £ 0.13 (solvent A) and 0.78 (solvent B); p.m.r. (CDC1 3): T 6.63 (3 H s i n g l e t , OMe), 7.86, 7.95, 8.01 (3 H s i n g l e t s , 3 OAc), 8.66 (3 H doublet, J 5 6 6 Hz, CH 3), 8.77 (3 H doublet, J $ & 6 Hz, CHj). 83 Methyl 2, 3-di-0-acetyl-4-0-(2, 3, 4~tvi-0-aoetyl-a-L-vhanmopyranosyl)-a-L--vhamnopyranoside (22) Crude compound _21_ (2.70 g) was acetylated with p y r i d i n e (25 ml) and a c e t i c anhydride (25 ml), f o r 5 h at room temperature. The sirup r e s u l t i n g from the usual work-up (page 68) c r y s t a l l i z e d from ethanol 169 (13 ml) when nucleated with a c r y s t a l obtained from a t . l . c . separation ; y i e l d 1.60 g, 3.0 mmoles (65% based on 3). R e c r y s t a l l i z a t i o n from ethanol gave pure 22^ m.p. 182 - 183°; [a]^ - 51.7° (c 2.3, chloroform); R^ . 0.51 (solvent A); p.m.r. (CDC1 3): T 5.02 (1 H doublet, J^, 2 , 1.4 Hz, H - l ' ) , 5.42 (1 H doublet, J1 2 1.2 Hz, H - l ) , 6.62 (3 H s i n g l e t , OMe), 7.88 - 8.03 (15 H, 5 OAc), 8.63 (3 H doublet, J 5 6 6 Hz, CH 3), 8.78 (3 H doublet, J 5 6 6 Hz, CH 3). Anal. Calcd. f o r c 2 3 H 3 4 ° i 4 : c> 51.68; H, 6.41. Found: C, 51.78; H, 6.52. Methyl 4-0-a-L-rh<mnopyranosyl-a-L-rhamnopyranoside (23) Compound 22_ (1.30 g) was deacetylated with sodium methoxide (0.2 M, 35 ml) i n the customary way (see page 69). The sirup obtained on evaporation showed one spot on t . l . c , R^ 0.19 (solvent B); y i e l d 0.73 g (97%); [ct] D - 109° (c 2.5, water); R g l u c o s e 4.0 (solvent C); p.m.r. (D 20, external tetramethylsilane) : 4.81 (1 H doublet, J j , 2, 1.8 Hz, H- l ' ) , 5.28 (1 H doublet, J j 2 1.0 Hz, H - l ) , 6.58 (3 H s i n g l e t , OMe), 8.64 (3 H doublet, J 5 6 6 Hz, CH 3), 8.68 (3 H doublet, J~5 6 6 Hz, CH-j) . The methyl glycoside 23 (0.30 g) was methylated by a method 84 previously described (see page 75), to give the corresponding hexamethyl ether 24_; y i e l d 0.30 g (82%). A small amount was p u r i f i e d by preparative 169 t . l . c . f o r a n a l y t i c a l purposes; 0.29 (solvent A); [ a ] Q - 81.5° [c 2.1, chloroform); p.m.r. (CDC1 3): T 4.77 (1 H doublet, J^, 2 , 1.7 Hz, H - l 1 ) , 5.26 (1 H doublet, J 1 2 1.6 Hz, H - l ) , 6.45 - 6.63 (18 H, 6 OMe), 8.69 (3 H doublet, J 5 & 6 Hz, CH3>, 8.74 (3 H doublet, J 5 & 6 Hz, CHj). Hydrolysis (as on page 76) of 24 (0.10 g) gave 2,3-di-0-methyl-L-rhamnose and 2,3,4-tri-O-methyl-L-rhamnose as shown by paper chromatography (solvent D) using authentic standards ( i . e . compound 6). The hydrolysate was reduced with sodium borohydride (0.1 g) i n water (10 ml) overnight. A c e t i c a c i d was added dropwise u n t i l the effervescence ceased and the mixture was neutral to litmus paper. The solvent was evaporated and successive por-tions of methanol (5 x 50 ml) were added and evaporated to remove the borate as methyl borate. The r e s u l t i n g residue (containing sodium acetate) was acetylated with a c e t i c anhydride (3 ml) f o r 1 h on a steam bath. The excess a c e t i c anhydride was removed as ethyl acetate by successive additions and evaporations of ethanol. The r e s u l t i n g residue was d i s s o l v e d i n e t h y l ace-tate, f i l t e r e d to remove the sodium acetate, and i n j e c t e d onto column c at 220°. Three peaks i n a 4:1:3 r a t i o were eluted at 14.0, 18.8, and 23.6 min r e s p e c t i v e l y . The peaks at 14.0 and 23.6 min cochromatographed with authentic standards of l,5-di-0-acetyl-2,3,4-tri-0-methyl-L-rhamnitol and l,4,5-tri-0-acetyl-2,3-di-0-methyl-L-rhamnitol (7) r e s p e c t i v e l y . Samples 173 were c o l l e c t e d and the mass spectra obtained corresponded to those of the 117 authentic standards The compound with retention time 18.8 min was c o l l e c t e d and had the following p.m.r. spectrum (CDCl^): x 6.51 - 6.56 (12 H, 4 OMe, expansion 85 indi c a t e d four separate methoxyl groups), 7.90, 7.94 (3 H s i n g l e t s , 2 OAc), 8.66 (3 H doublet, ^ 6 Hz, CH^). The mass spectrum obtained showed m/e 43, 45, 58, 71, 87, 101, 117, 130, 131, 143, 149, 161, 177, 191 and 203. This compound with r e t e n t i o n time 18.8 min when subjected to meth-anolysis (3% hydrogen ch l o r i d e i n methanol, prepared by adding 5 ml of acetyl chloride to 100 ml of methanol) overnight at r e f l u x , n e u t r a l i z a t i o n with Duolite A-4 (OH") r e s i n , evaporation, and subsequent a c e t y l a t i o n , cochromatographed (column c, 220°) with standard l,4,5-tri-<9-acetyl-2,3--di-O-methyl-L-rhamnitol (7). Periodate oxidation of 23_ by a procedure previously described (see page 77) showed a t o t a l consumption of 3.0 moles per mole of 23 i n 70 h. The r e a c t i o n sequence corresponding to that described previously (see page 77) gave 3-deoxy-L-glycerol diacetate and 4-deoxy-L-erythritol t r i a c e t a t e , i d e n t i f i e d by comparative g . l . c . (column b, 60 - 220°, programmed at 2°/min, r e t e n t i o n times 9.2 and 35.6 min, r e s p e c t i v e l y ) and mass-spectrometric analysis with authentic standards (i-©- 8). I, 2, Z-Tri-O-acetyl-4-0- (2, Z, 4-tri-O-acetyl-cL-L-rhamnopyranosyl)-a-L--vhamnopyvanose (25) Crude compound 21_ (2.70 g) i n a c e t i c anhydride (15 ml) was shaken with 1% (v/v) concentrated s u l f u r i c acid i n a c e t i c anhydride (30 ml) for 2 h at room temperature. The s i r u p r e s u l t i n g from the conventional work-up (see page 7 8) c r y s t a l l i z e d from ethanol (7 ml) when nucleated with a c r y s t a l 169 obtained from a t . l . c . separation ; y i e l d 1.70 g, 3.0 mmoles (65% based on 3). R e c r y s t a l l i z a t i o n from ethanol gave pure 25_, m.p. 162 - 163°; 86 [ a ] D - 63.6° (p 2.1, chloroform); R f 0.51 (solvent A); p.m.r. (CDC1 ): T 4.00 (1 H doublet, J J 2 1.7 Hz, H - l ) , 4.99 (1 H doublet, J j , 2 , 1.9 Hz, H - l 1 ) , 7.83 - 8.02 (18 H, 6 OAc), 8.62 (3 H doublet, J 5 6 6 Hz, CHj), 8.77 (3 H doublet, ^ 5 6 6 Hz, CH 3). Anal. Calcd. f o r C 2 4H 0^: C, 51.25; H, 6.09. Found: C, 51.27; H, 6.10. 4-O-a-L-Rhamnopyranosyl-L-rhamnopyranose (26) The peracetate 2_5_ (0.80 g) was deacetylated with sodium methoxide (0.2 M, 20 ml) f o r 1 h at room temperature. A f t e r the usual processing (see page 69), the r e s u l t i n g sirup (0.40 g, 91%) had [a] - 68° ( c 2.2, water); R . 2.2 (solvent C); p.m.r. (D o0, external tetramethylsilane): glucose r ' 2 x 4.82 (1 H doublet, 2, 1.7 Hz, H - l ' ) , 4.88 (0.64 H doublet, J j 2 1.3 Hz, H-l, a-L form), 5.14 (0.36 H doublet, 2 1.0 Hz, H-l, B-L form), 8.67, 8.68 (3 H doublets, J 5 6 6 Hz, 2 CH 3). G.l.c. (column a at 230°) of the p e r - O - ( t r i m e t h y l s i l y l ) disacchar-ide (prepared as on page 79) gave one peak (77.5%) at 7,2 min and a second 127 peak at 11.4 min [ p e r - O - ( t r i m e t h y l s i l y l ) sucrose, 19.4 min] 4-0-a-L-Rhamnopyranosyl-L-rhamnitoI (27) The free disaccharide 26. (0.20 g) was reduced with sodium boro-hydride (0.08 g) i n water (5 ml) for 6 h. The usual work-up (see page 72) gave 27 as a s i r u p ; y i e l d 0.19 g (95%); [ a ] D - 50° (c 2.1, water); R g l u c o s e 1.3 (solvent C); p.m.r. (D 20, external tetramethylsilane): T 5.02 (1 H doublet, J v 2 , 1.7 Hz, H - l 1 ) , 8.71 (6 H doublet, J $ 6 6 Hz, 2 CHj). 87 G.l.c. (column a at 230°) of the p e r - 0 - ( t r i m e t h y l s i l y l ) a l d i t o l (prepared by a previously described procedure, see page 79) gave one peak 127 at 11.6 min [ p e r - O - ( t r i m e t h y l s i l y l ) sucrose, 19.8 min] The a l d i t o l 27_ (0.19 g) was acetylated (see page 68) to give 1,2,3,5-tetra-0-acetyl-4-0-(2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl)-L--rhamnitol (28) which c r y s t a l l i z e d from ethanol (3 ml); y i e l d 0.35 g (95%). R e c r y s t a l l i z a t i o n from ethanol gave pure 28, m.p. 138.5 - 139.5°; [ a ] D - 67.2° (c 2.1, chloroform); R f 0.42 (solvent A); p.m.r. (CDC1 3): T 5.13 (1 H doublet, 2, 1.8 Hz, H - l ' ) , 7.83 - 7.99 (21 H, 7 OAc), 8.59 (3 H doublet, J 5 & 6-Hz, CH 3), 8.75 (3 H doublet, J 5 6 6 Hz, CH 3). Anal. Calcd. f o r C 2 6 H 3 8 ° 1 6 : C ' 5 1 > 4 8 > H> 6 - 3 2 - Found: C, 51.69; H, 6.18. G.l.c. (column a at 260°) of the peracetylated a l d i t o l 28 gave one peak at 6.4 min (sucrose octaacetate, 16.2 min). l j 2 j 3 3 4 , 6-Penta-O-benzoyl-a-D-mannopyranose (29) Compound 2£ was prepared e s s e n t i a l l y as described by Fischer 183 and Oetker . a-D-Mannose (25.0 g f i n e l y ground, dry) was added i n por-tions to a s o l u t i o n of dry chloroform (375 ml), benzoyl c h l o r i d e (125 ml) and dry pyridine (125 ml) at 0°. The mixture was s t i r r e d at 0° u n t i l a c l e a r s o l u t i o n r e s u l t e d and the s t i r r i n g was continued f o r an ad d i t i o n a l 3 h. The pyridine was removed by washing the r e a c t i o n mixture with s u l f u r i c a c i d (3 M, 5 x 200 ml) and then with water (3 x 200 ml). Ethanol (500 ml) was added to the chloroform extract which was then evaporated to a sirup that c r y s t a l l i z e d from ethanol (1600 ml); y i e l d 90.0 g (93%). R e c r y s t a l l i z a -88 t i o n from ethanol gave pure 29, m.p. 152 - 153°; [ a ] D - 19.9° [c 2.9, 184 chloroform); l i t . m.p. 152 - 153°, [ a ] Q - 18.6° [c 1.6, chloroform); R f 0.80 (solvent A); p.m.r. (CDC1 ): x 1.74 - 2.75 (25 H, 5 OBz), 3.35 (1 H doublet, J J 2 1.9 Hz, H - l ) . 2}3i436-Tetra-0-benzoyl-a-D-mannopyranosyl bromide (30) Compound 30_ was prepared by the procedure of Ness, Fl e t c h e r 184 and Hudson . A s o l u t i o n of 29_ (10.0 g) i n ethylene d i c h l o r i d e (20 ml) was treated with hydrogen bromide i n a c e t i c a c i d (30 - 32%, 20 ml, commercial preparation, from Eastman Kodak) overnight at room temperature. The r e a c t i o n mixture was d i l u t e d with ethylene d i c h l o r i d e (100 ml) and washed successively with ice-water (2 x 100 ml), saturated sodium hydrogen carbonate s o l u t i o n (2 x 100 ml), and ice-water (2 x 100 ml), d r i e d with sodium s u l f a t e and calcium s u l f a t e , and f i l t e r e d through a layer of c a l -cium oxide and s i l i c a g e l . The s i r u p obtained on evaporation showed one spot on t . l . c . (solvent A) with R £ 0.83 and on one occasion the sirupy pro-duct c r y s t a l l i z e d neat but i t was not possible to r e c r y s t a l l i z e i t from solvent; y i e l d 9.10 g (97%); [a] 11° (c 3.3, chloroform); l i t . 1 8 4 [a] 11.7° (c 2.8, chloroform). Methyl 23 3-0-isopropylidene-4-0-(2, 3, 4, 6-tetra-Q-benzoyl-a-D-mannopyranosyl)--a-L-rhamnopyranoside (31) The bromide 30 (15.0 g) i n dry a c e t o n i t r i l e (20 ml) was added to a s t i r r e d s o l u t i o n of 3_ (4.00 g, 18.3 mmoles) and dry mercuric cyanide 89 (4.0 g) i n dry a c e t o n i t r i l e (10 ml) during 2 h and the s t i r r i n g was continued f o r an a d d i t i o n a l 4 h. The standard work-up procedure (see page 74) gave a crude amorphous s o l i d (17.4 g) which showed a major compon-ent 80%) on t . l . c . (solvent A) with R^ 0.71. A small amount was 169 p u r i f i e d by preparative t . l . c . f o r a n a l y t i c a l purposes ; p.m.r. (CDCl^): x 1.82 - 2.78 (20 H, 4 OBz), 4.78 (1 H doublet, J p 2, 1.5 Hz, H - l 1 ) , 5.14 (1 H s i n g l e t , H - l ) , 6.63 (3 H s i n g l e t , OMe), 8.42, 8.66 (3 H s i n g l e t s , CMe 2), 8.63 (3 H doublet, J & & 6 Hz, CH 3). Methyl 233-0-isopropylidene-4-0-(23 334,6-tetra-O-acetyl-a-D-mannopyranosyl)--a-L-rhamnopyranoside (32) Compound 31_ (crude amorphous s o l i d , 17.3 g) was debenzoylated with sodium methoxide (0.2 M, 200 ml) f o r 1 h at room temperature. Follow-ing the usual work-up (see page 69) the remaining methyl benzoate was removed by steam d i s t i l l a t i o n under diminished pressure. The r e s u l t i n g sirup (R^ 0.31 (solvent B)) was acetylated with p y r i d i n e (100 ml) and a c e t i c anhydride (100 ml) overnight at room temperature. The acetate 32_, obtained on conventional work-up (see page 68), c r y s t a l l i z e d from ethanol (25 ml); y i e l d 7.60 g, 13.9 mmoles (76% based on 3). R e c r y s t a l l i z a t i o n from ethanol gave pure 32_, m.p. 119 - 120°; [ a ] D 30.7° (c 2.2, chloroform); R f 0.53 (solvent A); p.m.r. (CDC1 3): T 5.01 (1 H doublet, J ^ , 2, 1.7 Hz, H-l»), 5.14 (1 H s i n g l e t , H - l ) , 6.63 (3 H s i n g l e t , OMe), 7.84, 7.90, 7.97, 8.01 (3 H s i n g l e t s , 4 OAc), 8.48, 8.66 (3 H s i n g l e t s , CMe 3), 8.72 (3 H doublet, J 5 6 6 Hz, CH 3). Anal. Calcd. f o r C 2 4 H 3 6 ° 1 4 : C ' 5 2 - 5 5 > H» 6 - 6 2 - Found: C, 52.38; H, 6.49. 90 Methyl 4-0- (2,3} 4, 6-tetra-O-aaetyl-a-D-mannopyranosyl)-a-L-rhcarmopyranoside (31) A s o l u t i o n of 52_ (2.00 g) i n chloroform (90 ml) was deacetalated by a previously described procedure (see page 69). The r e s u l t i n g s i r u p showed one spot on t . l . c , R^ . 0.17 (solvent A) and 0.59 (solvent B); y i e l d 1.82 g (98%); p.m.r. ( C D C l ^ : T 5.04 (1 H doublet, J ^ , 2 , 1.7 Hz, H - l ! ) , 5.31 (1 H s i n g l e t , H - l ) , 6.62 (3 H s i n g l e t , OMe), 7.85, 7.90, 7.95, 7.99 (3 H s i n g l e t s , 4 OAc), 8.67 (3 H doublet, J & 6 6 Hz, CH 3). Methyl 4-Q-a-D-mannopyranosyl-a-L-rharmopyvanoside (34) Compound 33_ (1.83 g) was deacetylated i n the usual way (see page 69). The s i r u p obtained on evaporation showed one spot on t . l . c , R f 0.03 (solvent B); y i e l d 1.17 g (96%), [ a ] D 13° (e 2.2, water); R g l u c o s e 2.9 (solvent C); p.m.r. (D^O, external t e t r a m e t h y l s i l a n e ) : x 5.01 (1 H doublet, J , , ~, 1.8 Hz, H - l 1 ) , 5.29 (1 H doublet, J 1.5 Hz, H - l ) , 6.60 (3 H s i n g l e t , OMe), 8.67 (3 H doublet, J 5 6 6 Hz, CH 3). Methylation of 34_ by a previously described procedure (see page 75) gave the corresponding heptamethyl ether 3_5, which showed one spot on t . l . c . (solvent A) with R^ 0.14; [ a ] Q 10° (c 1.0, chloroform); p.m.r. (CDC1 3): x 4.93 (1 H doublet, 2 , 1.6 Hz> H - l ' ) , 5.27 (1 H doublet, 2 1.6 Hz, H-l), 6.46 - 6.63 (21 H, 7 OMe), 8.72 (3 H doublet, J*5 & 6 Hz, CH 3). Hydrolysis (as on page 76) of _35 gave 2,3-di-O-methyl-L-rhamnose and 2,3,4,6-tetra-O-methyl-D-mannose i d e n t i f i e d by paper chromatography (solvent D) using authentic standards ( i . e . 6). Subsequent reduction and a c e t y l a t i o n (see page 72) gave equimolar amounts of 1,4,5-tri-0-acetyl-2,3,-di-c?-methyl-91 -L-rhamnitol and 1 ,5-di-0-acetyl-2,3,4,6-tetra-(9-methyl-D-mannitol, iden-t i f i e d by g . l . c . (column c, 220°, retention times 33.0 and 41.0 min, 117 respectively) and mass-spectrometric comparison with authentic standards ( i . e . 7). Periodate oxidation of 34_ by a previously described procedure (see page 77) showed a rapid uptake of 2 moles of periodate per mole of 34 with the consumption becoming constant (3.0 moles per mole) i n 30 h. Subsequent reduction, methanolysis, and evaporation (see page 77) gave a sirup that showed two major spots on paper chromatography (solvent D) corresponding to standard 4-deoxy-L-erythritol (8) and g l y c e r o l . Acetyla-t i o n gave 4-deoxy-L-erythritol t r i a c e t a t e and g l y c e r o l t r i a c e t a t e , iden-t i f i e d by g . l . c . (column b, 140°, r e t e n t i o n times 7.2 and 10.0 min, respec-t i v e l y ) and mass-spectrometric comparison with authentic standards. Methyl 23 Z-di-O-aaetyl-4-0-(23 334,6-tetra-0-aeetyl-a-D-mannopyvanosyl)-a--L-rhamnopyranoside (26) Compound 33 (0.10 g) was acetylated by standard procedures (see page 68); y i e l d 0.11 g (94%). A p o r t i o n of the sirupy product was p u r i f i e d 169 by preparative t . l . c . f o r a n a l y t i c a l purposes; 4° (c 1.3, chloroform); R f 0.41 (solvent A); p.m.r. (CDC1 3): x 5.01 (1 H doublet, J p 2 , 1.6 Hz, H- l ' ) , 5.40 (1 H doublet, 2 1.5 Hz, H - l ) , 6.61 (3 H s i n g l e t , OMe), 7.84 - 8.01 (18 H, 6 OAc), 8.60 (3 H doublet, J"5 & 6 Hz, CHj) . 92 I, 2,3-Tri-0-acetyl-4-0-(2, 3, 4,6-tetra-O-aeetyl-a-D-mannopyranosyl)-a-L--rhamnopyranose (37) Compound 53 (1.00 g) i n a c e t i c anhydride (5 ml) was shaken with 2% (v/v) concentrated s u l f u r i c a c i d i n a c e t i c anhydride (10 ml) f o r 3 h at room temperature. The sirup r e s u l t i n g from the conventional work-up (see page 78) c r y s t a l l i z e d from ethanol (10 ml); y i e l d 0.95 g (78%). R e c r y s t a l l i z a t i o n from ethanol gave pure 3_7, m.p. 149.5 - 150.5°; [ a ] D -5.5° (e 4.3, chloroform); R £ 0.39 (solvent A); p.m.r. (CDClj) : x 4.00 (1 H doublet, J 1.5 Hz, H - l ) , 5.00 (1 H doublet, J 1.8 Hz, H - l ' ) , 7.82 - 8.00 (21 H, 7 OAc), 8.60 (3 H doublet, J 5 & 6 Hz, CHj). Anal. Calcd. f o r C 0, H,,0 • C, 50.32; H, 5.85. Found: C, 50.37; H, 5.98. 4-0-a-D-Mannopyranosyl-a-L-rhamnopyranose (38) The peracetate 37. (1-20 g) was deacetylated with sodium methoxide (0.2 M, 30 ml) f o r 1 h at room temperature followed by the usual work-up (see page 69). The sirup (0.60 g, 95%) obtained on evaporation c r y s t a l l i z e d neat a f t e r long standing. Pure 3_8_ was obtained by r e c r y s t a l l i z a t i o n from a 1:1 mixture of methanol and 2-propanol (20 ml/g), m.p. 143 - 145°; [a]p 53.0° mutarotating to 60.3° i n 2 h (c 1.0, water); R , 1.0 (solvent C); B K > J > g i U C o s e ^ p.m.r. (D 20, external tetramethylsilane): x 4.90 (0.64 H doublet, 2 1.5 Hz, H-l, a-L form), 5.03 (1 H doublet, J 1.8 Hz, H - l ' ) , 5.16 (0.36 H doublet, J x 2 1.0 Hz, H-l, g-L form), 8.71 (3 H doublet, J 5 & 6 Hz, CHj). Anal. Calcd. f o r C ^ H ^ O ^ : C, 44.17; H, 6.80. Found: C, 43.91; H, 6.86. 93 G.l.c. (column a at 240°) of the p e r - O - ( t r i m e t h y l s i l y l ) disaccharide (prepared by the procedure on page 79) gave one peak (80%) at 8.5 min and a second peak at 11.'7 min [ p e r - 0 - ( t r i m e t h y l s i l y l ) sucrose, 14.5 m i n ] 1 2 7 . The disaccharide 3_8 (0.10 g) was d i s s o l v e d i n sodium acetate b u f f e r (0.3 M, pH 4.5, 2 ml) and incubated at 37° with crude a-mannosidase 185 (2 ml) prepared from jack bean meal e s s e n t i a l l y by the procedure of L i The free disaccharide 38. was ^ 80% cleaved i n 10 h and completely cleaved i n 22 h as i n d i c a t e d by paper chromatography (solvent C) using authentic standards. Under these conditions methyl 8-D-mannopyranoside (46) was not cleaved but methyl a-D-mannopyranoside (commercial preparation, from Calbiochem) was hydrolyzed. The disaccharide r e a c t i o n mixture was p r e c i p i -tated into ethanol and centrifuged. The supernatant was deionized with Amberlite IR-120 (H +) and Duolite A-4 (0H~) r e s i n s . The r e s u l t i n g s o l u t i o n of mannose and rhamnose was reduced and acetylated by the standard procedure (see page 72). I n j e c t i o n of the mixture onto column b programmed from 150 to 220° at 2°/min gave a 1:1 r a t i o of peaks i d e n t i c a l to authentic standards of L-rhamnitol pentaacetate and D-mannitol hexaacetate. Samples were c o l -173 lected and the mass spectra obtained corresponded to those of the authentic standards. Furthermore, the D-mannitol hexaacetate had m.p. 120 - 122°, 186 undepressed by an authentic sample; l i t . m.p. 125°. 4-0-oL-D-Mcmnopyranosyl-L-rhamnitol (39) The free disaccharide 38_ (0.50 g) was reduced with sodium boro-hydride (0.20 g) i n water (25 ml) overnight. The routine work-up (see page 72 ) gave sirupy 39; y i e l d 0.49 g (97%); [ a ] D 57° (c 3.5, water); R g l u c o s e 94 0.5 (solvent C); p.m.r. (D 20, external tetramethylsilane) : T 4.89 (1 H doublet, J l t 2 , 1.9 Hz, H - l 1 ) , 8.73 (3 H doublet, J 5 & 6 Hz, CHj). G.l.c. (column a at 240°) of the p e r - 0 - ( t r i m e t h y l s i l y l ) a l d i t o l (preparation analogous to that on page 79) gave one peak at 11.5 min 127 [ p e r - O - ( t r i m e t h y l s i l y l ) sucrose, 12.9 min] The a l d i t o l 39 (0.30 g.) was acetylated with pyridine (10 ml) and a c e t i c anhydride (10 ml) to give 1,2,3,5-tetra-0-acetyl-4-C-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-L-rhamnitol (40), which was c r y s t a l l i z e d from ethanol (5 ml); y i e l d 0.56 g (92%). R e c r y s t a l l i z a t i o n from ethanol gave pure 40, m.p. 84 - 85°; [a] D - 4.4° (e 1.9, chloroform); R f 0.33 (solvent A); p.m.r. (CDC1 3): T 4.98 (1 H doublet, J ^ , 2 , 1.6 Hz, H - l ' ) , 7.84 - 8.02 "(24 H, 8 OAc), 8.63 (3 H doublet, J 5 6 6 Hz, CHj) . Anal. Calcd. f o r C_ oH._0 1 o: C, 50.60; H, 6.07. Found: 28 40 18 ' ' C, 50.39; H, 6.13. G.l.c. (column a at 270°) of the peracetylated a l d i t o l 40 gave one peak at 7.0 min (sucrose octaacetate, 10.0 min). Methyl 4,6-0-benzylidene-a-D-mannopyranoside (41) Compound 4_1_ was prepared e s s e n t i a l l y as described by Buchanan 187 ' 149 and Schwarz with the modification of Gibney . Methyl a-D-mannopyranoside (10.0 g, f i n e l y powdered, commercial preparation, from Calbiochem) was dissolved as r a p i d l y as possible i n formic a c i d (98 - 100%, 50 ml) and benzaldehyde (50 ml, f r e s h l y d i s t i l l e d ) was immediately added to the solu-t i o n . A f t e r standing 5 min with occasional shaking, the s o l u t i o n was poured with s t i r r i n g into a mixture of water (200 ml) and anhydrous potassium car-bonate (137 g). The excess benzaldehyde was immediately removed by steam 95 d i s t i l l a t i o n of the r e s u l t i n g s o l u t i o n . The aqueous phase was extracted with chloroform i n a continuous extractor. The chloroform extract was evaporated to a s o l i d residue that c r y s t a l l i z e d from benzene (210 ml); y i e l d 5.50 g (38%). R e c r y s t a l l i z a t i o n from benzene gave pure 41_, m.p. 146-147°; [ a ] D 64.3° {a 2.1, chloroform); l i t . 1 8 7 m.p. 140-143°, [ a ] 61° (e 1.84, chloroform), l i t . 1 8 8 m.p. 146-147°, [ a ] Q 71.7° (<? 1.185, chloroform); R f 0.23 (solvent A); p.m.r. (CDC1 ): T 2.44 - 2.66 (5 H, 0H), 4.44 (1 H s i n g l e t , 0CH), 5.27 (1 H doublet, ^ 2 0.9 Hz, H - l ) , 6.62 (3 H s i n g l e t , OMe). Methyl 4,6-0-benzylidene-2,3-0-carbonyl-a-D-mannopyranoside (42) Compound 42_ was prepared with some reference to Doane et al . Compound 41_ (3.00 g, dry) i n p-dioxane (15 ml) was treated with a s o l u t i o n of triethylamine (7.5 ml) i n benzene (45 ml) and subsequently cooled i n an ice-water bath. While the suspension was being magnetically s t i r r e d e t h y l chloroformate (25 ml) was added dropwise so as to keep the effervescence under c o n t r o l (during ^ 20 min). The rea c t i o n mixture (basic to litmus paper) was s t i r r e d i n the ice-water bath f o r an a d d i t i o n a l 20 min, then d i l u t e d with benzene (200 ml) and washed successively with ice-water (2 x 200 ml), hydrochloric acid (1 M, 200 ml), water (200 ml), saturated sodium hydrogen carbonate s o l u t i o n (200 ml), and water (2 x 200 ml). Evaporation of the solvent gave a s o l i d residue which was c r y s t a l l i z e d from ethanol (9 ml); y i e l d 3.10 g (94%). R e c r y s t a l l i z a t i o n from ethanol gave pure 42_, m.p. 125 - 127°; [ a ] D - 21.0° (c 2.1, chloroform); l i t . 1 5 1 m.p. 125 - 126°, [ a ] D - 19.3° (chloroform); R f 0.75 (solvent A); p.m.r. (CDClj): T 2.46 - 2.70 (5 H, 0H), 4.43 (1 H s i n g l e t , 0CH), 4.99 (1 H s i n g l e t , H - l ) , 96 6.62 (3 H s i n g l e t , OMe). Z-j 43 6-Tvi-O-acetyl-2, 3-0-carbonyl-a-D-mannopyranose (43) Compound 4_2_ (3.00 g) was suspended i n a c e t i c anhydride (15 ml) and shaken with 1% (v/v) concentrated s u l f u r i c a c i d i n a c e t i c anhydride (30 ml) for 2 h at room temperature. The sirup r e s u l t i n g from the conven-t i o n a l work-up (see page 78) c r y s t a l l i z e d from ethanol (10 ml) to y i e l d 2.00 g of c r y s t a l s . A f u r t h e r 0.60 g of sir u p containing some 3-anomer was 189 obtained by pressure column chromatography on s i l i c a gel H (from EM Reagents) and e l u t i o n with solvent A; t o t a l y i e l d 2.60 g (81%). Recrystal-l i z a t i o n from ethanol gave pure 43, m.p. 117.5 - 118.5°; [ct]p 15.6° (e 2.5, 95 chloroform); l i t . 7.5° (e 1.0, chloroform); R £ 0.21 (solvent A); p.m.r. (CDC1 3): x 3.60 (1 H s i n g l e t , H - l ) , 7.86, 7.88, 7.92 (3 H s i n g l e t s , 3 OAc). Anal. Calcd. f o r C^H^O • C, 46.99; H, 4.85. Found: lo i o 1U C, 46.90; H, 4.95. 4,6-Di-0-acetyl-233-0-carbonyl-a-D-mannopyranosyl bromide (44) Compound 44_ was prepared e s s e n t i a l l y as described by Gorin and 95 P e r l i n . Compound 4_3 (2.00 g) i n chloroform (200 ml) was s t i r r e d with hydrogen bromide i n a c e t i c a c i d (30-32%, 40 ml, commercial preparation, from Eastman Kodak) f o r 3 h at 0°. The chloroform s o l u t i o n was quickly washed successively with ice-water (2 x 200 ml), saturated sodium hydrogen carbonate s o l u t i o n (2 x 200 ml), and ice-water (2 x 200 ml), dr i e d with an-hydrous sodium s u l f a t e and calcium s u l f a t e , f i l t e r e d through a layer of 97 calcium oxide and s i l i c a g e l , and evaporated to a sirup that c r y s t a l l i z e d from ethyl ether (anhydrous, 25 ml) at -10°. Petroleum ether (b.p. 65-70°, 10 ml) was added i n portions to complete the c r y s t a l l i z a t i o n ; y i e l d 1.75 g (82%). R e c r y s t a l l i z a t i o n from ethyl ether gave pure 44, m.p. 79 - 80°; [ a ] D 89.8° (c 3.3, chloroform); l i t . [ a ] D 55° (c 1.0, chloroform); R f 0.63 (solvent A); p.m.r. (CDC1 3): T 3.33 (1 H s i n g l e t , H - l ) , 7.86, 7.92 (3 H s i n g l e t s , 2 OAc). Methyl 436-di-0-acetyl-2i3-0-carbonyl-$-D-mannopyranoside (45) Compound 44_ (1.60 g) was s t i r r e d with dry a c e t o n i t r i l e (10 ml), dry methanol (10 ml), and dry mercuric cyanide (1.60 g) for 4 h. Standard work-up procedures (see page 74) gave a sirup that c r y s t a l l i z e d from ethanol (12 ml); y i e l d 1.20 g (87%). R e c r y s t a l l i z a t i o n from ethanol gave pure 45, m.p. 137 - 138°; [ a ] D - 85.9° (c 2.7, chloroform); l i t . [ a ] D - 37° (c 1.0, chloroform); R £ 0.22 (solvent A); p.m.r. (CDC1 3): x 5.17 (1 H doublet, J x 2 3 Hz, H - l ) , 6.43 (3 H s i n g l e t , OMe), 7.89, 7.91 (3 H s i n g l e t s , 2 OAc). Anal. Calcd. f o r C 1 2 H 1 6 0 9 : C, 47.37; H, 5.30. Found: C, 47.49; H, 5.45. Methyl &-D-mannopyranoside 2-pvopyl alaoholate (46) Compound 45_ (0.50 g) was d e e s t e r i f i e d with sodium methoxide (0.2 M, 15 ml) f o r 1 h at room temperature, followed by the usual work-up (see page gg). The sirup obtained on evaporation c r y s t a l l i z e d from 2-propanol (15 ml) a f t e r addition and evaporation of 2-propanol (2 x 30 ml); y i e l d 0.36 g (86%). R e c r y s t a l l i z a t i o n from 2-propanol gave pure 46, m.p. 98 72 - 73°; [a] - 52.7° (a 2.2, water); l i t . 1 9 0 m.p. 74 - 75°, [ a ] D - 53.3° (c 4, water); 1U 0.02 (solvent B); R 1 1.62 (solvent C); p.m.r. (D„0, v > ± v J> glucose r 2 external tetramethylsilane): T 5.41 (1 H doublet, J 1 ? 0.9 Hz, H - l ) , 6.43 (3 H s i n g l e t , OMe), Methyl 233J436-tetra-0-acetyl-$-D-mannopyranoside (47) Compound 46_ (0.10 g) was acetylated i n the usual way (see page 68). The r e s u l t i n g product c r y s t a l l i z e d neat or from ethanol (3 ml); y i e l d 0.18 g (96%). R e c r y s t a l l i z a t i o n from ethanol gave pure 47, m.p. 163 - 164°; [ a ] D - 50.1° (c 2.2, chloroform); l i t . 9 5 m.p. 161 - 162°, l i t . 1 9 0 m.p. 159 - 160°, [ a ] D - 47.8°; R f 0.43 (solvent A); p.m.r. (CDC1 3): T 5.42 (1 H doublet, J 1 2 1.1 Hz, H - l ) , 6.47 (3 H s i n g l e t , OMe), 7.83, 7.92, 7.97, 8.02 (3 H s i n g l e t s , 4 OAc). Methyl 4-0-(4} 6-di-0-acetyl-233-0-carbonyl-^-D-mannopyranosyl)-2 3 3-0--isopropylidene-a-L-rhamnopyranoside (48) Compound _3 (1.00 g, 4.6 mmoles) was s t i r r e d magnetically i n the dark f o r 1 h at room temperature with s i l v e r oxide (3 g, f r e s h l y prepared, dry), calcium s u l f a t e (4 g, anhydrous), and chloroform (10 ml, dry, al c o h o l -f r e e ) . Compound 44 (3.00 g) i n chloroform (20 ml, dry, alco h o l - f r e e ) was added dropwise during 3 h and the s t i r r i n g was continued f o r an a d d i t i o n a l 0.5 h. The r e a c t i o n mixture was f i l t e r e d and evaporated to a crude s i r u p (3.75 g) which showed a major component (y 80%) on t . l . c . (solvent A) with R^ 0.36. For a n a l y t i c a l purposes a small amount was p u r i f i e d by preparative 169 t . l . c . ; [ a ] D - 65° (c 3.1, chloroform); p.m.r. (CDC1 3): T 5.15 (1 H 99 s i n g l e t , H - l ) , 5.19 (1 H doublet, J j , 2 , 1.4 Hz, H - l 1 ) , 6.65 (3 H s i n g l e t , OMe), 7.90, 7.92 (3 H s i n g l e t s , 2 OAc), 8.49, 8.66 (3 H s i n g l e t s , CMe 2), 8.63 (3 H doublet, J 5 6 6 Hz, CHj). Methyl 4-0-(43 6-di-0-acetyl-23 3-0-carbonyl-$-D-mannopyranosyl)-a-L--rhamnopyranoside (49) A s o l u t i o n of crude compound 4J3 (3.75 g) i n chloroform (135 ml) was deacetalated by a previously described procedure (see page 69). The r e s u l t i n g s i r u p (3.60 g) showed mainly one spot on t . l . c , R £ 0.04 (solvent A) and 0.62 (solvent B); p.m.r. (CDClj): T 6.65 (3 H s i n g l e t , OMe), 7.89, 7.92 (3 H s i n g l e t s , 2 OAc), 8.61 (3 H doublet, 3^ 6 6 Hz, CH^. Methyl 233-di-Q-acetyl-4-0-(436-di-0-acetyl-23 3-0-carbonyl-8-D-mannopyranosyl)--a-L-rhamnopyranoside (50) Crude compound 49_ (3.60 g) was acetylated with py r i d i n e (20 ml) and a c e t i c anhydride (20 ml) overnight at room temperature. The excess reagents were removed as before (see page 68). The r e s u l t i n g sirup c r y s t a l l i z e d from ethanol (25 ml); y i e l d 1.80 g, 3.37 mmoles (73% based on 3). R e c r y s t a l l i z a t i o n from ethanol gave pure _50_, m.p. 204.5 - 205.5°; [ a ] D - 61.8° (e 2.4, chloroform); R f 0.08 (solvent A); p.m.r. (CDC l ^ : T 6.65 (3 H s i n g l e t , OMe), 7.92, 7.94, 8.03 (3 H s i n g l e t s , 4 OAc), 8.61 (3 H doublet, 6 6 Hz, CH^). Anal. Calcd. f o r C 2 2 H 3 0 ° 1 5 : C ' 4 9 - 4 4 ' H> 5 * 6 6 ' F o u n d : C, 49.27; H, 5.69. 100 Methyl 4-0-&-D~mannopyranosyl-a-L-rharmopyranoside (51) Compound 50_ (1.40 g) was d e e s t e r i f i e d with sodium methoxide (0.2 M, 40 ml) f o r 1 h at room temperature. The c a t a l y s t was destroyed as formerly described (see page 69). The sirup (0.85 g, 95%), obtained on evaporation, showed one spot on t . l . c , R £ 0.03 (solvent B), and c r y s t a l -l i z e d from 2-propanol (50 ml) as an alcoholate a f t e r addition and evapora-t i o n of 2-propanol (2 x 50 ml); y i e l d 0.90 g of c r y s t a l s (97%). Recrystal-l i z a t i o n from 2-propanol gave pure 51_ alcoholate, m.p. 113-114.5° [ct]p -74.8° re 2.2, water); R , 1.44 (solvent C); p.m.r. (D o0, external ' ' glucose v K 2 ' tetramethylsilane): x 5.16 (1 H doublet, J 0.9 Hz, H - l 1 ) , 5.35 (1 H doublet, J „ 1.3 Hz, H - l ) , 6.64 (3 H s i n g l e t , OMe), 8.69 (3 H doublet, J 6 Hz, CHg). The 2-propanol of c r y s t a l l i z a t i o n was l o s t by exchanging the a c t i v e hydrogens with deuterium oxide. Anal. Calcd. f o r c 2 9 H 5 6 ° 2 i : C ' 4 7-02; H, 7.62. Found: C, 46.82; H, 7.81. The methyl glycoside 5_1 was methylated by a method previously described (see page 75), to give the corresponding heptamethyl ether 52. 169 A small amount was p u r i f i e d by preparative t . l . c . by a n a l y t i c a l purposes R f 0.17 (solvent A); [ a ] D - 87.5° (e 1.4, chloroform); p.m.r. (CDC1 3) : x 5.25 (1 H doublet, JJ, 2 1.6 Hz, H - l ) , 5.37 (1 H doublet, J ^ , 0.8 Hz, H-l' 6.40 - 6.63 (21 H, 7 OMe), 8.63 (3 H doublet, J 5 fi 6 Hz, CH 3). Hydrolysis (as described on page 76) of 52_ gave 2,3-di-0-methyl-L-rhamnose and 2,3,4,6 -tetra-O-methyl-D-mannose as i d e n t i f i e d by paper chromatography (solvent D) using authentic standards ( i - e . 6 ) . Subsequent reduction and a c e t y l a t i o n (see page 72) gave equimolar amounts of 1,4,5-tri-0-acetyl-2,3-di-0-methyl--L-rhamnitol and l,5-di-0-acetyl-2,3,4,6-tetra-<3-methyl-D-mannitol, i d e n t i -101 f i e d by g . l . c . (column c, 220°, r e t e n t i o n times 21.8 and 26.6 min, respec-117 t i v e l y ) and mass-spectrometric comparison with authentic standards ( i . e . 7). Periodate oxidation of 51_ by a procedure previously described (see page 77) showed a t o t a l consumption of 3.0 moles of periodate per mole of 5_1_ i n 50 h. The usual processing (see page 77) gave a mixture of 4-deoxy-L-erythritol and g l y c e r o l , i d e n t i f i e d by paper chromatography (solvent D) using authentic standards ( i . e . 8) and as the peracetates by comparative g . l . c . (column b, 130°, r e t e n t i o n times 8.4 and 12.8 min, res p e c t i v e l y ) and mass-spectrometric analysis with authentic standards. Methyl 2, 3-di-0-acetyl-4-0- (2> 33 4, 6-tetra-0-a.cetyl-$-D-mannopyranosyl)-a--L-rhamndpyranoside (53) Compound 51_ (0.20 g) was acetylated with py r i d i n e (10 ml) and a c e t i c anhydride (10 ml) f o r 5 h at room temperature. The customary work-up (see page 68) produced the sirupy product; y i e l d 0.33 g (95%). For 169 a n a l y t i c a l purposes a small amount was p u r i f i e d by preparative t . l . c . ; [ a ] D - 57° (c 2.1, chloroform); R f 0.36 (solvent A); p.m.r. (CDC1 3) : T 5.25 (1 H doublet, J ^ , 2 , 0.8 Hz, H - l ' ) , 5.42 (1 H doublet, 2 1.6 Hz, H- l ) , 6.65 (3cH s i n g l e t , OMe), 7.88 - 8.04 (18 H, 6 OAc), 8.64 (3 II doublet, J 5 > 6 6 Hz, CH 3). 102 Unsuccessful a c e t o l y s i s attempts Compound 5J_ or compound 5_3 (0.15 g) i n a c e t i c anhydride (0.75 ml) was shaken with 1% (v/v) concentrated s u l f u r i c acid i n a c e t i c anhydride (1.5 ml) f o r 2 h at room temperature. The siru p r e s u l t i n g from the usual work-up procedures (see page 78) showed two spots on t . l . c . i n approximately equal amounts, one with 0.36 (solvent A) and the other with R £ 0.42 169 (solvent A). The two compounds were separated by preparative t . l . c . and the p.m.r. spectra obtained. The compound with R £ 0.36 (solvent A) was i d e n t i f i e d as the desired 1,2,3-tri-0-acetyl-4-0-(2,3,4,6-tetra-O-acetyl--8-D-mannopyranosyl)-a-L-rhamnopyranose (55) . The compound with R^ 0.42 (solvent A) had the following p.m.r. spectrum (CDCl^): T 4.05 (2 H, H-l, H - l ' ) , 7.87 - 7.99 (27 H, 9 OAc, on expansion s i g n a l s f o r nine separate acetate groups were i n d i c a t e d ) , 8.80 (3 H doublet, J 5 6 6 Hz, CH^). The mass spectrum obtained showed m/e 43, 83, 85, 115, 127, 145, 157, 169, 229, 242, 259, 273, 289, and 331. Deacetylation of the compound with R f 0.42 (solvent A) by the usual procedure (see page 69) gave a mixture of rhamnose and mannose as i d e n t i f e d by paper chromatography (solvent C) using authentic standards. I, 2, 3-Tri-0-acetyl-4-0-(4,6-di-0-acetyl-2J3-0-carbonyl-$-D-mannopyranosyl)--L-rhamnopyranose (54) Compound 50_ (1.50 g) i n a c e t i c anhydride (7.5 ml) was shaken with 1% (v/v) concentrated s u l f u r i c a c i d i n a c e t i c anhydride (-15 ml) f o r 2 h at room temperature. The siru p r e s u l t i n g from the usual work-up-procedures (see page 78) showed e s s e n t i a l l y one spot on t . l . c , R f 0.07 (solvent A); 103 y i e l d 1.50 g (95%). For a n a l y t i c a l purposes a small amount was p u r i f i e d 169 by preparative t . l . c . ; [ a ] D - 64° (c 1.7, chloroform); p.m.r. (CDCl^): T 3.99 (1 H doublet, 9 1.6 Hz, H - l ) , 7.84, 7.85, 7.88, 7.90, 7.95 (3 H s i n g l e t s , 5 OAc), 8.58 (3 H doublet, J 5 & 6 Hz, CH^). a Zj 2, 3-Tri-0-acetyl-4-0-(23 33 43 6-tetra-0-acetyl-$-D-mannopyranosyl)-a-L--rhamnopyranose (55) Compound 54_ (1.50 g) was d e e s t e r i f i e d with sodium methoxide (0.2 M, 40 ml) f o r 1 h at room temperature. The standard work-up procedure (see page 69) was employed. The sirup obtained on evaporation was a c e t y l -ated with py r i d i n e (25 ml) and a c e t i c anhydride (25 ml) overnight at room temperature. The excess reagents were removed i n the usual way (see page 68 ). The r e s u l t i n g sirup was a n o m e r i z e d * 5 9 , w i t h zinc c h l o r i d e (0.10 g, fr e s h l y fused) i n a c e t i c anhydride (10 ml) f o r 4 h at room temperature. The r e a c t i o n mixture was d i l u t e d with chloroform (200 ml), successively washed with ice-water (2 x 200 ml), saturated sodium hydrogen carbonate s o l u t i o n (2 x 200 ml), and water (2 x 200 ml), and the remaining a c e t i c anhydride and/or a c e t i c a c i d was removed by addition and evaporation of ethanol. The r e s u l t i n g s i r u p c r y s t a l l i z e d from ethanol (9 ml) when nucleated 169 with a c r y s t a l obtained from a t . l . c . separation ; y i e l d 1.30 g (79%). R e c r y s t a l l i z a t i o n from ethanol gave pure 5_5, m.p. 164 - 165°; [ a ] Q - 67.8° (e 1.3, chloroform); R f 0.36 (solvent A); p.m.r. (CDC1 3): T 4.06 (1 H doublet, J j 2 1.7 Hz, H - l ) , 5.27 (1 H doublet, 2 , 1.0 Hz, H - l ' ) , 7.89 -8.04 (21 H, 7 OAc), 8.64 (3 H doublet, J $ fi 6 Hz, CHj). Anal. Calcd. f o r C„.H_,0._: C, 50.32; H, 5.85. Found: C, 50.06; H, 5.65. 104 4-0-$-D-Mannopyranosyl-L-rhamnopyranose (56) The peracetate 55_ (0.40 g) was deacetylated with sodium methoxide (0.2 M, 15 ml) f o r 1 h at room temperature. A f t e r the usual operations (see page 69), the r e s u l t i n g sirup (0.20 g, 95%), had [ a ] D - 46° (c 2.5, water); R , 0.63 (solvent C); p.m.r. (D„0, external tetramethylsilan e ) : glucose J > r K 2 T 4.97 (0.64 H doublet, 2 1.4 Hz, H-l, ct-L form), 5.20 (1 H doublet, J 0.9 Hz, H - l ' ) , 5.23 (0.36 H doublet, J 1.0 Hz, H-l, B-L form), 8.77 (3 H doublet, J" 5 fi 6 Hz, CHj). G.l.c. (column a at 240°) of the p e r - O - ( t r i m e t h y l s i l y l ) disacchar-ide (prepared by the procedure on page 79) gave one peak (73%) at 8.2 min 127 and a second peak at 10.8 min. [per-<9- ( t r i m e t h y l s i l y l ) sucrose, 11.2 min] 4-0-fc-D-Mannopyranosyl-L-rharnn-Ltol (57) The free disaccharide 56^  (0.10 g) was reduced with sodium boro-hydride (0.05 g) i n water (5 ml) overnight. The customary r e a c t i o n sequence (see page 72) gave 57; y i e l d 0.10 g (99%); [ a ] D - 36° (c 2.4, water); Rglucose 0 - 5 1 ( s o l v e n t c)» P- m- r- (D2°> external t e t r a m e t h y l s i l a n e ) : T 5.26 (1 H doublet, J J t 2 , 0.9 Hz, H - l ' ) , 8.77 (3 H doublet, J 5 fi 6 Hz, CH 3). G.l.c. (column a at 240°) of the p e r - O - ( t r i m e t h y l s i l y l ) a l d i t o l (preparation analogous to that on page 79) gave one peak at 14.5 min 127 [ p e r - 0 - ( t r i m e t h y l s i l y l ) sucrose, 11.2 min] The a l d i t o l 5_7 (0.10 g) was acetylated with pyridine (4 ml) and ac e t i c anhydride (4 ml) overnight at room temperature to give the corres-ponding octaacetate 58_ (0.20 g, 98%) as a s i r u p ; [ a ] D - 67° (c 2.6, chloro-form); R f 0.33 (solvent A); p.m.r. (CDC1 3): x 5.21 (1 H doublet, , 2 , 0.9 Hz, H - l ' ) , 7.86 - 8.02 (24 H, 8 OAc), 8.72 (3 H doublet, J g & 6 Hz, CH 3). 105 G.l.c. (column a at 270°) of the peracetylated a l d i t o l 58 gave one peak at 7.0 min (sucrose octaacetate, 9.2 min). 106 IV BIBLIOGRAPHY 1. I. DANISHEFSKY, R.L. WHISTLER, AND F.A. BETTELHEIM, The Carbohydrates, IIA (1970) 375. 2. W.M. WATKINS, Science, 152 (1966) 172. 3. T. UCHIDA, P.W. ROBBINS, AND S.E. LURIA, Biochemistry, 2 (1963) 663. 4. M. HEIDELBERGER AND G.G.S. 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Stand,, 24 (1940) 125. 115 APPENDIX In the course of the synthetic studies described i n t h i s thesis other peripheral topics became of i n t e r e s t and hence were in v e s t i g a t e d i n c o l l a b o r a t i o n with other members of the laboratory. Methods to f a c i l i t a t e the separation of compounds by g a s - l i q u i d chromatography are c o n t i n u a l l y under i n v e s t i g a t i o n . The u t i l i t y of sugars as acetates was being considered and i t was shown that although 2,3,4,6 - tetra-O-methyl-D-glucose and 2,3-di-O-methyl-L-rhamnose were d i f f i c u l t to separate as methyl glycosides or a l d i t o l acetates they could be conveniently resolved as acetates. The inadequacy of current a n a l y t i c a l methods f o r r e a d i l y deter-mining the number of free hydroxyl groups i n a molecule was emphasized during i n v e s t i g a t i o n s of c e r t a i n of the intermediates i s o l a t e d during the synthesis of disaccharides. The use of proton magnetic resonance spectroscopy 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 of these compounds was being examined and i t was shown that the proton magnetic resonance spectrum 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 of a product obtained by p a r t i a l methylation of methyl 4-0-acetyl--a-L-rhamnopyranoside ind i c a t e d one O-acetyl, two 0-methyl, and one 0 - t r i -- m e t h y l s i l y l (therefore one hydroxyl) group per rhamnose moiety. Integra-ti o n of the t r i m e t h y l s i l y l s i g n a l at x 9.92 thus furnishes a non-destructive method f o r the determination of hydroxyl groups on a microscale. Semimicro methods for determining the configuration (D or L) of sugars from i s o l a t e d polysaccharides by the c i r c u l a r dichroism of t h e i r a l d i t o l acetates were being explored. 4-0-Methyl-D-galactose (3-0-methyl--L-galactose) was prepared and the c i r c u l a r dichroism of i t s a l d i t o l acetate 116 shown to be opposite i n sign to that of 3 -0-methyl-D-galactdse. Proton magnetic resonance spectroscopy was being extended to polysaccharides with d i s t i n c t repeating units i n order to determine the number of pyruvate k e t a l s , O-acetyl groups, and 6-deoxy sugars per repeat-ing u n i t as well as the configurations of the linkages of the sugars. I t was shown that Klebsiella type 18 capsular polysaccharide contained no pyruvate k e t a l and no 0-acetyl groups but four a- and two B-linked sugars including two non-identical rhamnose residues per repeating u n i t . The four enclosed r e p r i n t s are evidence of the scope of these complementary i n v e s t i g a t i o n s . 117 Reprinted from Carbohydrate Research Elsevier Publishing Company, Amsterdam - Printed in The Netherlands Note Separation by gas-liquid chromatography of tetra-O-methylaldo-hexoses and other sugars as acetates G. M. B E B A U L T , G. G. S. D U T T O N A N D R. H. W A L K E R Department of Chemistry, University of British Columbia, Vancouver (Canada) (Received December 13th, 1971; accepted in revised form, February 15th, 1972) Gas-liquid chromatography was first applied in the carbohydrate field to methyl glycosides of methylated sugars1. Since that time, data on the retention times of many such compounds have been published2. The use of methyl glycosides means that, in most cases, one compound will produce several peaks; these complicate the chromatogram even though the pattern of the peaks for a particular sugar may be a useful diagnostic feature. The most common way of avoiding this problem is to reduce the methylated sugars and to separate them as methylated alditol acetates3. In situations where neither of these methods give satisfactory resolution of certain components of a mixture it has been possible, in many cases, to achieve separa-tions of methyl glycosides'*'1 and nee sugars0"2 as tneir O-trimethylsiiyl derivatives or as acetylated methyl glycosides9,10. Unfortunately none of these combinations gives satisfactory resolution of a mixture containing 2,3,4,6-tetra-O-methyl-D-glucose, -D-galactose. and -p-mannose. The necessity for achieving this separation arises in studies on galactoglucomannans. such as occur in wood 1 1 , 1 3 , where each of the tetra-O-methyluldohexoses in a mixture must be determined or, alternatively, the absence of one in the presence of the other two needs to be confirmed positively. Table I shows that these three ethers T A B L E I R E T E N T I O N TIMES OF M E T H Y L A T E D A L D O H E X O S E A C E T A T E S Compound (as peracetate) Retention time (min) Approximate ratio peak 1 peak 2 peak \ I peak 2 (a) Isothermal at 170' 2,3,4,6-Tetra-O-methylglucose 9.4 . 11.5 0.85 2,3,4,6-Tetra-O-methylgalactose 13.4 20.2 1.4 2,3,5,6-Tetra-O-methylgalactose 15.1 2,3,4,6-Tetra-O-methylmannose 17.9 23.2 20 (b) Isothermal at 185° 2,3,4,6-Tetra-O-methyIglucose 4.8 5.2 0.85 2,3,6-Tri-O-methylglucose ' 14.8 17.0 1.3 2 ,3 ,6-Tri-O-methylmannose 31.2 2 ,3-Di-O-methylglucose 34.8 39.7 0.3 Carbohyd. Res., 23 (1972) 430-432 118 NOTE 431 may be separated readily in the form of their acetates. Although this method causes each compound to give two peaks, the retention times of these peaks serve for characterization. Table 1 also shows that, at a slightly higher column-temperature, di-0-methyl-aldohexoses are sufficiently volatile to be separated as their acetates; particularly noteworthy is the marked difference in retention times between 2,3,6-tri-0-methy!-i> glucose and the corresponding D-mannose analog. This is particularly useful since these two compounds as alditol acetates1 2 , 1 3 and as methyl glycosides2 have similar retention times. These results demonstrate that, for the examples cited, separation of methylated sugars as their acetates is the preferred method. This system is thus a useful comple-ment to those previously described, and has the additional merit that the free sugar may be readily regenerated for further characterisation. The only other report of the use of acetates of methylated sugars appears to be that of H. G. Jones, J. K. N. Jones, and Perry14, who used this method in a study on the separation of mono-, di-, tri-, and tetra-c9-rnethyl-D-glucose derivatives. E X P E R I M E N T A L 2,3,4,6-Tetra-O-methyl-D-glucose, -D-galactose, and -D-mannose were prepared from the corresponding sugars by a standard procedure15. 2,3,6-Tri-c9-methyl-i>-giucose was obtained by hydrolysis of a commercial methylated cellulose1". 2,3,0-Tri-0-methyl-D-mannose'7 and 2,3-di-0-methy!-D-glucose'u were available from previous studies. All compounds and/or their derivatives had physical properties in accord with literature values' 9"' 0 . Sugars were acetylated by dissolving them in acetic anhydride-pyridine (i:l) and heating the mixtures under anhydrous conditions (conveniently in a sealed tube) for 15 min on a steam bath. The acetates were extracted with chloroform and the solution was washed twice with M hydrochloric acid, followed by aqueous sodium hydrogen carbonate, and water. The dried (CaCl2) extract was concentrated to smaii volume for injection. Omission of the washing procedure gave extraneous peaks. Gas-liquid chromatography was carried out on a F and M 720 instrument using dual column (8 ft x 3/16 in.) of 3% ECNSS-M on Gas-Chrom Q with a helium flow-rate of 86 m!/min. A C K N O W L E D G M E N T We thank the National Research Council, Ottawa and the University of British Columbia for financial support. The award of the MacMillan Bloedel Ltd. Scholar-ship to R. H. W. is gratefully acknowledged. R E F E R E N C E S 1 A . G . M C I N N E S , D . H . B A L L , F . P. COOPER, A N D C . T . BISHOP, J. Chromatogr., 1 (1958) 556. 2 C . T . BISHOP, Advan. Carbohyd. Chem., 19 (1964) 95. 3 H . BJORNDAI., B . LINDBI:RG, A N D S. SVENSSON, Acta Chem. Scand., 21 (1967) 1801. Carbohyd. Res., 23 (1972) 430-432 119 432 NOTE 4 T . S. STEWART , P. B. MENDERSHAUSEN, AND C. E. BALLOU, Biochemistry, 7 (1968) 1843. 5 P. A . J . GORIN , J . F. T . SPENCER, AND S. S. BHATTACHARJEE, Can. J. Chem., 47 (1969) 1499. 6 H . H . SEPHTON, J. Org. Chem., 29 (1964) 3415. 7 S. HAWORTII, J . G . ROBERTS, AND B. F. SAGAR, Carbohyd. Res., 9 (1969) 491. 8 A . KLEMER , E. BUHE, AND R. K U T Z , Liebigs Ann. Chem., 739 (1970) 185. 9 D. A . REES AND J . W . B. SAMUEL, J. Chem. Soc. (C), (1967) 2295. 10 S. J . SCOTT AND G . W. H A Y , Can. J. Chem., 20 (1965) 2217. 11 T . E. TIMELL, Advan. Carbohyd. Chem., 20 (1965) 410. 12 H . BJORNDAL, B. LINDBERG, AND S. SVENSSON, Carbohyd. Res., 5 (1967) 433. 13 G . G . S. D U T T O N AND R. H. WALKER, Cell. Chem. Teeh/i., in press. 14 H . G . JONES, J . K. N . JONES, AND M . B. PERRY, Abstracts Papers ACS Winter Meeting, Phoenix, Ar i zona , Jan. , 1966, C -17. 15 E. S. WEST AND R. F. HOLDEN, Org. Syntheses Coll. Vol., 3 {1965) 800. 16 J . KOPS AND C. SCHUERCH, J. Org. Chem., 30 (1965) 3951. 17 G . G . S. DUTTON AND K. H U N T , J. Amer. Chem. Soc, 80 (1958) 5697. IS G . G . S. DUTTON , K . B. GIBNEY, AND P. E. REID, Can. J. Chem., 47 (1969) 2494. 19 E. J . BOURNE AND S. PEAT, Advan. Carbohyd. Chem., 5 (1950) 145. 20 G . O . ASPINALL, Advan. Carbohyd. Chem., 8 (1953) 217. Note added in proof (11 A p r i l 1972). 2,3,4,6-Tetra-O-methyl-D -glucose and 2,3-di -O-methyl -L-rhamnose, a pair of compounds difficult to separate as methyl glycosides or alditol acetates, may be resolved conveniently as acetates. Using a column (4 f t * 1/4 in.) containing 5 % butanedio! succinate on Diatoport S (80-100 mesh) and a temperature program from I 10 to 210' at 2°,'min, the acetates of 2,3,4,6-tetra-O-methyl-D -glucose were eluted at 27.9 and 30.2 min, and those of 2.3-di-(9-methyi-L -rhamnose at 37.0 and 40.7 min. In addit ion, two unidentified peaks (ra 7 % of total peak area) were eluted at 16.1 and 18.5 min. Carbohyd. Res., 23 (1972) 430-432 ANALTYIPAL LETTERS, 5 ( 7 ) , U l 3 - U l 8 (1972) D E T E R M I N A T I O N O F H Y D R O X Y L G R O U P S B Y P . M . R . S P E C T R O S C O P Y O F T R I M E T H Y L S I L Y L E T H E R S K E Y W O R D S : P . m . r . s p e c t r o s c o p y , t r i m e t h y l s i l y l e t h e r s , h y d r o x y l g r o u p d e t e r m i n a t i o n . G . M . B e b a u l t , J . M . B e r r y , G . G . S . D u t t o n a n d K . B . G i b n e y * D e p a r t m e n t o f C h e m i s t r y , T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r , B . 0. , C a n a d a (* P r e s e n t A d d r e s s : C o l u m b i a C e l l u l o s e C o . L t d . , A n n a c i s I s l a n d , B . C . , C a n a d a ) A B S T R A C T T r i m e t h y l s i l y l e t h e r s a r e s h o w n t o g i v e s h a r p p . m . r . s i g n a l s a t 1^9.8-9.9 a n d t o h a v e d i s t i n c t c h e m i c a l s h i f t s . T h e t e c h n i q u e i s u s e f u l f o r d e t e r m i n i n g t h e n u m b e r o f h y d r o x y l g r o u p s i n a m o l e c u l e . B e c a u s e e a c h t r i m e t h y l s i l y l g r o u p c o n s i s t s 01 n i n e p r o t o n s s t r o n g s i g n a l s a r e o b t a i n e d f r o m s m a l l a m o u n t s o f s a m p l e . T h e m e t h o d i s t h u s s u i t a b l e f o r a n a l y z i n g e l u a t e s c o l l e c t e d b y g a s - l i q u i d c h r o m a t o g r a p h y . I N T R O D U C T I O N P^je/o-trimethylsilyl e t h e r s ( - O S i M e ^ ) a r e w i d e l y u s e d f o r t h e a n a l y s i s o f c a r b o h y d r a t e s b y g a s - l i q u i d c h r o m a t o g r a p h y f o l l o w i n g t h e i n t r o d u c t i o n o f t h i s t e c h n i q u e b y S w e e l e y a n d c o - w o r k e r s ^ . T h e s e a p p l i c a t i o n s h a v e b e e n r e v i e w e d b y D u t t o n ^ . M o r e r e c e n t l y ^^O-t r i m e t h y l s i l y l e t h e r s h a v e b e e n u s e d a s c o n v e n i e n t c a r b o h y d r a t e d e r i v a t i v e s f o r t h e p . m . r . s p e c t r o s c o p i c d e t e r m i n a t i o n o f t h e g e o m e t r y 3 4 5 o f t h e a n o m e r i c p r o t o n - i n m o n o - a n d o l i g o - s a c c h a r i d e s ' ' . I n t h e s e t u d i e s a t t e n t i o n w a s c o n c e n t r a t e d o n t h e r e g i o n o f t h e s p e c t r u m t : = 5 a n d t h e s i g n a l s d u e t o t h e ^ - t r i m e t h y l s i l y l e t h e r s w e r e d i s m i s s e d a s - h a v i n g T T ^ l O . ' ^ 3 Copyright © 1972 by Marcel Dekker, Inc. NO PART of this work may be reproduced or utilized in any form or by any means, electronic or mechanical, including xerography, photocopying, microfilm, and re-cording, or by any information storage and retrieval system, without the written permission of the publisher. S 121 BEBAULT ET AL. E X P E R I M E N T A L T h e h y d r o x y c o m p o u n d w a s d i s s o l v e d i n p y r i d i n e a n d r e a c 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 a n d c h l o r o t r i m e t h y l s i l a n e a c c o r d i n g t o S w e e l e y a n d c o - w o r k e r s ' . W h e r e t h e h y d r o x y c o m p o u n d w a s c r y s t a l i n e t h e s o l v e n t a n d e x c e s s r e a g e n t s w e r e r e m o v e d b y e v a p o r a t i o n i n v a c u o a t 40 ° a n d t h e r e s i d u e w a s d i s s o l v e d i n c h l o r o f o r m o r b e n z e n e f o r p . m . r . m e a s u r e m e n t s . I n t h e c a s e o f s t a r t i n g m a t e r i a l s w h i c h w e r e s y r u p s t h e O - t r i m e t h y l s i l y l a t e d p r o d u c t w a s d i s s o l v e d i n h e x a n e a n d p u r i f i e d b y g . 1. c . o n a c o l u m n o f 2 0 % S F - 9 6 o n D i a t o p o r t S ( 6 0 - 8 0 m e s h ) h e l d a t 130° f o r 6 m i n a n d t h e n p r o g r a m m e d a t 3 ° / m i n t o 2 0 0 ° . P o r t i o n s o f g t h e g . 1. c . e l u a t e w e r e c o l l e c t e d a n d u s e d f o r p . m . r . s p e c t r o s c o p y . P u r e o(.- P ~ g l u c o s e w a s m u t a r o t a t e d i n w a t e r a n d e v a p o r a t e d t o a s y r u p b e f o r e s i l y l a t i o n ( g . l . c . p r o g r a m 190 -220° a t 3 ° / m i n ) . P . m . r . s p e c t r a w e r e o b t a i n e d o n V a r i a n H A - 1 0 0 a n d X L - 1 0 0 i n s t r u m e n t s o p e r a t e d i n t h e f r e q u e n c y s w e e p m o d e a n d l o c k i n g o n t o t h e c h l o r o f o r m o r b e n z e n e s i g n a l . R E S U L T S A N D D I S C U S S I O N F i g u r e s 1 - 9 s h o w t h a t t h e p r o t o n s o f t h e ( - O S i M e ^ ) g r o u p g i v e a s h a r p s i g n a l a t c" 9 . 8 - 9 . 9 a n d , f u r t h e r m o r e , t h a t t h e c h e m i c a l s h i f t s o f n o n - i d e n t i c a l - O S i M e ^ e t h e r s a r e d i f f e r e n t . S u c h p . m . r . m e a s u r e -m e n t s t h u s p r o v i d e c l e a r e v i d e n c e o f t h e n u m b e r o f t r i m e t h y l s i l y l e t h e r g r o u p s , a n d h e n c e h y d r o x y l g r o u p s , i n a m o l e c u l e . T h e m a j o r i t y o f t h e s p e c t r a s h o w n w e r e o b t a i n e d i n c h l o r o f o r m s o l u t i o n b u t i t w a s s u b -s e q u e n t l y f o u n d t h a t r e s o l u t i o n o f t h e s i g n a l s w a s g r e a t e r i n b e n z e n e , c o m p a r e F i g s . 3 a n d 4 ; 5 a n d 6; a s i t u a t i o n s i m i l a r t o t h a t w h i c h e x i s t s f o r O - m e t h y l e t h e r s ^ . T h e u s e o f C ) - t r i m e t h y l s i l y l e t h e r s f o r t h e d e t e r m i n a t i o n o f • h y d r o x y l g r o u p s b y p . m . r . s p e c t r o s c o p y i s s i m i l a r t o o t h e r m e t h o d s 1 DETERMINATION OF HYDROXYL GROUPS F I G S . 1 - 4 P . m . r . s p e c t r a o f p e r - O - 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 o f : 1 . g l y c e r o l , 2. e r y t h r i t o l , 3 . m e t h y l a - D - g a l a c t o p y r a n o s i d e , 4 . a s 3 i n C g H r -F o r F i g s . 1 - 9 o n l y t h e s p e c t r u m i n t h e r e g i o n T 9 . 9 i s s h o w n . P o s i t i o n s o f p e a k s a r e g i v e n i n Hz d o w n f i e l d f r o m TMS. F I G S . 5 - 8 P . m . r . s p e c t r a o f p e r - O - 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 o f : 5 . 6 - 0 - ( B - D -g l u c o p y r a n o s y l ) - l , 2 : 3 , A - d i - i s o p r o p y l i d e n e - D - g a l a c t o s e , 6 . a s 5 in C^H 7 . a p - D - g l u c o f u r a n o s e , 8 . 8 _ n - g l u c o p y r a n o s e . i'l5 123 BEBAULT ET AL. 6 7 w h i c h h a v e e m p l o y e d O - a c e t y l o r O - t r i f l u o r o a c e t y l d e r i v a t i v e s b u t h a s c e r t a i n s i g n i f i c a n t a d v a n t a g e s . E v e r y - O S i M e ^ g r o u p i n t r o d u c e d i n c r e a s e s , i n e f f e c t , t h e n u m b e r o f p r o t o n s o n e a c h h y d r o x y l g r o u p b y a f a c t o r o f n i n e . I n t h i s s e n s e a t r i m e t h y l s i l y l g r o u p m a y b e c o n s i d e r e d t o a c t a s a " s i g n a l m u l t i p l i e r " . T h i s i s o f p a r t i c u l a r a d v a n t a g e w h e n o n l y s m a l l q u a n t i t i e s o f s a m p l e a r e a v a i l a b l e , a n d t h e s i g n a l s f r o m o t h e r p r o t o n s m a y b e l o s t in b a c k g r o u n d n o i s e . T h i s f e a t u r e i s i l l u s t r a t e d b y t h e f o l l o w i n g e x p e r i m e n t w h i c h a l s o d e m o n s t r a t e s t h e f e a s a b i l i t y o f e x a m i n i n g t h e n a t u r e o f O - 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 s e p a r a t e d b y g a s - l i q u i d c h r o m a t o g r a p h y b y p . m . r . s p e c t r o s c o p y . T h e g a s c h r o m a t o g r a m o f p e r - O ^ t r i m e t h y l s i l y l a t e d D - g l u c o s e o f t e n s h o w s a s m a l l p e a k , a m o u n t i n g t o a b o u t 1 % o f t o t a l p e a k a r e a , r e p r e s e n t i n g a n u n i d e n t i f i e d c o m p o u n d , i n a d d i t i o n t o m a j o r p e a k s o f fcL-D- a n d p - D - g l u c o p y r a n o s e . A l t h o u g h t h e u n k n o w n w a s t h o u g h t t o b e a f u r a n o s i d e i t c o u l d n o t b e r u l e d o u t t h a t i t w a s d u e t o u n d e r s i l y l a t i o r i or a n a n h y d r o f o r m o f D - g l u c o s e . R e p e a t e d i n j e c t i o n o f p e r - O -8 t r i m e t h y l s i l y l a t e d D - g l u c o s e p e r m i t t e d t h e c o l l e c t i o n o f 2 m g o f m a t e r i a l . T h e p . m . r . s p e c t r u m o f t h i s c o m p o n e n t ( F i g . 7) i n d i c a t e s t h e p r e s e n c e o f f i v e - O S i M e ^ g r o u p s . T h e r e s o l u t i o n of t h e s i g n a l s i s l e s s d i s t i n c t i n t h i s c a s e s i n c e , p r e s u m a b l y , t h e c o m p o n e n t w a s a m i x t u r e o f oCand p a n o m e r s . W i t h a s a m p l e o f t h i s s i z e t h e s i g n a l s f o r t h e o t h e r p r o t o n s i n t h e m o l e c u l e w e r e o b s c u r e d b y s p e c t r o m e t e r n o i s e . F o r t h e p u r e p y r a n o s e a n o m e r s t h e r e s o l u t i o n i s g o o d ( e . g . F i g . 8 I n t h e d o m a i n o f c a r b o h y d r a t e c h e m i s t r y it is o f t e n n e c e s s a r y t o k n o w t h e n u m b e r o f O - m e t h y l g r o u p s in a m o l e c u l e . I t is u s u a l l y n o t p o s s i b l e t o d o t h i s b y d i r e c t p . m . r. s p e c t r o s c o p y o f t h e - O C H ^ s i g n a l s i n c e t h e m e t h y l p r o t o n s r e s o n a t e a t f r e q u e n c i e s s i m i l a r to t h e p r o t o n s of t h e c a r b o h y d r a t e r i n g ( F i g . 1 0 ) . T h i s p r o b l e m m a y b e r e s o l v e d b y 1*16 1 2 4 DETERMINATION OF HYDROXYL GROUPS p 1 1 I I I r— 3 0 2 5 2 0 15 10 F I G S . 9 a n d 10 T 7 8 l O P . m . r . s p e c t r a o f p e r - O - 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 o f : 9 . m e t h y l 2 , 3 - d i -O - m e t h y l - a - D - g a l a c t o p y r a n o s i d e , 1 0 . Unknown m e t h y l O - a c e t y l - 0 - m e t h y l - a -L - r h a m n o s i d e (CDCI3). F i g . 10 shows t h e r e g i o n x 6 . 5 - 1 0 . A l l s p e c t r a w e r e r u n i n C H C l - j e x c e p t as n o t e d . c o n v e r s i o n o f t h e s a m p l e t o t h e ( D - t r i m e t h y l s i l y l e t h e r a n d d e t e r m i n a t i o n o f t h e n u m b e r o f u n m e t h y l a t e d h y d r o x y l g r o u p s ; t h e n u m b e r o f m e t h y l e t h e r s m a y t h e n b e d e t e r m i n e d b y d i f f e r e n c e . T h i s i s s h o w n i n F i g . 9 w h i c h r e p r e s e n t s t h e s p e c t r u m o f t h e d e r i v a t i v e o f m e t h y l 2 , 3 - d i - O -m e t h y l - Q ( . - D - g l u c o s i d e a n d a l s o b y F i g . 1 0 . S i n c e m e t h y l e t h e r s o f c a r b o h y d r a t e s a r e o f t e n s e p a r a t e d b y g . l . c . a s t h e i r O - 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 t h i s i l l u s t r a t e s a n o t h e r e x a m p l e o f a u s e f u l c o m b i n a t i o n o f t h e t w o t e c h n i q u e s . A f u r t h e r a d v a n t a g e o f - O S i M e ^ e t h e r s i s t h a t t h e y m a y b e u s e d f o r d e t e r m i n i n g t h e n u m b e r o f f r e e h y d r o x y l g r o u p s i n a m o l e c u l e i n t h e 9 p r e s e n c e o f O - a c e t y l g r o u p s . N o t o n l y a r e t h e p . m . r . s i g n a l s o f O - a c e t y l a n d C J - t r i m e t h y l s i l y l g r o u p s w i d e l y s e p a r a t e d b u t , m o r e • i m p o r t a n t l y , t h e l a t t e r g r o u p m a y b e i n t r o d u c e d a n d r e m o v e d u n d e r c o n d i t i o n s w h i c h d o n o t a f f e c t t h e f o r m e r . T h u s a n u n k n o w n m a t e r i a l i s o l a t e d i n o t h e r r e s e a r c h ' " w a s s h o w n ( F i g . 1 0 ) t o c o n t a i n o n e O - a c e t y l a n d o n e f r e e h y d r o x y l g r o u p b y c o n v e r s i o n t o t h e j O - t r i m e t h y l s i l y l e t h e r . T h e s e r e s u l t s s h o w c l e a r l y t h e u t i l i t y o f O - t r i m e t h y l s i l y l e t h e r s a s a n a n a l y t i c a l t o o l f o r d e t e r m i n i n g t h e n u m b e r o f h y d r o x y l g r o u p s i n .1*17 1 2 5 BEBAULT ET AL. o r g a n i c m o l e c u l e s . W h i l e t h e e x a m p l e s s h o w n a r e m a i n l y c a r b o h y d -r a t e s t h e t e c h n i q u e i s c l e a r l y o f g e n e r a l a p p l i c a t i o n . A C K N O W L E D G M E N T S W e g r a t e f u l l y a c k n o w l e d g e t h e s u p p o r t o f t h e N a t i o n a l R e s e a r c h C o u n c i l , C a n a d a a n d t h e C o m m i t t e e o n R e s e a r c h o f t h e D e a n o f G r a d u a t e S t u d i e s . W e t h a n k B e a t r i x K r i z s a n f o r t h e d r a w i n g s . R E F E R E N C E S 1 . C . C . S w e e l e y , R . B e n t l e y , M . M a k i t ' a , a n d W . W . W e l l s , J . A m e r . C h e m . S o c , 8 5 , 2 4 9 7 ( 1 9 6 3 ) . 2 | 2 . G . G . S . D u t t o n , A d v a n . C a r b o h y d . C h e m . B i o c h e m . , J i n , i n p r e s s ( 1 9 7 2 ) . 3 . J . P . K a m e r l i n g , D . R o s e n b e r g , a n d J . F . G . V l i e g e n t h a r t , B i o c h e m . B i o p h y s . R e s . C o m m u n . , 3 8 , 7 9 5 ( 1 9 7 0 ) . 4 . C . G . H e l l e r q v i s t , O . L a r m , a n d B L i n d b e r g , A c t a C h e m . S c a n d . , 2_5, 7 4 3 ( 1 9 7 1 ) . 5 . a ) T . E . A c r e e , R . S . S h a l l e n b e r g e r , a n d L . R . M a t t i c k , C a r b o h y d . R e s . , 6 , 4 9 8 ( 1 9 6 8 ) . b ) C . Y . L e e , T . E . A c r e e , a n d R . S . S h a l l e n b e r g e r , C a r b o h y d . R e s . , j>, 3 5 6 ( 1 9 6 9 ) . 6 . L • D . H a l l , A d v a n . C a r b o h y d . C h e m . . _1_9, 51 ( 1 9 6 4 ) . 7 . B . A . D m i t r i e v , A . V. K e s s e n i c h , A . Y a . C h e r n y a k , A . D . N a u m o v , a n d N . K . K o t c h e t k o v , C a r b o h y d . R e s . _1±, 2 8 9 ( 1 9 6 9 ) . 8 . G . G . S . D u t t o n a n d K . B . G i b n e y , J . C h r o m a t o g r . , i n p r e s s ( 1 9 7 2 ) . 9 . A . P . T u l l o c h a n d A . H i l l , C a n . J . C h e m . , 4 6 , 2 4 8 5 ( 1 9 6 8 ) . 1 0 . G . M . B e b a u l t a n d G . G . S . D u t t o n , t o b e p u b l i s h e d . R e c e i v e d : A p r i l 1 8 , 1 9 7 2 A c c e p t e d : J u n e 1, 1 9 7 2 Ul8 126 C O M M U N I C A T I O N S Semimicro Determination of Sugar Configuration by Measurement of Circular Dichroism of Alditol Acetates' G. M . BEBAULT, J. M. BERRY, Y. M. CHOY, G. G. S. DUTTON, N. FUNNELL, L. D. HAYWARD, AND A. M. STEPHEN 2 Department of Chemistry, The University of British Columbia, Vancouver 8, British Columbia Received July 13, 1972 The configuration (D or L) of a sugar may be determined conveniently by circular dichroism measure-ments at 213 nm on alditol acetates, or their methylated derivatives, where the acetoxy group acts as a chromophore. Only milligram quantities of material are required and the method is well suited to analyz-ing fractions obtained by gas-liquid chromatography. Structural information which may be derived from the c d . spectra is briefly discussed. La configuration (D or l.) d'un sucre peut etre etablie par le dichroisme circulaire a 213 nm soit des alditol acetates soit de leur derives methyles, ou le groupement acetate joue le role de chromophore. On ne necessite que de tres petites quantites de substance et la methode s'adapte tres bien a 1'analyse des fractions obtenues par chromatographic en phase gaseuse. On discute brievement des renseignments structuraux que fournissent les spectres d.c. Can. J. Chem.. 51. 32-4 (1973) Determination of the structure of a poly-saccharide requires that the nature and propor-tion of the constituent sugars be known as well as the positions through which the monosac-charide units are linked in the polymer. Such investigations are facilitated by the use of gas-liquid chromatography (g.l.c.) and sugar ratios are customarily determined as trimethylsilyl ethers or as alditol acetates (I). Similarly, parti-ally methylated sugars are conveniently separated by g.l.c. of their derived alditol acetates and the pattern of methylation is determined by mass spectrometry (2). The technique of mass spec-trometry does not readily differentiate between diastereoisomers but this problem may be re-solved by collection of the methylated alditol acetate from the effluent gas stream (3) followed by demethylation and characterization of the parent alditol as the peracetate (4). None of these chromatographic methods is capable of determining the configuration (D or L) of the constituent sugars of a polysaccharide, an aspect of structural studies which is sometimes 'Presented at the Vlth International Symposium on Carbohydrate Chemistry, Madison, Wisconsin, August 1972. 2 O n Jeave from the University of Cape Town, Ronde-bosch. South Africa. ignored. One great merit of procedures involving gas-liquid chromatography and mass spectrom-etry is the small quantity of materia! required and this is of particular value when only limited amounts of sample are available as, for example, with bacterial polysaccharides. The advantage of these techniques is lost if the determination of the configurational series must be made on macroscopic amounts of specimen. In the partic-ular cases of n-glucose and D-galactose specific oxidases are available (e.g. Glucostat and Galac-tostat reagents,' Worthington Biochemical Corpo-ration) but no such method is available for other commonly occurring sugars; even so these are destructive methods. We have now demonstrated that the sugar configuration (D or L) may be determined un-ambiguously on milligram quantities of mono-saccharide derivatives from the circular dichro-ism (cd.) band at ca. 213 nm of the correspond-ing alditol acetates. Just as a sugar of D-config-uration may have either a positive or negative specific rotation so also D-alditol acetates may have cd. bands of either sign. Thus, this sign must be determined empirically and a selection of such data is presented in Table 1. While the method is obviously inapplicable to meso-alditols, such as galactitol, it is readily extended 127 C O M M U N TABLE ]. Circular dichroisni of alditol acetates Alditol Configuration A ~ M c C N Threitol L -0 .60 Arabinitol D + 0.99 Arabinitol L -0 .96* Glucitol D + 0.33 Idito! L -0 .95 Mannitol D + 1.7 Talitol D + 0.73 Fucitol D t Fucitol L + 0.02 Rhanmitol L -1 .52 2-O-Methylxylitol D + 0.37 2-O-Methylglucitol D + 0.71 3-O-Methylgalactitol D -0.31 3-O-MethyJgalactitol L + 0.43 3-O-Methylmannitol D + 0.74 2,3-Di-O-methylgalactitol D + 0.26 4,6-Di-O-methylgalactitol D + 0.51 2,4,6-Tri-O-methylgalactitol D + 1.40 2,3,4,6-Tetra-O-methylgalactitol D + 0.54 *A weak band, Ac —0.11, appe; tNo reliable value obtainable. ired at 265 nm. to their chiral methylated acetates obtained in structural studies. Conversely, in order to avoid the necessity of measuring cd. bands for a!! possible methylated aiditois these compounds may conveniently be demethylated (4) where the parent alditol is chiral. Circular dichroism measurements were made using a Jasco J-20 automatic recording spectro-polarimeter, accurately weighed milligram quan-tities of the alditol acetate being dissolved in acetonitrile (0.15 ml, spectroscopic grade) and placed in a quartz spectrophotometer cell with path length of 0.1 cm. The spectrometer was calibrated using the value of E L — eR = +2.3 for ( + )-10-camphorsulfonic acid in water (5). The cd. curves obtained at a scan-rate of 5 nm/min were bell shaped and symmetrical as far as could be ascertained (measurements below 205 nm were not reproducible). While there is no doubt about the sign of the cd. curve in each case examined, the numerical values of As are some-times hard to reproduce particularly for those compounds which have small values of As. This is an instrumental problem since the acetate band lies near the extreme range of the instrument used. The initial experiments described here were carried out during an investigation, to be de-scribed later (6), into the structure of the capsular polysaccharide of Klebsiella K-type 7 and have C A T I O N S 325 subsequently been employed in other, related structural studies. Measurements have been made on the alditol acetates used for quantita-tive analysis of polysaccharide hydrolyzates (7), as for Klebsiella K-type 21, on methylated alditol acetates and on alditol acetates obtained by demethylation and acetylation. The first method is particularly convenient for mannose where no simple micromethod exists. The capsular polysaccharide of Klebsiella K7 contains glucose, galactose, mannose, and glu-curonic acid. From the methylated polysaccha-ride 2-O-methyl- and 2,4,6-tri-O-methylglucose are obtained as well as 2,4-di-c9-methylglucose which results from reduction of the glucuronic acid residue. These compounds were separated by g.l.c. of their alditol acetates. Demethylation and acetylation of the individual fractions yielded (g.l.c.) glucitol hexaacetate, each sample of which gave a positive cd. band with As values ranging from +0.28 to +0.33 depending on the amount of contaminating liquid phase caused by bleeding of the column. Thus each glucose resi-due in Klebsiella K7 is shown to be of D-configuration as is the glucuronic acid. Mannitol hexaacetate samples were obtained similarly from the methylated mannose derivatives, namely the 4,6-di- and 3,4,6-tri-O-methyl ethers, and each sample showed a strong positive cd. band (Ae +1.4 to +1.7) thus demonstrating that all mannose units in the polysaccharide are of D-configuration. The galactose in Klebsiella K7 gives only 2,3,4,6-tetra-O-methylgalactose which, on reduc-tion and conversion to the syrupy diacetate, gave a positive cd. band (As +0.54) similar to that obtained from an authentic sample of the methylated sugar of D-configuration and to that obtained by methylation of a wood galactogluco-mannan (8). One sample of galactitol hexa-acetate prepared by demethylation and acetyla-tion was shown to be achiral. In addition, a sample of 2-O-methyI-D-glucitoI pentaacetate obtained from K7 polysaccharide was recrystallized from ethyl acetate - petroleum ether (b.p. 30-60°) and had m.p. 56-57°. The cd. measurement showed a positive band with As +0.71, a value significantly greater than for D-glucitol hexaacetate. . Measurements of cd. spectra may also provide alternative means for determining the position of methoxyl substitu-tion, distinguishing between isomers and esti-mating the composition of mixtures. Thus, 128 C A N . J. C H E M . VOL . 51, 1973 326 because of symmetry, achiral methylated alditol acetates can be expected in polysaccharide structural studies only from 3-, 2,4-, or 2,3,4-0-methyl derivatives of ribose and xylose and 2,4-or 2,5-O-methyI derivatives of galactose or allose. The significance and utility of this is demon-strated by the following experiments. In structural studies on xylans both 2- and 3-0-mcthyl-D-xyloses are commonly encountered and this pair of compounds is not separable as alditol acetates by g.l.c. Collection of the mono-methyl pentilol acetate fraction from the g.l.c, in a study on western red cedar (8), and measure-ment of Ae showed that the 2-0-methyl deriva-tive was present to the extent of 35%, a value in close agreement with that (33%) obtained by integration of the well resolved methoxyl signals in the p.m.r. spectrum of the mixture of alditol acetates. A control experiment verified that 3-0-methyl xylitol is achiral. The symmetry of /Mcso-alditols may be de-stroyed on substitution thus leading to enantio-meric compounds. For example, whereas the sugars 3- and 4-O-methyl-D-galactose are dia-stereoisomers and reduction yields 3- and 4-0-methyl-D-galactitol, the latter compound is cor-rectly named S-O-metliyl-L-gaiaciiiGl indicating that the derived aiditois are etiantiomers. Mea-surement of the cd. spectra of these two alditols as the pentaacetates shows clearly that they are of opposite sign although the numerical agree-ment in the A'e values is not good (Table 1) for the reasons given previously. The signs of the cd. bands, however, suffice in these cases to identify the pattern of substitution in such monosac-charides. The 3- and 4-0-methyl-D-galactoses were syn-thesized by methylation of 1,2:5,6-di-0-isopro-pylidene-D-galactofuranose (9) and of methyl 2,3,6-tri-O-benzoyl-a-D-galactopyranoside (10), respectively. Each sugar was crystalline and did not depress the m.p. of authentic samples (10,11). Measurement of cd. spectra of chiral alditol acetates and their methylated derivatives is thus proposed as a convenient, non-destructive method for determining the configurational series of a sugar. The technique requires very small samples and is admirably suited to exam-ining individual fractions obtained by gas-liquid chromatography. By combining these techniques with mass spectrometry most partially methyl-ated monosaccharides may be identified com-pletely, including the configurational assignment. The symmetry rule relating molecular con-formation and the sign of the cd. band for alditol acetates is now under investigation as is the possibility that differences in rotational strength may be useful in distinguishing isomeric methyl-ated alditol acetates. The cd. spectra of sugars (12, 13) and of monoacetates of methyl glyco-sides (14) have been discussed recently. We are grateful to the Nat iona l Research Counc i l of Canada for financial support, to Professor J . K . N . Jones, F .R .S . for an authentic sample of 3 -O-niethyl -D -galactose and to M r s . E. Dumitrescu for skil led assistance in taking spectra. One of us ( A . M . S . ) is indebted to the University of Cape T o w n for study leave and to C . S . I .R . for the award of a bursary. 1. G . G . S. D U T T O N . Adv . Carbohydr. Chem. In press. 2. H . B J O R N D A L , C . G . H E L L E R Q V I S T , B . L I N D B E K G , and S. S V C N S S O N . Angew. Chem. int. Ed . 9, 610 (1970). 3. G . G . S D U T T O N and K . B . G I B N E Y . J . Chromatogr. 72 , 1 7 9 (1972). 4. G . G . S D U T T O N and Y . M . C H O Y . Carbohydr. Res. 21, 169 (1972). 5. J . Y . C A S S I M and J . T . Y A N G . B iochem. 8.1947(1969). 6. G . G . S. D U T T O N and A . M . S T E P H E N . T O be pub-l ished. 7. Y . M . C H O Y and G . G . S. D U T T O N . Can. J . Chem. 51. 198 (1973). 8. G . G . S. D U T T O N and N . F U N N E L L . T O be published. 9. H . P A U L S E N and H . B E H R E . Carbohydr. Res. 2, 80 (1966). 10. E. G . G R O S and 1. O. M A S T R O N A R D I . Carbohydr. Res. 10, 318 (1969). 11. D . J . B E L L . Adv . Carbohydr. Chem. 6, 11 (1951). 12. R. G . N E L S O N and W. C. J O H N S O N . JR. J . A m . Chem. Soc. 94. 3343 (1972). 13. R. N . T O T T Y . J . H U D E C , and L . D . H A Y W A R D . Car-bohydr. Res. 23, 152 (1972). 14. H . B . B O R E N , P. J . G A R E G G , L . K E N N E , h. M A R O N . and S. S V E N S S O N . Ac ta Chem. Scand. 26, 644(1972). 129 JOURNAL OF BACTERIOLOGY, Mar. 1973, p. 1345-1347 Vol. 113, No. 3 Copyright © 1973 American Society for Microbiology . Printed in U S.A. Proton Magnetic Resonance Spectroscopy of Klebsiella Capsular Polysaccharides G . M . B E B A U L T , Y. M . C H O Y , G. G. S. D U T T O N , N. F U N N E L L , A. M . S T E P H E N , A N D M . T . Y A N G Department of Chemistry, Uniuersity of British Columbia, Vancouver 8, B.C., Canada Y Received for publication 8 December 1972 The presence of acetate and pyruvate groups in Klebsiella capsular polysac-charides may be demonstrated and estimated quantitatively by running the proton magnetic resonance spectrum of the polysaccharide (as sodium salt) in deuterium oxide at 95 C. Such spectra also permit an assessment to be made of the number of a- and -^linkages in the repeat unit of the polysaccharide structure. Pyruvic acid is found covalently linked to a sugar residue in a variety of polysaccharides, especially those which form the capsule of Klebsiella bacteria (3). There is a lack, how-ever, of a nondestructive method for the esti-mation of pyruvate, it has been shown recently (Y. M. Choy et al, Anal. Lett. 5:675, 1972) that certain capsular polysaccharides (molecular "•eights 5 to 9 x 105) from Klehxiplln. after exchange- with deuteiium cxide (D20), give proton magnetic resonance (PMR) spectra in which the presence of a pyruvic acid ketal is clearly demonstrated by the signal at T 8.5, characteristic of CH 3—C. Derivatives of these ketals, prepared during structural studies. (Y. M. Choy et al., Anal. Lett. 5:675, 1972), also give signals at T 8.5 to 8.6. M A T E R I A L S A N D M E T H O D S In P M R spectra the chemical shift (i.e., position) of peaks is measured downCield from the signal given by tetramethylsilane (TMS) which is assigned a value of T = 10. The integral gives the area beneath a peak which is proportional to the number of protons resonating at that particular frequency. Labile hy-drogen atoms in poly- and oligosaccharides are deuterated by repeated exchange with D 2 0 . Further -details may be found in-many reviews (4. 5). Some difficulty was experienced in making quan-titative determination of the pyruvic acid content because of the large peak present due to partially deuterated water (HOD). This peak appeared at ap-proximately r 5 to 6, partially covering those regions of the spectrum associated with anomeric and ring protons. The magnitude of the H O D peak was largely due to the necessity of working with solutions of less than 2% concentration because of their viscosity. This same viscosity prevented the solutions from being cooled in order to move the H O D peak downfield. The H O D peak may be moved downfield by running the spectrum on a solution of polysaccharide in tri-fluoroacetic acid which permits integration of the pyruvic acid and ring proton signals (Y. M . Choy et al., Anal. Lett. 5:675, 1972). This is not very precise because of the disparity in size of the integrals being compared. The fact that the acid may degrade the polysaccharide is a further disadvantage. Conversely, the H O D signal is moved upfield when the polysac-charide (in free acid form) is dissolved in methyl sulfoxiHe-rfj and the spectrum is nin at 95 C. This method also suffers from the disadvantage of heating solutions of acidic polysaccharides (Y. M . Choy et al., Anal. Lett. 5:675, 1972). We have now found that excellent P M R spectra may be obtained by dissolving the sodium salt of the polysaccharide, after exchange, in D 2 0 and running the spectra at 95 C . This is currently used as a routine screening process from which the polysaccha-ride samples may be recovered unchanged. Some representative results are discussed and the figures illustrate spectra obtained from Klebsiella capsular polysaccharides containing O-acetyl or pyruvate ketal groups or both. Polysaccharides were isolated as described elsewhere (1; Y. M . Choy and G. G. S. Dutton, Can. J. Chem.. in press), converted to their sodium salts (It is convenient to use samples whose equivalent weight has been determined by titration.), and exchanged 2 or 3 times with D 2 0 . Spectra were run on solutions of approximately 2% concentration in DjO at 95 C using a Varian XL100 instrument with tetramethylsilane as the external standard. RESULTS Figure 1A shows the spectrum of K21 poly-saccharide and illustrates the sharp signal at r 8.5 for the CHa-—C of the pyruvate ketal. The four signals in the range T 4.5 to 5.5 are due to the anomeric protons and integration shows clearly that there are five anomeric protons to 1345 130 CH^C COOH i 1 1 1 1 1— 4 5 6 7 8 9 T FIG. 1. PMR spectra (100 MHz) run in D,0 at 95 C of capsular polysaccharides from Klebsiella: A, K21; B. K24; C K5. The numbers beside the integrals represent the areas under each peak in arbitrary units. Thus, in A the signal at T 8.5 is due to the methyl group of the pyruuic aciii ketal, and therefore represents three protons; hence one proton = 46/3 units ~ 15 units. It is then clear that the peaks around r 5 correspond to 1 + 2 + 1 + 1. or five protons. The signals around r 5 are those given by the anomeric protons, and therefore there are five sugar units and one pyruvic acid ketal per repeat unit. In C the signals at T 7.8 and T 8.5 each correspond to 3 protons (CH,— of acetate and pyruvate, respectively); thus, taking the average, 3 protons = 19 units and therefore the integral of the signals at r 5.5 shows that there are three anomeric protons (i.e., 3 sugar residues) per repeat unit. Case B is less precise but suggests 1-0-acetyl group (T 7.8, 3H = 50 units) for about 7 sugar residues. each pyruvate ketal. Furthermore, an anomeric signal above T 5.0 is indicative of a hexose unit linked in the /8-D-configuration (6). The PMR spectrum therefore suggests that in this K21 polysaccharide, only one of the five sugar units in the repeat unit has a /8-D-linkage. This polysaccharide contains u-galactose and »-mannose, in addition to D-glucuronic acid. The splitting of 7 Hz at the signal of T 5.15 shows that the protons on C-l and C-2 are trans-diax-ial; i.e. they have a dihedral angle of 180°. This signal therefore cannot be due to a n-mannose unit, since in this sugar the protons on C-l and C-2 are either trans-diequatorial (a-n-) or cis-axial-equatorial ((3-D-) and in each case have a splitting (coupling constant) of 2-3 Hz (4). 131 Vol . . 113, 1973 T h i s spl i t t ing is not dist inct when the spectrum is run in methyl sulfoxide-dc. Detailed exami-nation of K21 polysaccharide has subsequently confirmed that the structure has a repeat unit o f five sugar residues, of which one D-galactose unit is /3-linked (Y. M . Choy and G . G . S. Dutton, Can . J . Chem. , in press). Figure IB is the spectrum for K2<1 polysac-charide which shows the presence of an 0-acetyl group (T 7.8) and the absence of pyru-vate. The anomeric signals integrate for five protons, but in this case, the chemical shifts suggest that three sugar units are «-relinked and 2 are /3-D-linked. The acetate content corresponds to one O-acetyl group per seven or eight sugar units. This indicates a certain degree o f random character in the acetate substitution or loss of O-acetyl during the isolation of the polysaccharide. The latter is unlikely in view of the m i l d procedures used. Figure I C shows the spectrum for K 5 poly-saccharide and demonstrates that there are one O-acetyl group and one pyruvate ketal to every repeat unit of three sugars, each of which is l inked by a /3-D-bond. T h i s is in accord wi th the chemical structure as subsequently determined (2). . In a s imilar manner, the ratio of pyruvate to L-fucose in K 6 has been shown to be 1:1; K 7 has one pyruvate to 8 to 9 sugar residues; K18 has neither O-act-tyl nor pyruvate. In K02 , the ratio of pyruvate to L-rhamnose is 1 :A, and in K56 the ratio is 1:1, with the i.-rhamnose being one member of a five-sugar repeat unit . The sharpness of the signals due to the 1347 acetate and pyruvate groups, as well as those of the anomeric protons, may be taken to indicate that such structural features all have a con-stant environment in these bacterial polysac-charides. In other words, the nature of these signals is further evidence that Klebsiella cap-sular polysaccharides are indeed composed of true repeat units. A C K N O W L E D G M E N T S We are indebted to I. 0rskov. Copenhagen, for authentic Klebsiella cultures, to P. J . Salisbury of this Department for growing the bacteria, and to R. \V. Wheat lor a sample of K6 polysaccharide. The financial support of the National Research Council. Ottawa, and the award of N.R.C. scholarships to G.M.B. and N.F. and Killam Fellowship to Y.M.C. are gratefully acknowledged. L I T E R A T U R E C I T E D 1. Dutton, G. G. S „ and Y. M. Choy. 1972. Capsular polysaccharide from Klebsiella tvpe 21. Carbohyd. Res. 21:109-172. 2. Dutton, G. G. S., and M. T. Yang. 1972. 4,6-0-(l-Carboxyethylidene)-D-mannose as a structural unit in capsular polysaccharide of Klebsiella K-type 5. Can. J. Chem. 50:2382-4. 3. Gormus, B. J., R. W. Wheat, and J. F. Porter! 1971. Occurrence of pyruvic acid in capsular polysaccharides from various Klebsiella species. J . Bacteriol. 107:150-154. 4. Hall, L. D. 1964. Nuclear magnetic resonance. Advan. Carbohyd. Chem. li»:5i-'jo. 5. Rowe, J. J. M., J. Himon. and K. L. Roue. 1970. Nuclear magnetic resonance studies on the biochemistry of biopolymers. Chem. Rev. 70:1-57. 6. van der Veen, J. M. 1963. An N.M.K. study of the glyco-side link in glycosides of glucose and galactose. J . Org. Chem. 28:564-566. S P E C T R O S C O P Y O F KLEBSIELLA PUBLICATIONS G.M. Bebault, J.M. Berry, G.G.S. Dutton and K.B. Gibney, Determination of Hydroxyl groups by P.M.R. Spectro-scopy of T r i m e t h y l s i l y l Ethers, Analyt. L e t t . , 5(7), 413-418 (1972). G.M. Bebault, G.G.S. Dutton and R.H. Walker, Separation by g a s - l i q u i d chromatography of t e t r a -0 -methylaldohex-oses and other sugars as acetates, Carbohyd. Res.,23, 430-432 (1972). G.M. Bebault and G.G.S. Dutton, Synthesis of 4-0-B-D-Glucopyranosyl-L-rhamnopyranose, Can.J.Chem.,50, 3373-3378 (1972). G.M. Bebault, J.M. Berry, Y.M. Choy, G.G.S. Dutton, N.; Funnell, L.D. Hayward, and A.M. Stephen, Semimicro Determination of Sugar Configuration by Measurement of C i r c u l a r Dichroism of A l d i t o l Acetates, Can. J . Chem., 51, 324-326 (1973). . . G.M. Bebault, Y.M. Choy, G.G.S. Dutton, N. Funnell, A.M. Stephen, and M.T. Yang, Proton Magnetic Resonance Spec-troscopy of Klebsiella Capsular Polysaccharides, J . Bacteriology, 113, 1345-1347 (1973). G.M. Bebault and G.G.S. Dutton, Synthesis of 4-£7-ct-D-Mannopyranosyl-L-rhamnopyranose, Can. J . Chem., 52, 678-680 (1974). G.M. Bebault and G.G.S. Dutton, Synthesis of 4-#-ct-L-Rhamnopyranosyl-L-rhamnopyranose, Carbohyd. Res., i n press. G.M. Bebault and G.G.S. Dutton, Synthesis of 4-tf-g-D-Mannopyranosyl-L-rhamnopyranose, Carbohyd. Res., submitted. 

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