CARBOHYDRATE SULPHATES By E. G. V. PERCIVAL D.Sc. PH.D. F.R.I.C. (READER IN CHEMISTRY UNIVERSITY OF EDINBURGH) IT is not always realised how widespread is the occurrence of carbohydrate sulphates in Nature. In the plant kingdom many algal polysaccharides belong to this group the red seaweeds Chondrus crispus and Gigartina steZhta contain the carragheen polysaccharides Dilsea edulis and Iridez Zaminarioides contain galactan sulphates and the important polysaccharide agar-agar which occurs in many species of Bhdophycea is either a member or a close relative. In the still more common brown seaweeds (Pkophycea?) the polysaccharide sulphate fucoidin is found. Still greater interest is attached to the animal carbohydrate sulphates which include the chondroitin sulphate of cartilaginous tissue the mucoitin sulphate of the gastric mucosa which is probably identical with the hyalu- ronic acid sulphate of the cornea and the blood-anticoagulant heparin.The jelly coat of sea-urchin eggs contains polysaccharide sulphates,l and galactose sulphates have been reported in brain lipids. The functions of the compounds under review are not always well understood ; in the red algB the polygalactose sulphates usually make up the major part of the plant but this is not so with the fucoidin of the brown seaweeds where the principal polysaccharide is alginic acid and considerable amounts of laminarin and mannitol occur at the proper seasons. In the animal group the main purpose of heparin is clear and chon- droitin sulphate is believed to act as a cementing material in connective tissue probably as a gel-like network in association with protein.2 The animal carbohydrate sulphates in loose combination as salts with protein^,^ as in the mucins derived from mucoitin sulphate form viscous slimy layers with lubricating and protective properties ; furthermore by an inhibiting action on the action of trypsin and pepsin autodigestion of the gastric mucosa is prevented.Quite apart from their complexity as carbohydrates it is not surprising that our knowledge of the constitution of the natural carbohydrate sul- phates is scanty when one considers their distinctive physical properties such as solubility in water to form viscous solutions in some cases and gels in others. They also occur as mixtures of salts of several different metals and especially in the animal group are difficult to separate from proteins.The only means of purification are usually dialysis or electro- dialysis and fractional precipitation. Progress has been delayed too by an inadequate knowledge of the properties and reactions of simple mono- saccharide sulphat es. E. Vasseur Acta Chem. Scad. 1948 2 900. a G. Blix Biochem. J . 1946 40 vi. =Karl Meyer Cold Spring8 H w b w Symp. &w;cnt. BWl. 1938 6 91. 369 370 QUARTERLY REVIEWS Monosaccharide Sulphates Monosaccharide sulphates are usually prepared by treating the sugar derivative dissolved in pyridine with chlorosulphonic acid,4 sulphuryl ~hloride,~ or preferably,s with the pyridine-sulphur trioxide complex.' By choosing appropriate derivatives it is possible to prepare sulphates usually as the barium or brucine salts with this group in a definite posi- tion in a particular sugar molecule and to compare the properties of such products with those of the sulphates prepared directly from the parent sugar.Thus H. Ohle by the partial hydrolysis of 1 2-5 6-diisopropylidene glucose-3 sulphate obtained 1 2-isopropylidene glucofuranose-3 sulphate which differed from the monosulphate obtained by the direct sulphation of 1 2-isopropylidene glucofuranose. Removal of the isopropylidene group from the last -mentioned substance gave the glucose monosulphate which is obtained directly from glucose ; it is inferred therefore that the hydroxyl group on C(s) was sulphated in the last two preparations. On the other hand galactose-6 sulphate prepared through 1 2-3 4-diisopropylidene galactopyranose was shown to differ from the galactose sulphate prepared directly from galactose.Where more than one free hydroxyl group exists owing to the vigorous nature of the sulphating agents generally used and the difficulties of isolating crystalline derivatives the possibilities of the formation of mixtures of monosulphates and of polysulphates must be borne in mind so that it is necessary to use the direct method with caution. In any event the synthetic method of attack is only of limited application and other methods have been sought in attempts to answer the outstanding question of the location of the sulphate groups. It has been claimed lo that hydrolysis by acids may be used to distin- guish poly- from mono-sulphates since the former are hydrolysed more rapidly but differences in the rate of hydrolysis of selected monosaccharide sulphates with acid reagents 59 9 9 10 have been shown to be too small t o act as a guide More useful although not by any means always decisive is a study of the behaviour of the unknown sulphate under alkaline conditions.The Eydrolysis of Carbohydrate Sulphates with AlkaU.-A salt such as sodium methyl sulphate is relatively stable to hot aqueous alkali so that the observation 9 that diisopropylidene galactose 6-sulphate was not attacked by sodium hydroxide solution ( 2 ~ . ) during 6 hours at 100' is hardly sur- prising. On the other hand the glucose and galactose sulphates prepared directly from the free sugars yielded all the sulphate in ionisable form within five minutes at 100" in N/lO-sodium hydroxide but since disruption of the monosaccharide residues occurred this observation is of no diagnostic value.When a-methylglucopyranoside sulphate was examined heating 4C. Neuberg and L. Liebermarm Biochem. Z . 1921 121 326. 5 P. A. Levene and G. M. Meyer J . BioZ. Chem. 1922 53 437. 6 R. B. Duff J. 1949 1597. 8 Biochem. Z . 1922 131 601 ; 1923 136 428. * E. G. V. Percival and T. H. Soutar J. 1940 1475. S O T . Soda and W. Nagai J . Chem. SOC. Japan 1936 56 1268. P. Baumgarten Ber. 1926 59 1166 1977. PERCNAL CARBOHYDRATE SULPHATES 371 with alkali caused rapid hydrolysis of the sulphate group with the produc- tion of an anhydromethylhexoside and on further investigation l1 it was established that the sulphates of a- and p-methyl-glucopyranosides and -galactopyranosides and a-methylmannopyranoside gave the corresponding 3 6-anhydromethylhexosides.It was concluded therefore that unless the removal of the sulphate group can lead to the production of an anhydro- ring for which a suitably placed hydroxyl group is necessary hydrolysis of the sulphate group with alkali proceeds only slowly. E'urther evidence in support of this view was collected in the glucofuranose series.12 The hydrolysis of 1 2-isopropylidene glucofuranose-3 sulphate (I) with alkali was found to be exceedingly slow-in fact just as slow as for 1 2-5 6- diisopropylidene glucose-3 sulphate (12% hydrolysis in 47 hours at 100" in 2*8~-sodium hydroxide). On the other hand the corresponding 6-sulphate (11) was readily hydrolysed to give almost equal amounts of 1 2-isopropyl- H O-CMe 1 1 O-CMe (I.) m.) (II 1 idene 3 6-anhydroglucofuranose (111) and 1 2-isopropyfidene gluco- furanose (IV).Leaving aside for the moment the fact that the primary alcoholic group in (I) is apparently suitably placed for 3 6-anhydride formation (3 6- anhydro-derivatives have been isolated from methylglucofuranoside-3 sul- phates l2) it is necessary here to draw attention to the close parallel between the behaviour of sulphates and of such sulphonic esters as toluene-p-sul- phonates and methanesulphonates. Thus diisopropylidene galactopyranose 6-toluene-p-sulphonate diisopropylidene glucofuranose 3-toluene-p-sulphon- ate,l3 and 1 2-isopropylidene glucofuranose 3-toluene-p-sulphonate 1 4 are hydrolysed with comparative difficulty by alkali and no 3 6-anhydride is produced from the last substance. On the other hand methylhexoside 6-toluene-p-sulphonates are readily converted into the 3 6-anhydromethyl- hexosides,15 and a knowledge of the properties of these compounds was of the first importance in developing the subject under discussion.l1 Duff and Percival J . 1941 830. l3 J. W. H. Oldham and G. J. Robertson J . 1935 885. l* Ohle and H. Wilcke Ber. 1938 71 2316. l5 See S. Peat " Advances in Carbohydrate Chemistry " Academic Press New l2 Percival J . 1945 119. York 1946 vol. 2 p. 37. 372 QUARTERLY REVIEWS There is however a property of the sulphonic esters of even greater fundamental importance which is observed when the sulphonic ester group is adjacent to a free hydroxyl group and in trans-relationship to it. Treat- ment of such compounds with an alkaline reagent usually sodium methoxide causes an ethylene oxide ring to appear between the carbon atoms concerned with Walden inversion on the atom which carried the sulphonic ester residue.To give but one of many examples 3 4 6-triacetyl /&methyl- glucoside 2-toluene-p-sulphonate (V) on treatment with sodium methoxide CH,*OAc CH,*OH C H,-OMe {-'FMeNaOMe OAc H '4TgMe A920 Me1 $kqMe AcO H HO H Me0 6 .I H H OTs H H H H (n) (md (rn) JNaOMe C H,.O Me I C H . 0 Me Me0 OMe H + gives 2 3-anhydro-~-methylmannopyranoside (VI) 17 The dimethyl derivative (VII) of (VI) by the further action of sodium methoxide gives 2 4 6-trimethyl /?-methylglucoside (VIII) and 3 4 6-trimethyl ,&methyl- altroside (IX) in practically equal am0unts.l' At the point of entry of the new methyl groups Walden inversion takes place and the formation of two products shows that the ethylene oxide ring may be broken in either of the two ways indicated by the dotted line in (VII).It should CH,-OH $H,*OH 4-F. H OH (X) ( X I ) CUI) be pointed out however that two products cannot invariably be recog- nised in such a reaction the chances of the ethylene oxide ring breaking in a particular way depending presumably on steric factors. If sodium hydroxide instead of sodium methoxide acts on (VII) a mixture of 4 6- dimethyl p-methyl-glucoside and -altroside is produced. When the sul- phonic ester group is cis- to the neighbouring hydroxyl group no anhydride l6 W. N. Raworth E. L. Hirst and L. Panizzon J . 1934 154. 17 W. H. G. Lake and Peat J. 1938 1417. PERCIVAL CARBOHYDRATE SULPHATES 373 formation takes place and hydrolysis of the ester is comparatively difficult.Thus 4-methanesulphonyl b-methylgalactoside (X) is unaffected by sodium methoxide under conditions where the corresponding glucoside (XI) is readily converted into 3 4-anhydro-~-methylgalactoside (XII).18 With such examples in mind it became of obvious interest to determine whether carbohydrate sulphates could also take part in transformations cm cxm (xp) involving the intermediate formation of ethylene oxide rings especially as the interconversion of sugars in Nature might in aome cases depend on such processes. A superficial examination of the examples of the hydrolysis of sulphates already cited would appear to indicate that 3 6-anhydro-rings but not ethylene oxide rings were formed. There is evidence however that the formation of a 3 6-anhydride is in some cases preceded by the production N a0 Me 9 0' of an ethylene oxide derivative.Thus E. Seebeck A. Meyer and T. Reich- stein l9 showed that 1 2-isopropylidene 5 6-anhydroglucofuranose (XIV) prepared from the corresponding 6-toluene-p-sulphonate (XIII) under- went a transformation into the more stable 3 6-anhydride (XV) even on storage in a desiccator and that contrary to an earlier claim,20 no L-idose derivatives could be obtained on the fission of (XIV) with alkaline reagents. Clearly therefore the observation that alkaline hydrolysis of the corresponding sulphate gave (XV) could be interpreted in a similar fashion. In order to arrive at a decision barium 3-methyl 1 2-isopropyl- idene glucofuranose-6 sulphate (XVI) was prepared. The inclusion of a methyl residue a t C(3) not only prevented the formation of a 3 6-anhydride but also conferred sufficient solubility in methanol to permit the use of l8 A.Miiller M. Moricz and. G. Verner Ber. 1939 72 746. ID Helv. C h h . Acta 1944 27 1142. 2o Ohle and L. von Vaxgb Ber. 1929 62 2435. 374 QUARTERLY REVIEWS sodium methoxide as a hydrolytic agent; normally the barium salts of sulphate esters are soluble only in aqueous media. Treatment with sodium methoxide at 40" gave 3-methyl 1 2-isopropylidene 5 6-anhydrogluco- furanose (XVII) (50% yield) and by more vigorous treatment 3 6-dimethyl 1 2-isopropylidene glucofuranose (XVIII) was obtained. The production of (XVIII) is explained by the fact that the entering methoxyl anion attaches itself apparently exclusively to the primary carbon atom in an ethylene oxide derivative of this type.22 Although the experiment outlined proves beyond doubt that the hydro- lysis of a sulphate ester can give an ethylene oxide derivative because the sulphate group was not attached to an asymmetric centre and the fission of the ethylene oxide ring takes place in only one direction the Walden inversion so characteristic of the hydrolysis of sulphonic esters could not be observed.An attempt to settle this point by the hydrolysis of 6-methyl @-rnethylgalactopyranoside-Z sulphate 21 failed because of extensive decom- position with the production of reducing substances on treatment with sodium methoxide the glycosidic methoxyl group apparently becoming labile to alkali when a sulphate group is adjacent. By taking advantage of the well-known stability to alkali of 1 6-anhydrides this difficulty has been overcome and the analogy between the sulphate and sulphonic esters is now complete.1 6-Anhydro-~-~-galactopyranose-2 sulphate (XIX) was converted by means of sodium methoxide into the known 1 6-2 3-dianhydro-p-~-talopyranose (XX). CH,-O H *so (xnc.) (XXS The failure to isolate an anhydro-derivative from 1 2-isopropylidene glucofuranose-3 sulphate (I) mentioned on page 370 can now be explained to some extent. There is no hydroxyl group available for the production of an ethylene oxide derivative but one occurs in the most nearly compar- able case of the methylglucofuranoside-3 sulphate (ap-mixture) which was shown l2 to give 3 6-anhydromethylglucofuranosides together with methyl- glucofuranosides on hydrolysis with alkali.The toluene-p-sulphonates furnish a similar pair of examples,*4 for 3-toluene-p-sulphonyl 1 2-480- propylidene glucofuranose gives no anhydro-derivative with alkali whereas 5 6-dibenzoyl 3-toluene-p-sulphonyl 2-acetyl ,!I-methylglucofuranoside (XXI) gives 5 6-dibenzoyl 2 3-anhydro-~-methylallofuranoside (XXII) on treatment with alkali in aqueous acetone and this with boiling sodium hydroxide ( 2 ~ . ) produces 3 6-anhydro-~-methylgalactofuranoside (XXIII). 2 1 Duff and Percival J. 1947 1675. 22 Ohle and K. Tsssmar Ber. 1938 71 1843. PERCIVAL CARBOHYDRATE SULPHATES 375 It will be clear from the above that the isolation of a particular 3 6- anhydro-derivative by the hydrolysis of an ethereal sulphate does not of itself establish at once the position of the original sulphate group and it ___ H OAc H OH (Xxf.1 c!cxII) is not always an easy task to identify small quantities of the isomers which may have been formed by the fission of any ethylene oxide rings produced a t an intermediate stage.It is possible to say however that if the sul- phate group is readily hydrolysed with alkali then an adjacent trans- hydroxyl group is present or that the sulphate group is on (&) (in a hexose) and that a free hydroxyl group is present at C(5) or C(,) or both. If the sulphate group is stable to alkali then none of these conditions applies and by taking into account also the results of methylation experiments etc. it is sometimes possible to deduce the position of the sulphate group in a polysaccharide ethereal sulphate. Natural Carbohydrate Sulphates The representation of an unknown substance as a sulphuric ester may be deduced from the fact that while no sulphate ions can be detected in solution until after hydrolysis cations such as calcium are detectable e.g.by precipitation with ammonium oxalate. Another important check 23 is the fact that the amount of sulphate estimated in the whole polysaccharide either by hydrolysis or by fusion with sodium peroxide is double the amount found in the ash e.g. 2RO*SO,K -+ K,SO + SO + products of combus- tion. In practice this 2 1 ratio is seldom realised because of the reduction of sulphate to sulphide during the ashing process. Although the complete structure of a natural polysaccharide sulphate has in no case been worked out fully some progress has been recorded in a few instances.Some of the difficulties involved in the isolation and purification of these sulphates have been mentioned already. For experi- mental purposes they are only stable as salts since the free acids are rela- tively strong acids. Since unlike alginic acid the acids are soluble in water there is no simple method of isolating the free acid apart from dialysis against mineral acids or of converting them from the naturally- occurring mixtures of salts into the salts of a single cation. None of the salts is insoluble so the facile double decomposition reactions like the conversion of sodium into calcium alginate cannot be applied as a means of isolation and purification. A further handicap in structural investiga- tions lies in the difficulty of acetylation and of methylation-the former because of the presence of an inorganic ion attached to the sulphate group 23 P.Haas Biochem. J. 1921 15 469. 376 QUARTERLY REVIEWS making the resulting salt difficult to disperse in pyridine and the latter for two reasons first the methylated derivatives are soluble in water and insoluble in organic solvents so that dialysis is necessary for purifica- tion at each stage and secondly the presence of the sulphate groups hinders the methylation process itself. If it were possible to remove the sulphate groups without affecting the glycosidic links an examination of the products of hydrolysis of the methylated desulphated polysaccharide in comparison with those from the original methylated substance would fix the positions of the sulphate groups. Unfortunately the sulphate groups cannot be removed by alkaline hydro- lysis under ordinary conditions and acid hydrolysis splits the polymeric links.The ideal process would be the use of enzymes but apart from T. Soda’s alleged glucosulphatase 24 isolated from Charonia lampas little work has been done in this field; preliminary experiments in Edinburgh with certain molluscs show however that some hydrolysis of sulphate groups can be induced. Polysaccharide Sulphates of Marine Algae.-(a) Carragheenin.-The material extracted by water from the red seaweeds Chondrus crispus and Gigartim stelkta (Irish moss carragheen) of some importance as a thicken- ing and emulsifying agent and as an article of diet has been the subject of chemical studies for at least eighty years. F. A. Fluckiger 25 obtained mucic acid by oxidation with nitric acid and B.Tollens et aZ.26 confirmed the pres- ence of galactose. At various times the presence of fru~tose,~’ pentoses (or methylpentoses 28) and 2-ketogluconic acid has been rep0rted.2~ The work of Haas 3% 32 and of B. Russell-Wells 31 showed that two polysaccharides appeared to be present the product extracted from Chondrus crispus by cold water containing less calcium but more sodium and potassium than the hot extract. The significant observation was also made that the sul- phate residues were very stable to alkali. Nova Scotia chondrus extracts 33 were found to have a much higher potassium content and a 3 1 ratio of total to ash sulphate attributed to the presence of ammonium salts but by dialysis against appropriate solutions pure calcium and potassium salts were obtained which gave the correct 2 1 ratio.In none of the above investigations was any progress made towards deciding the constitution of the polysaccharides concerned but a step in this direction was made by T. Dillon and P. O’Colla 34 who by treating carragheenin with acetic anhydride and sulphuryl chloride isolated an acetylated galactan devoid of sulphate residues although considerable There is also another difficulty. 24 Soda et al. Bull. Chem. Soc. Japan 1931 6 258 ; 1933 8 148; 1934 9 83. 25 L c Repertorium of Pharmacie ” 1868 p. 350. 26 J. Hadecke R. W. Bauer and B. Tollens AnnuZen 1887 238 302. 27 F. Bente Bey. 1875 8 416. 28 A. Muther and B. Tollens ibid. 1904 37 302. 29 E. G. Young and F. A. H. Rice J . Biol. Chem. 1946 164 35. 30 Biochem. J . 1921 15 469. 31 B. Russell-Wells ibid.1922 16 578. asM. R. Butler ibid. 1934 28 759. Haas and Russell-Wells ibid. 1929 23 426. 3 4 Nature 1940 145 749. PERCIVAL CARBOHYDRATE SULPHATES 377 degradation occurred. Workers in Edinburgh 35 studying the hot- and cold-water extracts of ChoncErus crispw demonstrated that the polysac- charides were essentially identical although differing in their mineral con- stituents and that the principal sugar unit was D-galactose.* Both extracts could be methylated with sodium hydroxide and methyl sulphate without this loss of sulphate residues and 2 6-dimethyl and 2-methyl galactose were recognised as the principal products of hydrolysis of the methylated polysaccharides. The red alga Qigartiruz stellata which closely resembles Chndrus crisps waa also in~estigated.~~ The material extracted with hot water (ash 17.5 ; Cay 3.7 ; Mg 1.0 ; SO4 23.9%) gave on methyla- tion a product (ash 18.2; OMe 18.6; Ca 3-8; Mg 0-9; SO, 2407%) from which on hydrolysis a good yield of crystalline 2 6-dimethyl galac- tose was obtained.Assuming the sulphate groups to be linked directly to the galactose residues it is possible to decide how these are arranged in the polysaccharide which is considered to be fundamentally the same from both sources. Clearly the hydroxyl groups on C(2) and C(s) are free which leaves four possible structures Quite apart from the improbability that the units occur in the furanose form in the polysaccharide a conclusion based on the rate of hydrolysis by acids ( A ) is the only possible formulation (B) on treatment with alkali would lose the sulphate group to form a 2 3- and probably also a 3 6-anhydride (C) would give a 5 6- and (D) a 2 3- and 3 6-anhydride.In all these cases the sulphate group would be easily removed in sharp distinction to the facts. On the other hand ( A ) could not give rise either to a 3 6-anhydride or to an ethylene oxide ring and would be expected to resist alkaline hydrolysis. For these reasons it was concluded 3 5 9 36 that s5 J. Buchanan E. E. Percival and E. G. V. Percival J . 1943 51. 36 E. T. Dswar and Percival J . 1947 1622. * Recently by chromatographic separation on a cellulose column methyla,ted L-galactose derivatives have been isolated from the hydrolysis products of certain methylated carragheenin fractions. 37 378 QUARTERLY REVIEWS the galactose units in carragheenin are galactopyranose residues linked through the 1 and 3 positions and carrying the sulphate group on C(4).Confirmation of this view has been obtained 37 by the isolation of 2 4 6- trimethyl galactose from the hydrolysis of methylated partly-degraded specimens of carragheenin from which the sulphate residues had been removed and Dillon 3* has also supported the allocation of the 1 3-linkages. Much remains to be done before the full constitution of carragheenin is settled for the yield of galactose obtained on hydrolysis represents only about two-thirds of the organic matter present. Fructose has been stated to be a c o n s t i t ~ e n t ~ ~ ~ 28 and certainly ca. 20% of a constituent giving the colorimetric tests of a ketose can be detected.35* 36 The isolation of a crystalline derivative of 2-keto-~-gluconic acid has been recorded,29 but it is by no means certain that this product estimated to make up about 3% of the polysaccharide is present as such in the original material.Two possibilities exist one that carragheenin is a mixture of a poly- galactan sulphate and a labile polysaccharide or mixture of polysaccharides which has eluded characterisation so far and the other that the unidentified residues are present with galactose in the same polymeric structure. (b) The galactan sulphate of Dilsea edu2lis.-7. C. Barry and Dillon 39 isolated a galactan sulphuric ester from the red seaweed Dikea edulis by extraction with dilute acids. The sulphate content is much lower than for carragheenin and corresponds to one sulphate group in four or five galactose residues.A tentative formula (XXIV) has been advanced for the repeating unit from oxidation experiments with periodic acid only one galactose unit in five being attacked. The sulphate residue was found to be stable to alkali ..- I I CH,. OH I ' CH,. OH 3 - 1 L.. n and is therefore assigned to C(41 instead of to C(s) and 1 3-linkages pre- dominate in accordance with the evidence that most of the galactose residues are untouched by periodate. (c) Agar-agar.-There is some doubt whether agar-agar the important polysaccharide extracted from Gelidiam spp. and related algz can be regarded strictly as a sulphuric ester. Various estimates of the sulphur 37 R. Johnston and Percival unpublished. 38 Proc. Chern. Soc. 1949 34. 39 Proc. Roy. Irish Acad. 1945 50 349. 379 content of commercial agar give values approaching 2yo but specimens 4 0 prepared from Gracibria confervoides (S 0.43y0) Gelidium crimle ( S 0*47%) and Gelidium kctifolium 41 ( S 0.36y0) contain much less.There is a strong suspicion however that because agar contains 3 6-anhydro-~-galactose units in its molecule sulphate residues were present a t an earlier stage. The main structural feature of agar is a chain of /?-D-galactopyranose units linked through the 1 3-positions (XXV) since the chief hydrolysis PERCIVAL CARBOHYDRATE SULPHATES product of methylated agar was shown to be 2 4 6-trimethyl D-galac- tose. Subsequent investigations 43 showed that 3 6-anhydro-~-galactose residues were present also and this was thought a t the time to be related to the discovery that hepta-acetyl DL-galactose could be isolated on the acetolysis of agar.44 It was shown subseq~ently,~~ however that the acetolysis of 3 6-anhydro-/3-methyl-~-galactoyyranoside gave the same DL-galactose derivative and that the racemisation was due to a rearrange- ment made possible by the special symmetry of the galactose series.W. G. M. Jones and S. Peat 46 isolated 2 5-dimethyl 3 6-anhydro-~- galactonic acid from methylated agar which had been dialysed in acid solution remethylated and hydrolysed the production of the free acid being attributed to the formation of a free aldehyde by the opening of the pyranose ring in the 3 6-anhydro-~-galactose residue (a typical feature of 3 6-anhydrogalactopyranosides 4 7 followed by atmospheric oxidation. 2 5-Dimethyl 3 6-anhydro-~-galactonic acid was also isolated 48 by the acetolysis of methylated agar fdllowed by oxidation remethylation and hydrolysis of the mixture of disaccharide esters produced.It is certain therefore that the 3 6-anhydro-~-galactose residues are joined to the main chain through C(4). Jones and Peat 46 interpreted their results as showing that agar is made up of repeating units which are composed of a chain of nine D-galacto- pyranose units linked through 1 3-positions terminated by an L-galacto- pyranose residue linked through C(4) carrying a sulphate group on c(6) 40 Percival Nature 1944 154 673. 41V. C. Barry and T. Dillon Chem. and Ind. 1944 63 167. 42 Percival and 5. C. Somerville J . 1937 1615. 43 S. Hands and Peat Chem. and Ind. 1938 57 937 ; Nature 1938 142 797 ; Percival Somerville and I. A. Forbes ibid.p. 797 ; Percival and Forbes J . 1939 1844. 44 N. W. Pirie Biochem. J. 1936 30 369. 4 5 T. L. Cottrell and Percival J . 1942 749. 47 W. N. Haworth Jackson and F. Smith J . 1940 625. 48 Percival and T. G. H. Thomson J . 1942 750. Ibid. p. 225. 380 QUARTIoRLY REVIEWS (XXVI) from which the 3 6-anhydride is produced during methylation. This view has been contested 419 429 48 as an over-simplification of the problem and on the ground that the sulphur content of natural agar is too low to account for the yields of 3 6-anhydro-~-galactose derivatives actually isolated. It is possible though that the 3 6-anhydro-ring was formed at some stage in the elaboration of the polysaccharide by the alga. A speculation is made by Peat 46 as to the possible origin of the L-galactose residue linked through C(4).He points out that a D-galactopyranose residue (XXVII) linked through C(3) with a sulphate group on C(l) could by fol- lowing the sequence of events depicted below involving an oxidation- reduction process become transformed into an L-galactose residue (XXVIII) OH OH kH-OH C H.H SO ti H n ti OH lo OH fo H OH 1 (XxPcI) carrying the sulphate on It is also suggested that the sulphate group plays a part in the synthesis of agar in the same way as the phosphate group does for starch but evidence in support of either suggestion is likely to be difficult to obtain. H. Kylin 4B isolated from various common brown sea- weeds a soluble polysaccharide called fucoidin which gave a methylpentose on hydrolysis. This substance was shown to be a sulphuric ester by M. Bird and P.Haas 6* since the sulphate contained in the ash (15.1%) was half the total sulphate (30.3%). A similar polysaccharide was isolated from and linked through C(4). (a) Fucoidin. 2. physiol. Chem. 1913 83 171. 6o Biochem. J. 1931 25 403. PERCfVAL CARBOHYDRATE SULPHATES 381 Macrocystis pyrifera 51 and the principal building unit was identified as ~-fucose.6~ G. Lunde and his co-workers 53 prepared fucoidin from Lamin- aria digitah and estimated that 33-37% of the polysaccharide was L- fucose and that 35*5-37*7% of the sulphate was present in combination with sodium together with smaller quantities of calcium and magnesium. Since 20% of the molecule could not be accounted for the formula R,R'O*SO,*ONa was suggested where R = L-fucose R' = unknown. Several different sources of fucoidin have now been examined and it is now possible to account for 99% of the components of the poly~accharide.~~ One of the difficulties in the analysis of fucoidin is that the substance stubbornly retains water and ethanol but a specimen from Himanthalea Zorea after correction for these components gave fucose 56-7 ; galactose 4.1 ; uronic acid 3.3 ; xylose 1.5 ; sulphate 38.3 ; and metals (chiefly calcium) 8.2%.A calcium polyfucosan monosulphate (C,H,O,SCa,.,) would give fucose 66.9 ; sulphate 39.2 ; and Ca 8.2%. It is not possible to say at present whether the small quantities of carbohydrate material other than L-fucose are combined in a single polysaccharide but the broad pic- ture of fucoidin as a polyfucosan ethereal sulphate seems to be a good working hypothesis for future studies.The sulphate residues in fucoidin are very stable 1 4-linkage since with a sulphate group on 6CH3 to alkali; this excludes the possibility of a either C(a) or C(3) in a 1-substituted L-fucose HO (XXIX) there would be a tram-hydroxyl group in the reciprocal position. The polymeric link must therefore be on or C(3). Animal Carbohydrate Sulphates.-(a) Chondroitin sulphate. Nasal septa trachea aorta tendons sclera etc. contain chondroitin sulphate which is the only member of the animal group in which progress has been made towards determination of structure. Said to comprise some 40% of dried cartilage it is best extracted with calcium chloride,55 a method which gives less-degraded products than that used for the original isolation in 1861.56 P. A. Levene and F. B.LaForge 57 showed that equimolecular quantities of chondrosamine (2-amino-2-deoxy-~-ga~actose) acetic acid D-glucuronic acid and sulphuric acid were produced on hydrolysis and these ratios have been confirmed recently. 58 A degraded chondroitin sulphate of small mole- cular weight and devoid of sulphate has been examined by H. G. Bray J. E. Gregory and M. Stacey. 59 Evidence is presented based on methylation and hydrolysis that this product contains a terminal group of D-gluco- 44??... (-I OH H 61 D. R. Hoegland and L. L. Lieb J. Biol. Chem. 1915 23 287. 5 2 W. L. Nelson and L. H. Cretcher ibid. 1931 94 147. 53 G. Lunde E. Heen and E. Oy 2. physiob. Chem. 1937 247 189. 54 A. G. Ross Thesis Edinburgh 1949. 56Kar1 Meyer and E. M. Srnyth J. Biol. Chem. 1937 119 507. 66 C. Fischer and C.Boedeker Annulen 1861 117 111. ST J. Biol. Chem. 1914 18 237. 68 M. L. Wolfrom D. J. Weisblat J. V. Karsbinos W. H. McNeely and J. McLean J . rimer. Cltern. Soc. 1943 65 2077. 50 Biockm. J . 1944 88 142. 382 QUARTERLY REVIEWS pyruronic acid (isolated as the amide of 2 3 4-trimethyl a-methyl-D- glucuronoside) associated with doubly-linked glucuronic acid and acetyl chondrosamine residues (appearing as dimethyl derivatives). A branched- chain structure is suggested for chondroitin sulphate but no conclusions are drawn as to the position of the sulphate groups or the linkages involved. Kurt H. Meyer and his associatesYGo however propound a straight- chain structure. The relation between viscosity and molecular weight (27-33 x 103) as estimated by methods dependent on the presence of a free reducing group is held to indicate a straight-chain molecule containing about 120 monosaccharide residues and the specific rotation ([a]% - 31") is taken as evidence of @-linkages between them as in (XXX).The evidence for the 1 3-linkages and the allocation of the sulphate group to c@] in the acetyl galactosamine residue is briefly as follows. One macro-molecule (120 units) reduces only four periodic acid molecules so that large numbers of adjacent hydroxyl groups are excluded. By cautious hydrolysis it is claimed that half the sulphate groups can be re- moved without attacking more than 3% of the glycosidic links and the product obtained still requires only four molecules of periodate for complete oxidation. The sulphate group is therefore not adjacent to a free hydroxyl group and the possibility that a high proportion of the glucuronic acid residues are present as sulphated terminal groups is rendered remote.After methylation and hydrolysis followed by periodate oxidation ammonia is evolved almost quantitatively ; therefore a free hydroxyl is adjacent to the amino-group in the partly methylated chondrosamine which means that C(3! is either a main linking point or is blocked by a sulphate group in the mtrogenous unit. Furthermore after the same treatment one mole- cule of formaldehyde is liberated per disaccharide period which shows that c(6) is not methylated and that the methylated chondrosamine is the 4-methyl derivative. The consumption of periodate by the hydrolysed methylated chondroitin sulphate-three molecules per disaccharide period -is accounted for by the 4-methyl galactosamine so that the dimethyl glucuronic acid fragment uses no periodate from which it is concluded that it is 2 4-dimethyl glucuronic acid.The glycosides produced on methanolysis consumed one molecule of periodate per disaccharide period with a quantitative elimination of ammonia in agreement with this result. (XXX) requires that the sulphate residue should be stable to alkali since there is no possibility of anhydride formation ; this fits in with the observa- Kurt H. Meyer M. E. Odier and A E. Siegrist Helv. Chim. Acta 1948 31 1400. PERCIVAL CARBOHYDRATE SULPHATES 383 tion that repeated methylations with sodium hydroxide and methyl sulphate do not eliminate the grouping. Mucoitin sulphate occurs in gastric mucosa 61 and has also been isolated from ox cornea.62 The constituents are N-acetyl- glucosamine sulphuric acid and D-glucuronic acid in molecular propor - ti on^,^^ although it was only recently 63 that D-saccharic acid was obtained by the oxidative degradation of mucoitin sulphate to confirm the latter constituent.The arrangements of the building stones of the molecule are quite unknown a t present. It is of interest that the substance hyaluronic acid,g4 isolated from vitreous humour and present in umbilical cord synovial fluid etc. is thought to be mucoitin sulphate devoid of the sulphate residues. (c) Heparin. The blood-anticoagulant heparin first isolated in 1918 by W. H. Howell and E. Holt 6 5 from dog liver was early recognised to be carbohydrate in nature arid to contain nitrogen shown later 66 to reside in D-glucosamine.The presence of a uronic acid was inferred by the usual method and this was eventually proved to be D-glucuronic acid.67 That heparin was a polysulphate was suggested by E. Jorpes 68 in 1935 and A. F. Charles and D. A. Scott's crystalline barium salt 69 was shown to be a salt of a sulphuric ester. Charles and A. R. Todd 70 sub- mitted evidence that heparins from different sources e.g. lung and liver appeared to be analytically identical and also that conversion into the ammonium salt followed by reconversion into the barium derivative did not impair the physiological activity. It was also observed that periodic acid did not attack heparin and the substance was represented as a mucoitin sulphate containing five sulphuric ester residues in a tetra- saccharide unit (C2,H3,03,N2S5),Ba,,24H,0 with two carboxyl and two ncetamido-residues.Charles and Todd also observed a diminution of physiological activity with the progressive removal of sulphate groups. tJorpes,71 on the other hand considers heparin to he a mixture of poly- saccharides containing variable amounts of sulphate residues such as di- and tri-sulphuric esters of a glucuronic-glucosamine disaccharide unit of which the crystalline component is a relatively small fragment. In con- riexion with this view two components have been detected on electro- ph~resis.'~ According to &I L. Wolfrom 58 the crystalline barium acid heparinate is the same both analytically and biologically whether isolated from such varied sources as beef lung or dog pork or beef liver. The sulphate residues are all combined with barium but the carboxyl groups (b) Mucoitin sulphate.61 Levene and J. Lbpez-Suarez J . BioE. Chem. 1916 25 511. R 2 Karl Meyer and E. Chaffee ibid. 1941 138 491. 6 3 Wolfrom and Rice J . Amer. Chem. SOC. 1946 68 532. B4 Karl Meyer and J. W. Palmer Amer. .7. Ophthalmol. 1936 19 859. 65 Amer. J . Physiol. 1918 47 328. 66 E. Jorpes and 5. Bergstrom 2. physiol. Ghem. 1936 244 253. R7 Wolfrom and Rice J . Amer. Chem ~ o c . 1946 68 532. 68 Biochem. J . 1935 29 1817. Ibid. 1940 34 112. 71 Ibid. 1942 36 203. 7 2 Wolfrom and Rice J . Amer. Chena. SOC. 1947 69 2918. 69 Ibid. 1936 30 1927. c c 384 QUARTERLY REVIEWS are free. The claim is made that heparin contains no acetyl residues and that the glucosamine residues are joined to the glucuronic acid units of the polymer by >CH*NH*CH< links to the potential reducing groups of the latter.There is no direct evidence to support this unique type of linkage or to indicate any other structural features of the heparin molecule. The apparent connexion between '' heparin activity '' arid sulphate content 70 has led to a search for more accessible materials for physio- logical studies. An extract from Chondrzis crispus 73 has been shown to be about 40% as active as heparin but fucoidin is ina~tive.7~ Polysac- charide sulphates have been synthesised and heparin activity demonstrated in many of them although no product of practical value has been reported upon until recently since most of the synthetic products have the dis- advantage of high toxicity not possessed by heparin. Thus sulphated agar,75 cellulose starch and glycogen sulphates 76 have been prepared.A new clot-inhibitor " Paritol," has been reported 77 which is presumably an alginic acid sulphate and resembles heparin in containing carboxyl and sulphate groups but differs from it in the absence of glucosamine residues. This product is claimed to be no more toxic than heparin and since its physiological action lasts twice as long may be substituted for heparin for short -term administration to human beings although clinical test!s are not complete. 7 3 ,4. R. Todd private communication. 7 4 A. F. Charles private communication. 75 C. Neuberg and C. H. Schweitzer Monatsh. 1937 71 46. 7 6 E. Chargaff F. W. Bancroft and M. Stanley-Brown J. Bid. Chem. 1936 115 155; Bergstrom 2. physiol. Chem. 1936 238 163; P. Karrer H. Koenig and E. Usteri Helv. Chiin. Acta 1943 ; H. Gebauer-Fulnegg and 0. Dingler J. Amer. Chena. SOC. 1930 52 2849 ; W. Traube. B. Blaser and E. Lindemann Ber. 1932 65 603. 77 Chem. and Eng. News 2949 27 2162.