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The methylation of sugar mercaptals Yates, Keith 1957

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THE METHYLATION OP SUGAR MERCAPTALS by KEITH YATES B.A., University of B r i t i s h Columbia, 19^6 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Chemistry We accept th i s thesis as conforming to the required standard THE. UNIYERSITY OF BRITISH COLUMBIA October, 19^7 i i ABSTRACT It was reported i n 1934 by Lieser and Leckzyck that D-glucose d i e t h y l mercaptal could he s e l e c t i v e l y methyl-ated by use of methyl iodide and s i l v e r oxide at 0°C. to give exclusively 2-0-methyl-D-glucOse d i e t h y l mercaptal i n 5>2$ y i e l d . They stated that the mercaptals of D-galactose.-,. L-arabinose, D-xylose and L-rhamnose f a i l e d to react i n a similar way and isolated no products. The. present Investi-gation was undertaken to determine to what extent these statements are correct,, and i f methylation does occur with the mercaptals of other sugars,,, to determine the nature of the methylated products. It was also of interest to determine i f reaction predominates at any one p o s i t i o n as i n D-glucose mercaptal. The o r i g i n a l observations of Lieser and Leckzyck were substantially confirmed by a r e p e t i t i o n of t h e i r experi-ments. The present work, however, has shown that some methylation does occur with mercaptals other than that of D-glucose but to a much smaller extent, and that this i s p a r t l y due to the i n s o l u b i l i t y of these; mercaptals i n methyl iodide. Tetrahydrofuran was found to be a suitable methyl-ation solvent i n which the d i e t h y l mercaptals of D-glucose, D-galactose, L-arabinose and D-mannose have comparable solu-b i l i t y . These mercaptals were methylated i n t h i s solvent by a modification of Lieser*s procedure. Investigation of the i i i products by paper chromatography indicated that a l l four gave similar r e s u l t s . Further there did not appear to. be any s i g n i -f i c a n t difference in the reactions when they were carried out at 3 ° , 22° , and f?0°C. These experiments were a l l on an exploratory scale. Suitable conditions were established f o r larger scale methylations and separations of products„ One such large scale methylation and separation was carried out on D-gluCose d i e t h y l mercaptal and the products hydrolysed and separated as the free sugars by means of column chromatography. Six d i s t i n c t methylated glucoses were sep-arated showing that the reaction i s by no means as selec t i v e when tetrahydrofuran i s used as reaction solvent. The largest product, but no longer dominantly so., was p o s i t i v e l y i d e n t i f i e d as the 2-0_-methyl derivative, again showing that r e a c t i v i t y i s greatest at the 2-position. This was found to be the only monomethyl ether produced. A second large scale methylation and separation was carried out In an exactly s i m i l a r manner on D-^mannose d i e t h y l mercaptal. Eight d i s t i n c t methylated mahnoses were separated with the extent of methylation being comparable with that above. Two major monomethyl products were isolated and iden-t i f i e d by periodate oxidation studies and t h e i r osazones as the 6-p_-methyl and ^-0_-methyl derivatives. The res u l t s i n d i -cate that the 6-posltion i s most reactive i n D-mannose d i e t h y l mercaptal. A t h i r d s i m i l a r methylation of L-arabinose d i e t h y l mercaptal, followed by separation of products by a combination i v of column and paper chromatography, yielded seven d i s t i n c t methylated arabinoses. There were no major monomethyl products, reaction appearing to have gone large l y to the dimethyl stage. Three monomethyl arabinoses were, separated in n e g l i g i b l e amounts and could not be i d e n t i f i e d . Reaction apparently does not predominate at any one p o s i t i o n i n L-arabinose d i e t h y l mercaptal. Preliminary experiments on D-galactose d i e t h y l mercaptal indicated methylation to be of a highly random nature. An exploratory separation of products showed that no one monomethyl ether predominated. The large scale methylation and separation was not repeated on th i s mercaptal. Of the compounds investigated, unusual r e a c t i v i t y of the 2-hydroxyl group i s only c l e a r l y manifested by D-glucose mercaptal. No marked r e a c t i v i t y of t h i s p o s i t i o n i s shown by the other mercaptals, although there i s evidence that the 6- and 5~hydroxyls of D-mannose mercaptal are. unusually reactive. In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements f o r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representative. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of CHEMISTRY  The University of B r i t i s h Columbia, Vancouver 8, Canada. Date September- 10th.. 1957. y ACKNOWLEDGE]^ ICT Thanks are extended, to Dr. G..G.S. Dutton f o r his patient and invaluable, d i r e c t i o n of t h i s research, and also to the. National Research Council f o r f i n a n c i a l a s s i s -tance. y i TABLE OF CONTENTS Page HISTORICAL INTRODUCTION 1 DESCRIPTION AND .RESULTS OF PRESENT RESEARCH 6 I. Preliminary Investigation of Methylation Reactions. 6 I I . Large Scale Methylations and Separations „ . . . < , 11 I I I . Conclusions and Theoretical Implications . „ . . . 1$ EXPERIMENTAL 19 ,1.. Preparation of Diethyl Mercaptals . . . . . . . . . . 19 I I . Methylation of Mercaptals i n Methyl Iodide . . . . 19 H I . Chromatographic Investigation of the Reaction Products „ „ 20 'XV. Investigation of the E f f e c t of Individual c? Reagents . . . . 21 V. Hydrolysis of the Reaction Products . . . . . < > . . 22 VI. Chromatographic Investigation of the Hydrolysates . 22 VII. Methylation of Mercaptals. i n Tetrahydrofuran . » . 23 VIII. Chromatographic Investigation of the Methylations i n Tetrahydrofuran . . . . . . . . . . . . . . . 23 IX. Large Scale Methylation of D-Glucose Diethyl Mercaptal . . . . . . . . . . . . . 23 X. Large Scale Methylation of D-Mannose Diethyl Mercaptal . . . . . . . . . . . . . . . . 27 XI. Periodate Oxidation of Mannose Monomethyl A F r a c t i o n . » . . ' . . . . . ' 28 XII. Periodate Oxidation of Mannose Monomethyl B F r a c t i o n . . . . . . . . . . . . . . . . . . . . . 29 XIII. Preparation of Derivatives of Monomethyls A. and B . . XIV. Large Scale Methylation of L-Arabinose Diethyl Mercaptal . , . . „ . . . „ BIBLIOGRAPHY . . . . . . 0 . . . . . . . APPENDICES . . . . . . . . . . . . . o . . . . o . o c . . c T i n TABLES Page Io Physical Constants of Diethyl Mercaptals . . „ . „' . 19 I I . Results of Chromatograms of the Products of Methylations In Methyl Iodide . . . . . . . . . . 21 I I I . Results of Chromatograms of Hydrolysed Products of Methylations i n Methyl Iodide 22 IVo Results of Chromatograms of the Products of Methylations i n Tetrahydrofuran . . . . . . . . . . . . . . 23 V. Result's: of Chromatograms of Hydrolysed .Products of Methylations i n Tetrahydr.ofuran „ „ . 2'5? VI... ..Glucose Fractions Separated on Cellulose-Hydrocellulose (1:1) Column . . . . . . . . . . . . 26. VII.. Mannose Fractions Separated on Cellulose-Hydrbcelluldse (1:1) Column . . . . . . 28 VIII o Theoretical .and Obtained Results of Period ate Oxidation of Monomethyl Mannoses 30 EC. Arabinose Fractions Separated on Whatman No.. 3MM i x APPENDICES Page A. Diagram of Chromatographed Products, of Methylations i n Methyl. Iodide <> . . <> 35> B. Diagram of Chromatographed Hydrolysates from Methyl-.-* at.ions i n Methyl Iodide . . . . . . > . . . . . - 6 . 36 C. Diagram of Chromatographed Products of Methylations In Tetrahydrof.uran . .* . . . . . . . .- . . » 37 D. Diagram of Chromatographed Hydrolysates from Methyl-ations. i n Tetrahydrofuran 38 E. Infra-Red Spectra of Diethyl Mercaptals . . . . . . . 39 1 HISTORICAL INTRODUCTION In l 8 9 l | , F i s c h e r ^ prepared a new series of carbo-hydrate derivatives, the thioacetals, or as they are more usually c a l l e d , the mercaptals. These compounds were formed by the addition of mereaptans to carbonyl compounds i n the presence of acid c a t a l y s t s . An example of t h i s reaction i s shown below. CHO CH(SC-He) , , 2 5 .2 HCOH ' HCOH 1 H + 1 HOCH + 2C2H^SH — * HOCH + H 20 HCOH HCOH HCOH HCOH 1 1 CH20H CH20H D-glucose ethyl mereaptan D-glucose (aldehyde form) d i e t h y l mercaptal Though the reaction i s by no means peculiar to carbohydrate aldehydes and ketones, i t i s i n t h i s f i e l d that i t has found i t s widest application. The mercaptals have in t e r e s t i n g properties i n that they are necessarily a c y c l i c since they no longer possess a carbonyl function. For the same reason they are quite stable to bases and mild oxidants and can therefore be used in such reactions as the Purdie methylation with methyl iodide and s i l v e r oxide, unlike the simple sugars. Since none of t h e i r 2 hydroxyl groups are involved i n r i n g formation,, they lend themselves to the preparation of p a r t i a l l y substituted sugars whose syntheses would be unnecessarily complicated s t a r t i n g from the various c y c l i c forms. (12) I t was reported i n 193^  by Lieser and Leckzyck that D-glucose d i e t h y l mercaptal (I) could be s e l e c t i v e l y methylated by the use of methyl iodide and s i l v e r oxide at 0°C. to give exclusively 2M>methy1-D-glucose d i e t h y l mer-captal (II) In 5>2$ y i e l d . Similar r e s u l t s were also obtained with D-glucose dibenzyl mercaptal ( I I I ) . The corresponding 2-p_-methyl-D-glucose (IV) could e a s i l y be obtained by acid hydrolysis of the mercaptal groups.. For t h i s reason i t i s the most e a s i l y accessible glucose methyl ether. CH(SR) i HCOH i HOCH i HCOH l HCOH i CH2OH CH(SR) 2 HCOCH-L3 i HOCH + 2CHoI+Ag20 -* 2AgI+H20 + « J HCOH HCOH I CH20H H 20 CHO I . HCOCH. i ^ HOCH I +2RSH HCOH I HCOH. I CH20H I R = -C2H^ III R •= -CH2C6H^ II. Since the method showed promise, of an easy route to s p e c i f i c a l l y substituted sugars, they attempted the same reaction with the mercaptals of D-galactose, L-arabinose, D-xylose and L-rhamnose„ They stated that these f a i l e d to react i n a simi l a r way, since they were able to is o l a t e no products, and implied that the 3 reaction "was peculiar to D-glucose mercaptals, although there appears to be no a p r i o r i reason why t h i s should be so. They did not investigate further, since at that time the problem of separating the complex mixtures of products which may have resulted was almost insuperable. This was unfortunate f o r i f the reaction, a l b e i t an incomplete one, could haye been extended to other sugars i n general, the usual lengthy and d i f f i c u l t introduction and removal of blocking groups could be avoided i n the preparation of p a r t i a l l y methylated sugar derivatives. The l a t t e r have assumed great importance i n the. determination of polysaccharide structure-. „ However, since the introduction of p a r t i t i o n . (13) chromatography i h I9J4-I by Martin and Synge a valuable technique i s available f o r the separation of complex mixtures of carbohydrates such as might r e s u l t from incomplete methyl-ations. Apart from the above p r a c t i c a l s i g n i f i c a n c e , Lieser"? s work has considerable t h e o r e t i c a l interest with respect to the study of r e l a t i v e r e a c t i v i t i e s of hydroxyl groups i n sugars. . (18) This subject has been extensively reviewed by Sugihara. The problem i s by no. means c l a r i f i e d however, and t h i s applies i n p a r t i c u l a r to the r e a c t i v i t i e s of the hydroxyl groups i n sugar mercaptals, upon which r e l a t i v e l y l i t t l e work has been d one. In t h i s l a t t e r respect, L i e s e r ^ - ^ has shown that i n the methylation of D-glucose d i e t h y l and dibenzyl mercaptals, k the r e a c t i v i t y of the 2-hydroxyl so f a r exceeds any other that i t s methyl ether r e s u l t s exclusively. This i s i n contrast to the more random nature of methylation i n the free sugars or glycosides, but his work has. not indicated that t h i s .unusual r e a c t i v i t y i s generally exhibited by mercaptals. Further, Z i n n e r h a s recently shown that i n p a r t i a l e s t e r i -f i c a t i p n s of sugar mercaptals, the terminal or primary hydroxyl group i s considerably more reactive than the secondary hydroxyls, a phenomenon which i s not as markedly exhibited by other types (7) of carbohydrates. In other reactions, such, as t r i t y l a t i o n , the mercaptals appear to follow the general trends established f o r c arbohydrate s. A f a c t o r which r e s u l t s i n the mercaptal hydroxyls exhibiting d i f f e r e n t r e l a t i v e r e a c t i v i t i e s compared to the free sugars, or glycosides i s t h e i r a c y c l i c nature. Barker and co-workers^ have shown that f o r the polyols at l e a s t , there i s a marked tendency f o r the carbon chain to adopt the planar zig-zag form, r e s u l t i n g i n s e l e c t i v i t y i n c e r t a i n reactions. It i s reasonable to suppose that the mercaptals are similar i n nature and that the resultant differences i n conformation of t h e i r hydroxyl groups compared to those already established f o r c y c l i c sugars w i l l affect t h e i r r e a c t i v i t i e s . Further, the proximity of the bulky, e a s i l y p o l a r i s a b l e s u l -phur atoms i s bound to. exert an e f f e c t on the r e a c t i v i t y of the remainder of the mercaptal molecule. F i s c h e r ^ has shown that D-glucose d i e t h y l mercaptal i s weakly a c i d i c , giving a 2-sodium s a l t i n aqueous alkaline solution. In view of the above considerations, the aims of the present work are as follows.: (1) A r e i n v e s t i g a t i o n of Lieser's o r i g i n a l work, to determine, f i r s t l y i f methylation i s exclusive at the 2-position and secondly, i f any reaction does occur with, mercaptals other than those of D-glucose, and i f so, to what extent. (2) An i n v e s t i g a t i o n into the generality of the reaction. (3) The i d e n t i f i c a t i o n of the p r i n c i p a l monomethyl products of the reaction to e s t a b l i s h which, are the most h i g h l y reactive positions, i f any such c l e a r l y e x i s t as i n D-glucose mercaptals. The establishment of whether with, an e f f e c t i v e separation technique, an incomplete reaction such as the. above can be p r o f i t a b l y extended to the preparation of s p e c i f i c methylated sugars. The success of the investigation i n achieving these aims w i l l depend on the complexity of the mixtures of reaction products obtained and the ada p t a b i l i t y of the separation techniques of p a r t i t i o n chromatography. 6 DESCRIPTION AND RESULTS, OF PRESENT RESEARCH Io Preliminary Investigation of Methylation Reactions The d i e t h y l mercaptals of D-glucose, D-mannose, D-galactose and L-arabinose were methylated on an exploratory (12) scale by the method of Lieser and Leckzyck. Upon sep-aration of the. products from excess methyl iodide arid s l i v e r oxide, syrups were obtained<, The syrup from the reaction of D-glucose d i e t h y l mercaptal c r y s t a l l i s e d spontaneously to give a i|6$ y i e l d of 2-p_-methy 1 -D-glucose d i e t h y l mercaptal, the others f a i l i n g to y i e l d any c r y s t a l l i n e material except unchanged mercaptal. Thus i n a general way Lieser's r e s u l t s were confirmed. The mother l i q u o r from the above 2-0-methyl-D-glucose mercaptal and the remaining three reaction mixtures were investigated, by means of paper p a r t i t i o n chromatography. The developed and detected chromatograms gave a s t r i k i n g l y s imilar pattern of spots, as shown i n Appendix A, i n d i c a t i n g q u a l i t a t i v e l y that the reaction i s more general than had been supposed. The presence of a further amount of. 2-p_-methyl-D-glucose d i e t h y l mercaptal i n the mother liquor i s also indicated, suggesting that i f desired, the y i e l d of t h i s product could be improved to approach Lieser' a $2.%. It can be seen from the chromatograms arid Table II that the reaction i n each case gave products of two types. Those products with considerably lower rates of t r a v e l than the unsubstituted mercaptals are presumably strongly polar 7 compounds and are of unknown con s t i t u t i o n . However, i t i s clear that they r e s u l t from a modification of the mercaptal groups i n some way, since reaction at the hydroxyl groups would not .be expected to give r i s e to an increased p o l a r i t y . The mercaptal; groups have not been removed completely, since, these compounds gave no spot f o r a reducing sugar with p-anisidine hydrochloride. I t i s also.apparent that these compounds are probably carbohydrate i n nature since t h e i r Rp values vary from sugar to. sugar, suggesting that the mole-cule contains part of the o r i g i n a l sugar moiety. On hydrolysis with acid, however, they did riot regenerate the parent sugars. The most intense- of these "slow" spots can be produced by reacting the mercaptal with methyl iodide alone, which i n d i -cates that t h i s major "slow" component may possibly be a type of methyl sulphonium iodide. This would account f o r the increase i n p o l a r i t y . However, any attempt at postulating structures f o r any of these compounds i s highly speculative and i s i n e f f e c t a separate problem. It can be said d e f i n i t e l y from t h e i r rates of t r a v e l , t h e i r d i f f e r e n t colour r e a c t i o n with Iodine vapour and t h e i r f a i l u r e to y i e l d sugars on acid h y d r o l y s i s , t h a t these compounds are not mercaptals and were not Investigated further at t h i s time. Prom comparison with known standards, the " f a s t " spots whieh gave a yellow colour reaction with iodine-vapour are obviously mercaptals and methylated mercaptals. Prom the d i s t r i b u t i o n and i n t e n s i t y of these i t appeared that reaction had been most extensive i n the case of D-glucose since no. spot 8 f o r unchanged mercaptal was detected. Somewhat less r e a c t i o n appeared to have occurred i n the case of D-galactose and only a small amount i n the cases of the other two mercaptals 0 Due to the high Rp. values of the mercaptals and con-sequent low degree of chromatographic separation, the products were hydrolysed and reinvestigated by paper chromatography as the free sugars thus produced.. The results, of t h i s are shown in Appendix B. From a comparison with the c h a r a c t e r i s t i c RQ. values of known compounds ^  It appears that the f i r s t row of spots correspond to unsubstituted sugars, the second to t h e i r monomethyl derivatives and the remainder to more, highly sub-st i t u t e d sugars. (Table III.) From t h i s i t can be seen that some monomethylation has occurred i n each case, which i s i n apparent disagreement with Lieser*s statement that the reaction f a i l s f o r sugar mercaptals other than those of D-glucose. In- the l a t t e r case the reaction gives exclusively monomethylation and since no unsubstituted D-glucose was detected, a l l the o r i g i n a l mercaptal must hay© reacted. This agrees with the findings above and also explains the absence of a spot of R x = 0.9i? i n the glucose column In Table I I . In the reaction of D-mannose arid L=arabinose mercaptals methyl-ation appears to have gone to. the monomethyl stage also, but to a much l e s s e r extent since large amounts of the unchanged sugars"were detected. However, i n the reaction of D-galactose mercaptal some polymethylation has also taken place,. These r e s u l t s suggest the reason f o r Lieser's f a i l u r e to i s o l a t e any c r y s t a l l i n e products except i n the case of D-glucose; 9 i n two instances the small amounts of products seem to be responsible and i n the other, the complexity of the reaction mixtur e. Prom the above evidence i t again appeared that the s e l e c t i v i t y of the reaction, i f not the, extent, was more general than had been supposed, at l e a s t as f a r as D-glucose, D-mannose and L-arabinose were concerned. The evidence from the D-galactose hydrolysate was somewhat i n disagreement. During t h i s preliminary work i t was observed that there were considerable differences In the s o l u b i l i t i e s of the various mercaptals i n methyl iodide. Under the conditions used, i t was found that the extent of methylation corresponded approximately to the degree of s o l u b i l i t y of the p a r t i c u l a r mercaptal being reacted. In this respect D-glucose diethy1 mercaptal was found to be most soluble and i t s unusual r e a c t i v i t y appeared i n part due to the fo r t u i t o u s choice of methyl iodide as reaction solvent. Large amounts of D-mannose and L-arabinose mercaptals were found undissolved at the end of the reaction and these mercaptals appeared to have reacted to the lea s t extent. In order to eliminate t h i s s o l u b i l i t y e f f e c t i t was decided to incorporate an i n e r t reaction solvent i n which a l l four mercaptals had comparable s o l u b i l i t i e s and compare the reaction of each again on a more equal basis. The most s u i t -able of the common organic solvents which are in e r t to methylation was found to be tetrahydrofuran. Since the 10 presence of a large amount of inert solvent was found to quench the reaction almost e n t i r e l y , increased amounts of methyl iodide and s i l v e r oxide were used. In order to deter-mine i f changing the o r i g i n a l reaction temperature of 0°G„ had any c r i t i c a l e f f e c t on the nature of the products, three p a r a l l e l reactions were carried out on each mercaptal at 3°, 22°, and £0°C. The syrupy products were separated from the reaction, mixtures as before, however i n this case none of them c r y s t a l l i s e d spontaneously. They were again investigated by paper chromatography, the res u l t s being shown i n Appendix 0 and Table IV. The generality of the reaction was again Indicated from the s i m i l a r i t y of the pattern of spots obtainedo The unusual r e a c t i v i t y of D-glucose d i e t h y l mercaptal was not now so markedly apparent since a spot was obtained f o r unchanged mercaptal at a l l three temperatures. The products were hydrolysed and re chromat ographe d as before, the re s u l t s of t h i s being shown i n Appendix D and Table V. Some monomethylation has again occurred i n each case and also polymethy1at1on which was previously absent i n three of the reactions when carried out i n methyl iodide. The extent of methylation appeared more comparable i n each case-. The follow-ing conclusions were drawn from these results„ F i r s t l y , the peculiar s e l e c t i v i t y of the reaction of D-glucose d i e t h y l mercaptal was no longer apparent when carried out i n t e t r a -hydrofuran. Secondly, the reaction appeared more general i n thi s solvent. Thi r d l y , the e f f e c t of varying the reaction 11 temperature did not seem to be of c r i t i c a l importance. I I o Large Scale. Methylations and Separations In the l i g h t of the above evidence i t was decided to repeat the methylations on a larger scale to obtain work-able quantities of the products and attempt a separation and i s o l a t i o n of the products as q u a n t i t a t i v e l y as p o s s i b l e . Although complete recovery of the products i s not f e a s i b l e due to chromatographic losses, the r e l a t i v e y i e l d s which Can be obtained are of value i n esta b l i s h i n g which are the major products. I t was further decided to investigate the mono-methyl f r a c t i o n s most thoroughly to determine i f p r e f e r e n t i a l methylation had occurred at the 2-position. A large scale methylation of D-glucose d i e t h y l mercaptal was carried out i n tetrahydrof uran at 22°C. It was decided to remove the undesired "slow" components which might l a t e r contaminate the other products. The syrupy r e a c t i o n products were therefore subjected to a crude preliminary separation b y colu»n c h , o m a t o g r a p h y ( i 9 ) Into a m e r c a p t a l and non-mereaptai f r a c t i o n . The former was hydrolysed as before and the mixture of f r e e sugars separated by a more refined application of column chromatography„ The f r a c t i o n s collected were investigated systematically by paper chromatography and polarimetry to locate i n d i v i d u a l sugars. The r e s u l t s given i n Table VI show that there are s i x d i f f e r e n t methyl glucoses i n addition to. some unchanged sugar. The Rp values and methoxyl contents were, obtained to determine the degree of methylation 12 In each of the products. The monomethyl f r a c t i o n was shown to be one. component only by i t s chromatographic p u r i t y and constancy of o p t i c a l r o t a t i o n throughout the f r a c t i o n . It i s thus the largest single product of the reaction. This f r a c t i o n c r y s t a l l i s e d spontaneously and f a i l e d to depress the melting point of an authentic sample of 2>0-methyl-D-glucose on admixture w i t h ' i t . I t was further characterised as 2 - p_-me thy 1 -D - glue o s e through i t s p-toluidide d e r i v a t i v e . The polymethyi f r a c t i o n s were not further i n v e s t i -gated o The y i e l d s of the o r i g i n a l products of methylation were estimated and are given below. TFnreacted D-glucose d i e t h y l mercaptal 2.02$ Monomethyl " " " 8.36$ Dimethyl " " " 10.$$% Trimethyl " " " $.19% I t should again be emphasized that these do not take into account mechanical losses and are only of value i n showing the general extent of methylation. The important r e s u l t Is that although the only mono-methyl derivative r e s u l t i n g from the methylation i s the 2-0-methyl, the reaction i n t h i s solvent i s no longer so selective as to y i e l d t h i s exclusively. The addition of a solvent seems to favour polymethylation, possibly due to the increased s o l u b i l i t y of the reactants. Hence the s i n g u l a r i t y of glucose mercaptals i n Lieser's work appears to be a 13 consequence of the reaction solvent used. The above methylation and separation of products was repeated on D-mannose d i e t h y l mercaptal. The r e s u l t s are given i n Table VII, showing that there are eight d i s t i n c t methyl mannoses i n addition to some unchanged D-mannose. The degree of methylation i n each product was determined as before. Of the four monomethyl mannoses, two were present i n neg l i g i b l e amounts and were considered to be r e l a t i v e l y unim-portant. The two predominant monomethyl sugars were sub-jected to periodate oxidation studies. The r e s u l t s of t h i s , given i n Table VIII, indicated that the larger was 6-0-methyl-and the lesser 5>-0-methyl-D-mannose. These were further characterised through t h e i r phenylosazones„ The y i e l d s of the o r i g i n a l products were estimated as before and are given below. Unreaeted D-mannose d i e t h y l mercaptal 0*$3% Monomethyl " " " 12„07$ Dimethyl " " " 3.88$ Trimethyl " " " 21.i|£# The r e s u l t s i n t h i s case show that reaction does hot pre-dominate at the 2-position nor at any one p o s i t i o n . Never-theless the primary hydroxyl i s apparently most reactive, with the ^- p o s i t i o n also being highly .reactive. Further, the t o t a l y i e l d of methylated products i s now more comparable with that obtained from D-glucose d i e t h y l mercaptal than when methyl Iodide was used as rea c t i o n solvent. However, the monomethyl derivatives are not the largest products of the reaction, since methylation has apparently proceeded l a r g e l y to the trimethy1 stage. A large scale methylation and preliminary crude f r a c t i o n a t i o n was carried out on L-arabinose d i e t h y l mercaptal exactly as before. The f i n a l refined' separation, however, was effected by paper chromatography, followed by recovery of the i n d i v i d u a l sugars from the developed chromatograms by e l u t i o n of the appropriate zones. The r e s u l t s are given i n Table IX, showing that there are seven d i s t i n c t methyl arabinoses pre-sent but no unchanged L-arabinose. The y i e l d s of the o r i g i n a l products were estimated as before and are given below. Monomethyl L-arabinose d i e t h y l mercaptal 2.1$ Dimethyl " 11 " .26.8$ The r e s u l t s do not show that methylation predominates at any one p o s i t i o n . Apart from the less s p e c i f i c nature of methylation, reaction appears to have gone l a r g e l y to the dimethyl stage, with three of the four possible monomethyl arabinoses being present only i n n e g l i g i b l e amounts. The t o t a l y i e l d of methylated products Is now more comparable with that from D-glucose mercaptal than when methyl iodide was used as reaction solvent. Prom the d i v e r s i t y of spots obtained i n the pre-liminary inv e s t i g a t i o n of D-galactose d i e t h y l mercaptal i t did not appear that any p a r t i c u l a r s e l e c t i v i t y of reaction was exhibited. Indeed, an exploratory separation of the products showed that the monomethyl f r a c t i o n was not homogeneous, in d i c a t i n g that reaction did not predominate at any one p o s i t i o n . It was f e l t that the value of the r e s u l t s to be obtained did not j u s t i f y a r e p e t i t i o n of the above large scale work on D-galactose d i e t h y l mercaptal at t h i s time. I l l o Conclusions and Theoretical Implications Gf the mercaptals investigated both by Lieser and. i n the present work, only the D-glucose derivatives exhibit unusual r e a c t i v i t y of the 2-hydroxyl group. Under L i e s e r 1 s conditions methylation has been shown to occur exclusively at t h i s p o s i t i o n and although reaction i s less s e l e c t i v e when an i n e r t solvent i s used, the 2-_0-methyl i s the sole monomethyl ether formed. The reasons f o r t h i s p e c u l i a r r e a c t i v i t y are not clear. L i e s e r ( l ^ ) stated that the. r e a c t i v i t y of the 2-hydroxyl i s p a r t i c u l a r l y increased by the neighbouring mercaptal groups, presumably by an electronic e f f e c t . I f t h i s Is true-, i t i s d i f f i c u l t to see why t h i s e f f e c t i s not equally present i n other mercaptals which are i d e n t i c a l with: those of D-glucose except f o r the configuration of t h e i r hydroxyl groups. That there i s such an e f f e c t appears to be established by the a c i d i t y of the 2-hydroxyl as demonstrated by F i s c h e r ^ , and also by the f a c t that non-mercaptalated glucoses do not show t h i s peculiar r e a c t i v i t y of the 2-position i n methylation. It Is doubtful, however, that t h i s e f f e c t i s s o l e l y responsible f o r producing the unusual r e a c t i v i t y of the 2-hydroxyl. 16 The p o s s i b i l i t y of a purely s t e r i c e f f e c t operating i n conjunction with the above electronic e f f e c t was consid-ered. I t i s reasonable to assume that the proximity of the mercaptal groups confers p o t e n t i a l r e a c t i v i t y on the 2-position i n a l l the sugar mercaptals„ However, i f the a v a i l a b i l i t y of this p o s i t i o n to attack were reduced due to blocking by e i t h e r the bulky mercaptal groups or the rest of the molecule, t h i s p o t e n t i a l r e a c t i v i t y would be diminished arid perhaps no longer apparent. A study of models of the four mercaptal molecules was made. Various s p a t i a l arrangements of the groups were considered In an attempt to minimize non-bonded int e r a c t i o n s , but In no case did these reveal any pronounced differences between the various mercaptals In the a v a i l a b i l i t y of the 2-position. Hence i t appears that t h i s s t e r i c e f f e c t i s not responsible f o r the marked difference between glucose and the other mercaptals. A consideration of the conformation of the hydroxyl groups 'in the planar zig-zag form, established by Barker^ f o r the polyols, suggested the p o s s i b i l i t y of differences i n hydrogen-bonding being responsible f o r the s i n g u l a r i t y of glucose mercaptals. The greater the extent of hydrogen-bonding, the less r e a c t i v i t y would be expected from an alco-h o l i n a nucleOphilic substitution reaction such as. methyl-ation. The infra-red spectra of the four mercaptals (Appendix E) show marked differences i n the p o s i t i o n of the hydroxyl peak. The peak f o r glucose mercaptal at 339^ cm"-*-17 i s shifted the furthest towards the high frequency region of the spectrum ind i c a t i n g least hydrogen-bonding, with progres-s i v e l y more for' galactose, mannose and arabinose mercaptals as indicated by the positions' of t h e i r peaks at 3320, 3280 and 3275> cm re s p e c t i v e l y o Thus the greater freedom, of the hydroxyl groups i n cooperation with the e f f e c t of the neigh-bouring mercaptal groups suggests an explanation f o r the unusual behaviour of glucose mercaptal compared to the other mercaptals„ A complication i s introduced when tetrahydrofuran i s the reaction solvent. Since i t i s capable of solvent-solute hydrogen bonding whereas methyl iodide i s not, i t might be expected that differences i n the i n t e r - and intramolecular hydrogen-bonding of the various mercaptals would tend to be smoothed out i n the presence of a large number of bonding solvent molecules. This" would r e s u l t i n more s i m i l a r r e a c t i -v i t y of the 2-hydroxyls. The presence of tetrahydrofuran does indeed reduce the marked r e a c t i v i t y of the 2-hydroxyl over the other hydroxyls i n glucose mercaptal as evidenced by the occurrence of polymethylation, but apparently does not s i g n i f i c a n t l y increase the r e a c t i v i t y of thi s p o s i t i o n i n other mercaptals. The e f f e c t of the d i e l e c t r i c constant of the solvent used has been considered, but since a l l the reactions involve methyl iodide they probably take place by the same mechanism. Hence changes i n the d i e l e c t r i c constant of the medium would 18 not be expected to af f e c t p a r t i c u l a r hydroxyl groups. The complexity of the various electronic,, e l e c t r o s t a t i c and conformational e f f e c t s simultaneously operative makes any complete explanation of the p e c u l i a r i t y of glucose: mercaptals impossible without recourse to further i n v e s t i g a t i o n . A study of the r e l a t i v e importance of these factors seems des i r -able . A knowledge of these factors would also be of value i n extending the reac t i o n to the preparation of s p e c i f i c a l l y substituted derivatives of sugars other "than. D-glucose. There Is promise of t h i s i n the results from the methylation of D-mannose mercaptal. Adjusting the reaction conditions should lead to more p r a c t i c a l y i e l d s of the £-j3-methyl and 6r0-methyl ethers. Although the r e s u l t s from the methylations of D-galactose and L-arabinose mercaptals do not show any promise under the conditions used, p a r t i a l methylations of mercaptals of these and other sugars not yet investigated may be of preparative and t h e o r e t i c a l value i f suitable, con-d i t i o n s can be determined. 19 EXPERIMENTAL A l l melting points were taken .by means of a L e i t z e l e c t r i c a l l y heated melting point block and are corrected. A l l evaporations were done i n vacuo at l | .0 oC. Methoxyl analyses were carried out by the method of Viebock and Schwappach as described by Clark. (2). I. Preparation of Diethyl Mercaptals The following mercaptals were prepared by the method of Fischer ^  , and t h e i r p h y s i c a l constants are given i n Table I . , Table I Physical Constants of Diethyl Mercaptals PO PO Mercaptal. M.P. Lit.M 0P. [«]^(lf). Y i e l d D-glucose 128-129°C . 1 2 7 - 1 2 8 ° C. (ij.). - 2 7°(H 2 0 ) * - 2 9 „ 8 ° ( H 2 0 ) * D-mannose 133-13i | - 0 C. 13 i | - 0 Co(10) 0 ° (Pyridine) - $3% D-galactose 3 i j .0-1^2 o C o ll ) . 0-llj . 2 0 C. (i*.) -9°(H 2 0) - 1 0 ° ( H 2 0 ) Q\% L-arabinose 1 2 l | . - 1 2 5 0 C o 12^-126°C. (Ij.) Pyridine) - 60$ * o Rotation of this, compound was measured at 5>0 C „ 1 1 •' Methylation of Mercaptals i n Methyl, Iodide D-glucose d i e t h y l mercaptal was methylated by the method of Lieser arid L e c k z y c k ^ . The mercaptal (0v9Ji gm.) was shaken with s i l v e r oxide (1.5> gm„) and methyl iodide (10 ml.) f o r 21 hours at 0°C. under anhydrous conditions. F i n a l l y , the mixture .was allowed to a t t a i n room temperature (one hour) and f i l t e r e d . The residue was extracted succes-s i v e l y with b o i l i n g chloroform, acetone .and methanol.. The f i l t r a t e and extracts were combined and evaporated to a syrup which was taken up i n a small amount of methanol. On standing th i s solution deposited c r y s t a l s (0.1|6 gm.) of 2-0-methy 1 -D-glucose d i e t h y l mercaptal, m.p. lj?i|.-li?£0C. Li,t.m.p„ 'l££-15>60C. (12) Anal: Gale, f o r C ^ H ^ O ^ : -OCH^ 10.37$° Found: -OCH3 = 10.99. Y i e l d Ij.6$. The d i e t h y l mercaptals of D-mannose, D-galactose and L-arabinose were also methylated by the above procedure, but the syrups obtained f a i l e d to y i e l d any c r y s t a l l i n e product. I I I . Chromatographic. Investigation of the Reaction Products Chromatograms of the four reaction products above were run on Whatman No. 1 paper using n-butanol-e.thanol-water (14.0:10:^0) as developer. Detection of the chromatograms with iodine vapour (5>) showed the pattern of spots represented i n Appendix A. Table: II gives the R x values and descriptions of the spots produced. Further duplicate chromatograms were detected with p-anisidine hydrochloride but f a i l e d to y i e l d any spots f o r reducing sugars. 21 Table II Results of Chromat ograras of the Products of  Methylations i n Methyl Iodide D-Glueose D-Mannose D-Galactose L-Arabinose mercaptal mercaptal mercaptal mercaptal Ry?f Description R x Description R x Description R x Description 0.13 OF 0.10 OF 0.13 OF 0.13 OF 0.21 OF 0.18 OF 0.21 OF 0.21 OF 0.36 01 0.36 01 O.ifO 01 O.I4.O 01. - - O.lj.7 OF 0.i|6 OF - 0.924- YI YF o.?5** YI 1.00 TI I.07 YF 1.03 YI 1.0£ YF "'^R- i s based on the arb i t r a r y standard 2 - 0_-me thy 1 -D - glue o s e d i e t h y l mercaptal whose rate i s set equal to 1.00. '""These values correspond to standards of t he unsubstituted mercaptalS i Key; 0 = Orange. Y = Yelloxv I = Intense F = F a i n t . IV. Investigation of the E f f e c t of, Individual Reagents Two samples of D-glucose d i e t h y l mercaptal (10 mgm.) were dissolved i n carbon tetrachloride•(5> mli) and shaken f o r 21 hours at 0°C. with methyl iodide and s i l v e r oxide r e s p e c t i v e l y . After f i l t e r i n g , the solutions were chromatographed with stand-ards and detected as before. The r e s u l t s are given below. A. Me thy1. io.dide .treated mereaptal.: Three spots of R x = 0.21, 0.35* (Intense) and O.9I4. (unchanged mercaptal). 22 B. S i l v e r oxide treated mercaptal: One spot of R x = 0.95> (unchanged mercaptal). V. Hydrolysis of the Reaction Products The reaction products from II were dissolved i n 20$ aqueous ethanol containing hydrogen chloride. The s o l u t i o n was heated to r e f l u x , a stream Of nitrogen being bubbled through continuously„• When chromatography of the solutions f a i l e d to detect any unchanged mercaptals (jj>~6 hours), they were passed through a column of Duolite A-l}. anion exchange r e s i n and con-centrated by evaporation,, VI. Chromatographic Investigation of the Hydrolysates The: above hydrolysates, were chromatographed as i n I I I and detected with p-anisldlne hydrochloride, the r e s u l t s being shown i n Table I I I . Table I I I Results of Chromatograms,of Hydrolysed Products of Methylations i n Methyl Iodide Glucose Mannose .Galactose Arabinose Hydrolysate Hydrolysate .Hydrolysate Hydrolysate 0.11 I 0.08 F 0.12 I 0;23 I 0.28 F 0.22 F 0.3k F 0.39 P 0.72 P 0.86 F (The values given are RQ. values based on 2 ,3 ,4 ,6-tetra-O-methyl-D-glucose = 1.00) "" 23 VII. Methylation of Mercaptals i n Tetrahydrofuran The mercaptal (0.3 gm.) was dissolved i n dry peroxide free tetrahydrofuran (20 ml.). Afte r s i l v e r oxide (1 gm.) and methyl iodide (10 ml.) were added, the whole was shaken vigorously f o r 22 hours. Three p a r a l l e l experiments were car-r i e d out on each mercaptal at 3°, 22° and j?Q°C. The products were then extracted as before, no c r y s t a l l i n e materials, being obtained. VIII. .. Chromatographic Investigation ..of the Methylations in. Tetrahydrofuran Chromatograms of the syrupy products from the above were run as before and detected with iodine, vapour. The mixtures were then hydrolysed as, i n V., rechromatographed i n n-butanol-ethanol-water (1|0:10:50) and detected with, p-anisidine hydro-chloride. The r e s u l t s are presented i n Tables, IV and V res -pe c t i v e l y . IX. ' Large Scale Methylation. of D-Glucose Di e t h y l Mercaptal S i l v e r oxide (6.7 gm.) and methyl Iodide (67 ml.) were added to a solution of D-glucose d i e t h y l mercaptal (2.0 gm.) i n p u r i f i e d tetrahydrofuran (100 ml.). The vessel was flushed with nitrogen, sealed and shaken at room temperature f o r 22 hours.. The products were extracted as i n I I , an amber syrup (.2.07 gm.) being obtained. Table I V . Results of Chromatograms of the Products of  Methylations i n Tetrahydrofuran* D-Glucose D-Mannose D-Galactose L-Arabinose 3° 22° g Q ° 3° 22° £0° ,3,.o .= 22° £0° 3° 22P.. £0.° 0.11 0,10 0.12 0.10 0.10 0.10 0.12 0.13 0.12 0.12 0.13 O.llj. 0.20 0.20 0.20 0.17 0.18 0.20 0.21 0.21 0.22 0.21 0.21 0.22 0.36 0.36 0.36 0.36 0.36 O.36 0.39 0.39 O.lj.0 0.38 O.ifO O.la 0.9$ 0o9$ 0.9^ 0.9^ 0.96 0.95 0.96 0.96 0.96 -** 0.97 I c O O l o O O 1.00 1.01 1.00 1.01 I c O l 1.02 1.01 1.02 1.03 1.03 The values given are R x values based on 2-0-methy1-D-glueose d i e t h y l mercaptal = 1.00 "~ The absence of a spot f o r unreacted mercaptal corresponding to the spots f o r L-arabinose produced on hydrolysis may be due to the greater s e n s i t i v i t y of the p-anisidine. Paper chromatography of the above syrup using methyl ethyl ketone-water azeotrope as developer gave.essentially the same r e s u l t s as shown i n the f i r s t column of Table I V . An aliquot (1.96. gm.) was placed on a c e l l u l o s e column (2.8 cm. diam. x ij.0 cm. length) and developed with the same solvent. Fractions were collected at one hour' intervals, f o r eight hours. (Two column front times) Two f r a c t i o n s were then collected at two hour i n t e r v a l s and the remaining material was removed from the column by developing f o r a further 80 hours, (20 f r o n t times) without any attempt at fractionation'. Paper chromato-grams of the f i r s t eight f r a c t i o n s developed with, methyl e t h y l 2$ Table ,V Results of Chromatograms of the Hydrolysed Products of  Methylations i n Tetrahydrofuran D-Glucose  Hydrolysate , 3° 22° ,gQ° 0.09 0.09 0.1.1 0.23 0.23 0.26 O.I4.O O.lj.2 0.J+3 0.46 0.47 0.£2 D-Mannose  Hydroylsate 3° -22° g0° 0.12 0.13 0.13 0.2£ 0.26 0.28 0.1+2 0.4S 0.4s D-Galactose.  Hydrolysate 3,o 22°- $0° 0.:07 0.08 0.08 0.18, 0.20 0.23 0.36 0.38 0.40 based on 2,3 ,l)-,6 L-Arablnose  Hydrolysate 30. 22° $0°. 0.13 0.12 0.12 0.32 0.32 Ov34 0.1)4 0-k% 0.I4.3 o.$o o.$o 0.49 0.62 0.6^ 0.6$ -tetra-Q-methy1-The values given are R^ values D-glucose - 1.00 ketone-water and detected with iodine vapour indicated f a i r l y good separation had been achieved. Fractions 2-$ were found to contain a l l the: mercaptalated products with s l i g h t traces of undesired impurity. These were combined (0.879 gm.) and designated as mercaptal f r a c t i o n . The higher fractions" were discarded. The mercaptal f r a c t i o n was hydrolysed as before, passed through a Duollte A-I4. column and the eluate concentrated to a syrup (0„42^ gm.) by evaporation. Two chromatograms of the l a t t e r were run i n methyl et h y l ketonerwater and detected with iodine vapour and p-anisidine hydrochloride respectively. The f i r s t chromatogram showed no spots corresponding to glucose mercaptals in d i c a t i n g hydrolysis, was complete. The; second 26 showed four spots of Rp'a OoOlij., 0.06, 0.21 and 0.1+7 correspond-(3) ing to the c h a r a c t e r i s t i c R F ranges of glucose, mono-, d i -and trimethyl glucoses respectively. An aliquot (O.I4.O6 gm.) of the above, hydrolysate i n the minimum amount of methyl ethyl ketone was placed on a column (2.8 x 1+2 cm.) of cellulose-hydrocellulose (1:1) and developed with methyl ethyl ketone-water azeotrope. Fractions were c o l -lected at 30 minute in t e r v a l s at a flow rate of I4.O ml. per hour. After 300 f r a c t i o n s had been .collected no further carbohydrate: material could be detected i n the eluate by application of the Molisch test and f r a c t i o n a t i o n was terminated. The f r a c t i o n s were investigated systematically by paper chromatography, mea-surement of o p t i c a l r o t a t i o n and determination of methoxyl value to obtain the following c l a s s i f i c a t i o n . Table VI. Glucose Fractions, Separated on Cellulose-Hydrocellulose (1:l) Column C l a s s i f i c a t i o n Tube .No. Wt0(mgm.) [oc] 2 0 R ^ * % OCR*. Hexose 238-270 .23 +ij.8°(H20) 0.017 Monomethyl l69r235 lOij. +59°(H"20) 0o0> l£.7 D.ijnethyl A $l~7k $0 + 8'2o.( Acetone) 0.28 26.£ Dimethyl B 86-108 1+7 +32°(Acetone) 0.18 2J4..8 Dimethyl C 30-^0 1+2 +6l°(Me0H) 0.1+7 27.3 Trimethyl A 28-29 11 +91°(Me0H) 0.50 36.6 Trimethyl B 12-19 63 +20°(H20) 0.80 36.7 ",R values determined i n methyl ethyl ketone-water azeotrope. F """^Calculated methoxyl values, f o r mono,-, d i - and trimethyl hexoses respectively are 16..0, 29.8 and 1+1.9$. 27 A l l the above f r a c t i o n s were found to be chromato-graphic a l l y pure except f o r some overlap between Dimethyls A and B. The monomethyl f r a c t i o n also showed constancy of o p t i c a l r otation throughout the f r a c t i o n and yielded a c r y s t a l l i n e pro-duct melting at l £ 3 - l £ 7 ° C . Admixture with an authentic sample of 2-0-methy 1-D-glucose gave a melting point of l$l±-l$Q0C. (17) The p-Toluidide was prepared by the method of Smith- 1 , m.p. nljlj.-146°C. L i t . m.p. l£0-l£l°C. (14). X. Large Scale .Methylation. of D-Mannose D i e t h y l Mercaptal D-Mannose d i e t h y l mercaptal (2,1 gm.) was methylated i n an exactly s i m i l a r manner to D-glucose mercaptal, y i e l d i n g a syrup (2.21 gm.) which was fractionated into a mercaptal -f r a c t i o n and a non-mercaptal f r a c t i o n as before. Hydrolysis of the mercaptal f r a c t i o n yielded a syrup (0.79 gm.). Two chroma-tograms were run on t h i s syrup as before. One detected with Iodine yapour gave negative results,, whereas the other detected with p-anisidine hydrochloride gave three discrete spots of R F's 0.023, 0.08 and 0.20 and a t r a i l i n g spot of R F range 0 . £ 0 - 0 . 8 0 . The mannose mercaptal f r a c t i o n hydrolysate was sep-arated and the f r a c t i o n s Investigated exactly as f o r D-glucose. The r e s u l t s are given i n Table VII. 28 Table VII Hydrocellulose (1:1) Column C l a s s i f i c a t i o n Tube No. Wt. (mgm.) M 2 0 D Rp(MEK) $ 0 C H 3 Hexo.se 200-225 7 -. 0.023 -Monomethyl A ll|2ri7^ 107 +30°(H 20) 0..-08 15.4 Monomethyl B 102-139 53 +17o3°(H20) Go 09 15.3 .Monomethyl C* 89-99 9 +11.6°(MeOH) 0.13 -Monomethyl D 83-87 .3 •- 6.15 -Dimethyl A 27 +26°.(Me OH) .0,23 28.1 Dimethyl B 3$-kl. 32 +59°(MeOH) .0.28 2 7 . 2 Trimethyl A 13-22 ii+l +38.3°(H20) 0.53 34-.1 Trimethyl B 7-11 208 +36oO°(MeOH) 0.75 3 .^1. C l a s s i f i c a t i o n i s based c onf irmat ion. on Rp values only and Is subj ect to All., the above f r a c t i o n s were found to be chromatographi-c a l l y pure except f o r some overlap: between Trimethyls A and B. XI. Periodate Oxidation of Mannose Monomethyl A. F r a c t i o n .Solutions containing monomethyl A (21.76 mgm., 6.112 mmole.) and sodium metaperiodate (103.2 mgm., O.I4.82 mmole.) were mixed and immediately made up to 50 ml, with d i s t i l l e d water and maintained at 20°C. Aliquots (5 ml.) were, withdrawn at i n t e r v a l s and excess sodium bicarbonate and potassium iodide added > the solutions being l e f t to stand f o r l5 minutes to allow complete iodine formation. ..Consumption of periodate was then determined by adding a measured excess of 0.1N sodium arsenite (9) solution and bac k - t i t r a t i n g with 0.1N iodine solution . When perlodate consumption had reached a constant value j formic acid production was determined by ne u t r a l i s i n g an aliquot to methyl red with standard a l k a l i . A further aliquot was treated with (9) dime-done' reagent - to determine formaldehyde-, but f a i l e d to y i e l d any p r e c i p i t a t e even afte r prolonged standing. The resu l t s are tabulated below. A. Perlodate Consumption Time (minutes) $ 10 1$ 30 60 120 Consumption (mmoles.) O.40I4. O.lj.10 O.lp.3 Cl-pi}. O.Ip.7 O.lp.7 Consumption (moles per mole) 3.60 3 . 6 6 3 . 6 9 3 . 7 0 3 . 7 3 3 . 7 3 B. Formic Acid O.I4.O3 mmole. ( 3 . 6 moles per mole sugar.) C. Formaldehyde None. XII. Perlodate Oxidation of Mannose Monomethyl,B. F r a c t i o n Monomethyl B ( 1 6 . 2 mgm., 0.08ij . mmole.) was treated with sodium metaperiodate (62 .8 mgm., 0 . 2 9 4 mmole.) exactly as before, the following results being obtained. A. Perlodate Consumption Time (minutes) $ 10 l£ 30 60 90 120 Consumption (mmoles.) 0 . 0 3 0 . 1 3 0„l£ 0 . 1 9 0 . 2 3 0.26 0 . 2 6 Consumption (moles per mole) O .36 \ . $ $ 1 .79 2.26 2 „ 7 4 3=09 3 . 0 9 B* Formic Acid 0.2lj.2 mmole. ( 2 . 8 8 moles per mole sugar.) C. Formaldehyde None. The values obtained i n XI and XII ;are compared with the t h e o r e t i c a l values f o r the monomethyl mannoses in. Table VIII. Table VIII Theoretical and Obtained Results of Perlodate Oxidation of Monomethyl Mannoses Monomethyl Moles Perlodate Moles: Formic Moles Formaldehyde Mannose Consumed Acid Produced Produced 2- 0-methyl 3-0 2.0 1.0 3- 0-methyl 3.0 2.0 1.0 ij.-0-methyl 3.0 2.0 1.0 £-0-methyl 3-0 3.0 0.0. 6-0-methyl i{..0 If. 0 0.0 Monomethyl A 3.7 3.6 0.0 Monomethyl B 3°1 2.9 0.0 XIII. Preparation of Derivatives of Monomethyls A and B Treatment of the above sugars with phenylhydrazine by the method of H a m i l t o n ^ yielded c r y s t a l l i n e phenylosazones. Their melting points are- co mpared below with those of the corre spending methyl glucose phenylosazones which are id en- -• t i c a l i n structure with the 6-0-methyl- and £-0-methyl-D-mannose phenylosazones„ 31 Qsazone Monomethyl A 6 -0 -me thy 1 -D-g l u co s e Monomethyl B 5~ p_-rae thy 1-D-glue ose XIV. Large Scale Methylation M.p. l 8 l - i 8 5°c. 181 |-187°G 0 (11) l26 - 1 3 0°Go 120°C o (15) of L-ArablnOse D i e t h y l Mercaptal L-Arabinose d i e t h y l mercaptal (3.0 gm.) was methyl-ated by the same procedure as f o r the D-glucose and D-mannose mercaptals, y i e l d i n g a syrup (2.757 gm.) which was crudely fractionated as before. Hydrolysis of the mercaptal f r a c t i o n yielded a syrup (0.863 gm.) which when chromatographed i n n-butanol-ethanol-water-ammonia (liO :10 :li9:1) gave no spots with iodine vapour and seven spots with p-anisidine hydro-chloride of R G's 0.18, 0.22, 0.25, 0.33, 0.37, 0.39 and 0.1x5. The arabinose mercaptal f r a c t i o n hydrolysate' was streaked on the st a r t i n g l i n e s of sheets of Whatman No. 3 MM paper which had been pre-run i n n-butanol-ethanol-water-ammonia. The load of material was approximately 3 mgm. per cm. of paper width. The st r i p s were developed f o r l 5 hours using the same solvent. Marker strips. 1.5 cm. wide were then cut from the edges of the dried developed chromatograms. Detection of these with p-anisidine hydrochloride located the zones containing products. The products were recovered by Soxhlet extraction of the separated zones using $% aqueous acetone. Rotations, methoxyls and Rp values were determined as before and are given i n Table IX. 32 Table IX Arabinose Fractions Separated on  Whatman Mb. 3MM Paper Wt. C las s i n e at Ion (mm.) [oc} 20 D r £ . 6 13.4 1.8. $ 9 1 4 147.9 243 »2 + 1 0 6 °(H 2 0 ) +^2.5'°(H20) +37°(H20) +£8°(H 2 0) +77°(H20) + 0°(MeOH) Rp(MEK) % OCH 0.09 - ~ 0.16, 18.5 0.23 16.6 ca0 .4 l (Trails.) 33.9 caO.^ .O ( T r a i l s ) 36.1 0.80 34o4 3 Monomethyl A.' Monomethyl B Monomethyl C Dimethyl A Dimethyl B Dimethyl C Calculated methoxyl values f o r mono- and dimethyl pentoses respectively are 18.9 and 3 4 ° 8 $ . C l a s s i f i c a t i o n , of thi s product i s based on Rp only. A l l the above f r a c t i o n s were found to be chromato-graphic a l l y pure except Dimethyl B which was an overlap f r a c t i o n of two components which f a i l e d to separate. The monomethyl fr a c t i o n s were*not Investigated further since pre-sent i n only n e g l i g i b l e amounts. 33 BIBLIOGRAPHY 1. Barker, S.A., Bourne, E.J. and Whiff en,, D.H. J . Chem. Soc. 3 8 6 5 . 1 9 5 2 . 2 . Clark, E.P. Semimicro Quantitative Organic Analysis. Academic Press Inc., New York, 191+3. 3 . Dutton, G.G.S. Personal Communication. It. Fischer, E. Ber., 2 7 : 6 7 3 . I89I+. 5 . Greeriway, R.M., Kent, P.W. and Whitehouse> ,M.W. Research (London) 6 , Suppl. No. 1 : 6 5 . 1 9 5 3 . 6 . Hamilton, R.H. J . Am. Chem. Soc. 56:1+87. 1931+. 7. H e l f e r i c h , B. In Advances i n Carbohydrate Chemistry, 3 , ed. C.S. Hudson. Academic Press Inc., New York. 191+8. 8 . H i r s t , E.L. and Jones, -J.K.N. In Discussions of the Faraday Society 7, Chromatographic Analysis. .Gurney :and Jackson> London. 191+9 = 9 . Jackson, E.L. In Organic Reactions, 2 , ed. R. Adams.. John Wiley and Sons-, Inc., New York. 191+2+. 1 0 . Levene, P.A. and Meyer, G.M. J. B i o l . Chem. 7l+:695. 1 9 2 7 . 1 1 . Levene, P.A. and Raymond, A.L. J . B i o l . Chem, 9 7 : 7 5 l . 1 9 3 2 . 1 2 . Lieser, T. and Leckzyck,, E. Ann. 5 1 1 : 1 3 7 . 1 9 3 4 . 1 3 . Martin, A.J.P. and Synge, R.L.M. Biochem. J . 3 5 : 1 3 5 8 . 191+1. ll+. Mitts, E. and Hixori> R.M. J. Am. Chem, S,oe. 66:1+83. 191+1+. 1 5 . P e r c i v a l , E.E. and Pe r c i v a l , E.G.V. J. Chem. Soc. 1 3 9 8 . 1 9 3 5 . 1 6 . Purdie, T. and Irvine, J„C. J. Chem. Soc. 8 3 : 1 0 2 1 . 1 9 0 3 . 17 . Smith, F. J, Chem. Soc, 7 5 3 . 1939. 1 8 . Sugihara, J,M. In Advances In Carbohydrate Chemistry, 8 , ed. C„S. Hudson and M.L. Wolfrom, Academic Press Inc., New York.- 1 9 5 3 . 19. Wolfrom, ,M.L.<, and Binkley, W.W,. Chromatography' of Sugars arid Related Substances. Sugar Research Foundation, Inc., New York. 191+8. 20, ZInner, H. Angew. Chem. 69s238,. 1957. APPENDIX A 35 Diagram of Chrptnatographed. Products of Methylations i n M e t h y l I o d i d e S t a r t Unlaiown Polar Products Mercaotals Solvent Front APPENDIX B Diagram o f CQiromator-raphed H y d r o l y s a t e s from M e t h y l a t i o n 3 i n Methy l I o d i d e . S t a r t Unchanged Sugars Monomethyl Sugars i-'olymethyl So lvent F r o n t APPENDIX C Diagram of Chromatographed Products of Methylations i n Tet rahydro furan Start Unlmown Pola Products Mercaptals Solvent Front This chows products at 50 only, APPENDIX D DiaF.ran of Chroiaatoprraphca HjdroI"satos from Methyl.itions in Tet rahydrofuran D-Glucose D-Mannose D-Galactose L-Arabinose 0 0 0 0 0 0 0 0 0 o 0 o 0 0 Start Unchanged Sugars Monometh3rl Sugars Polymethyl Sugars 0 Solvent Front This shows products at 50 only. APPENDIX E Infra-Red Spectra of Diethyl Mercaptals APPENDIX A Diagram of Chromatographed Products of Methylations i n Methyl Iodide 0 0 0 0 0 A o 0 0 0 0 o 0 0 0 0 o o 0 0 Start Unknown P o l a r Products Mercaptals. Solvent Front APPENDIX B Diagram, of Chromatographed Hydrolysates from Methylations i n Methyl Iodide o 0 0 0 0 0 Start Unchanged Sugars Monomethyl Sugars Polymethyl Sugars 0 0 Solvent Front APPENDIX C Diagram of OhroEatographed Productg of Methylations i n * Tetrahydrofuran Start Unknown P o l a r Products Mercaptals Solvent Front This shows products at 50 only* APPENDIX D Diagram of Chro mat o graphed Hydrolysates from Methylations i n Tetrahydrofuran Start Unchanged Sugars Monomethyl Sugars Polymethyl Sugars Solvent Front This etiows products at 50 only. APPENDIX E  Infra-Red Spectra of Diethyl Mercaptals D-G-lucose D-Galactose D-Mannose L-Arabinose 3 2 0 0 2400 1 9 0 0 1 7 0 0 Wave numbers (cm~^) The i n f r a - r e d absorption was measured i n a Perkin-Elmer Recording Spectrophotometer using pressed potassium bromide pellets'! 

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