chem rev vol 1 041_71

7/28/2019 Chem rev VOL 1 041_71

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THE CONSTITUTION O F POLYSACCHARIDES’

JAMES COLQUHOUN IRVINE

Un i v e r s i t y of St. Andrews

In the construction of this paper I have endeavored to sum-marize the results of researches which have extended over twenty-

one years so as to display the consecutive evidence upon which

the structural representation of polysaccharides can now bebased. This treatment appears necessary although no rapidsurvey can be made, no adequate synopsis can be given of themanifold problems of carbohydrate constitution which have

already been solved or which still await adequate expression.To discuss even in broad outline the structure of a single sugardemands the consideration of a bewildering series of reactionsand relationships, and to deal within the limits of a single paperwith all classes of carbohydrates is to court disaster.

My excuse in attempting what I believe to be the impossibleis tha t, t o me, the carbohydrates are among the most fascinatingsubstances which engage the attention of the chemist. To thestudent who brings to bear on our science the special appreciationwhich comes from tracing historical developments there must beabundant satisfaction in discerning how our knowledge of carbo-hydrates has expanded in response to the stimulus conveyed byfresh views on structure. The pioneer researches, and earlyexplorations of the fate of carbohydrates in the living organism,must not be passed over lightly; the experimental difficulties

encountered to-day serve but to remind us of the skill andpatience devoted to this work. Yet, go back rather more than 8

century and you will find the “old masters” struggling with thesugars and giving in a few lines of print all that was then knownregarding them. Bridge a gap of seventy years from their time

1An essay prepared in connection with t he dedication of the Sterling Chemistry

Laboratory, Yale University, New Haven, Conn.

41

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42 JAMES COLQUHOUN IRVINE

and you will find little in addition; a few more sugars had been

recognized and a greater variety of derivatives had been pre-pared, but, in the absence of structural views, the information

provided is purely descriptive and empirical. Then began thatbrilliant period of constitutional carbohydrate chemistry whichculminated in the great awakening we owe to Emil Fischer.

It was indeed a marked advance t6 be able to expand theformula C6&06 so as to display not only the significant groupsbut also their configuration, and thereby to account for theisomerism and mutual relationships of the simple hexoses.Almost inevitably this progressive phase was followed by a

resting stage and it is not surprising that , during the past twenty-years the study of sugars has shown alternations of activity andstagnation. The present is a period of revival, and in sympathywith this growing interest, one is tempted to include manyaspects of carbohydrate constitution, but, with the space at mydisposal, it seems prudent to confine myself almost exclusively toproblems which have engaged the attention of my co-workersand of my colleagues.

Although with the information now available an elaborate

classification of carbohydrates is possible, we may, in the interestsof simplicity, adhere to the present system and may divide theminto three groups:

a. Reducing sugars which are unaffected by hydrolysis.b. Glucosidic compounds which, on hydrolysis, yield at least

(Di- and n-saccharides may be regardedne reducing sugar.as special examples of this class.)

c. Polysaccharides.The above groups are inter-related and the constitutional

problems presented by glucosides, n-saccharides, and poly-saccharides are obviously dependent on the structure of thesimple reducing sugars. Lack of knowledge of the mono-saccharides is at once reflected in the complex saccharides.

In order to arrive at the fundamental aspects of carbohydratestructure we may consider the special case of glucose and tracethe stages through which our present views regarding the hexosemolecule have evolved. These stages are expressed in historical

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CONSTITUTION OF POLYSACCHARIDES 43

sequence in the scheme shown below, which displays the transi-

tion from an empirical formula to the butylene-oxide representa-tion of a- and p-glucose now in general use.

r - i

1 0

I1

Y O H 1 2

C6IlaOs 3 (CH0H)r + 1 3 ..ACHO

II*C.OH1

I 3

CFIO II

€I.c O B

HO C - HP0.C.H

H*C*OH

H*C*OH 5

6

H*C*OH1

I

CHsOH

CH,OHHyOH

I I1 IT1 I V

The cyclic formula for reducing sugars has proved of real serviceand its adoption has been justified, but it is necessary to pointout that , until recently, less than one half of the moleculararchitecture of glucose had been studied. When we rememberthat position 1 in the above formula plays a part in mutarotation,in the formation of glucosides, and in oxidation processes it willbe agreed that this particular alcohol group has placed no ob-stacles in the way of thorough investigation. The propertiesof the group indexed as position 2 are not so well-defined but arerevealed to a limited extent in the formation of osazones, whileour knowledge of the group in position 6 is restricted to a fewreactions such as oxidation to saccharic acid and the formation

of dibromo-derivatives. Speaking generally, however, it may beclaimed that the essential reactions of the three hydroxyl groupsspecified are already known.

On the other hand, the asymmetric systems indexed as 3, 4,

and 5 represent areas in the sugar molecule which remainedalmost entirely unexplored. There are good reasons for this gapin our knowledge, yet a proper understanding of the glucosemolecule cannot be obtained if, as is generally the case, attentionis focussed exclusively on the reducing group; each hydroxylgroup must be studied, for each possesses its own particularproperties and contributes to the reactions of the molecule as a

whole. This consideration leads directly to another point which

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44 J A M E S COLQUHOUN IRVINE

I believe to be of even greater importance. It is evident that thelinkage of the ring-forming oxygen atom in the molecule need notremain exclusively in one position but may connect different pairsof carbon atoms, and thus, all the groups from 1 t o 6 must be

regarded as variables. For example :

Position 1 may possess either the a- or 6- configurationPosition 2 may be involved in an ethylene-oxide ringPosition 3 may be involved in a propylene-oxide ringPosition 4 may be involved in a butylene-oxide ringPosition 5 may be involved in an amylene-oxide ringPosition 6 may be involved in a hexylene-oxide ring

It follows, therefore, that if we include an aldehydic variety,d-glucose may react in any one of eleven forms or as a mixtureof these isomerides. These complicated possibilities must betaken into account in speculating on the reactions of the so-called“simple” sugars, in tracing their origin, their metabolism, andtheir relationship to the di- and polysaccharides.

These are preliminary considerations and I turn now to anaccount of how the intimate structure of carbohydrates has beenadvanced through the agency of one particular method of enquiry,which, for reasons already given, was applied in the first instanceto the monosaccharides. I refer to the process of methylation,

a reaction which has been employed to introduce stable methylgroups into all the hydroxyl positions of reducing sugars or intoany hydroxyl groups which remain unsubstituted and exposedin a sugar derivative. Taking an example which has been very

fully studied, the sequence of typical operations leading to a fullymethylated glucose may be expressed by the following scheme:

f2HO H +HOMe f2HOMe F H O H

1 CHOH ~ CHOH I dHOMe 1 JHOMe

CHOMe0 1

I

1

0 1

I

I

CHOMe0 1

II

CHOH01

II

CHOH

I ,CH Gluccaide-4CH Methylationb H Hydrolysis+ I 1 LCH

CHOMe

CHzOMe

CHOMe

CHiOMe

formation

CHOHHOH

CHzOHHzOH

v VI VI1 VI11

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CONSTITUTION O F POLYSACCHARIDES 45

The first stage is the formation of methylglucoside, a reaction

which has the effect of protecting the reducing group, and thisis followed by the introduction of methyl groups into the remain-ing hydroxyl positions. It is important to note that acid hy-

drolysis of the methylated glucoside affects the molecule onlyin one position, the glucosidic alkyl group being eliminatedwhilst the remaining methyl groups survive. In the tetramethylglucose thus produced, four hydroxyl groups have been maskedand it is thus possible to study the properties of the reducinggroup in the absence of many complicating factors. Extendingthe above principles, it will be clear that if a sugar is sub-stituted by any group or groups capable of subsequent removalby hydrolysis, it is possible to methylate the unoccupied

hydroxyl positions and ultimately t o obtain definite partlymethylated sugars. In these products the position of the OHgroups coincides with the position in which the substitutinggroups were attached to the original sugar residue. For example,starting from benzylidene methylgucoside, only two hydroxylgroups are available for methylation and consequently, on com-pleting the series of reactions, the final product obtained is a

dimethyl glucose. Reference to the published papers will showthat the method is of wide application and that a large varietyof completely and partly methylated aldoses and ketoses havebeen prepared and examined.

In general, it may be said that alkylated sugars are extremely

suitable for exact and critical experimental study. In manycases the compounds crystallize readily in highly characteristicforms and, in addition, the sugars or their glucosides can be

effectivelypurified by distillation in a high vacuum. Moreover,the presence of the alkyl groups increases the solubility in organic

solvents enormously, and thus the reactions of the compoundscan be studied under a variety of conditions. Characterized byproperties such as I have described, alkylated sugars are well-adapted as reference compounds, in that they can be isolated andidentified with certainity, even when only small quantities areavailable. The same remark applies to the alkylated sugarswhich are liquids, as, in the majority of cases, they yield crystal-

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line anilides or glucosides upon which characteristic physical

constants may be determined accurately.I have referred to the opportunities afforded by alkylated sugars

to restrict the attack of reagents to a limited number of selected

hydroxyl groups, but a more obvious application of these com-pounds and one which is superficially of more importance liesin the use which has been made of them to solve the structure of

typical glucosides, disaccharides and polysaccharides. If weascribe to any sugar derivative the general formula S -- G,

where S is a sugar residue and G the group with which i t is con-densed, then methylation followed by hydrolysis will give as a

minimum two products, one of which will be a methylated sugar.Determination of the number and distribution of these methyl

groups in each of the cleavage products gives the completestructure of the parent compound, as the mode of attachment ofthe individual constituents is thereby revealed.

In the special case where the group G is also a sugar residue,the compound under consideration would be a disaccharide andthe general formula may be written S - I. Precisely the

same structural study can evidently be applied to it with thesingle difference that two methylated sugars will be obtained

on hydrolysis in place of one. The method is equally applicableto the general type S, which includes polysaccharides.

This exploitation of methylated sugars in solving constitution

was recognized at an early period in our wrork, but the policywas deliberately adopted of postponing such an extension untilthe standard xnethylated sugars required as reference compoundshad been examined and definite constitutions assigned to them.Once this was done, results have come quickly and I now proposeto refer to the conclusions which have been reached in a fewtypical cases selected from the classes into which carbohydratesare divided.

CONSTITUTION O F NATURAL GLUCOSIDES

As already explained, the application of the methylationmethod to a- and B-methylgulcoside results finally in the forma-tion of one of the five different forms in which a tetramethylglucose can exist. The sugar actually obtained is a crystalline

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CONSTITUTIOS OF POLYSACCHARIDES 47

pr , H

0 1

I IT O M e

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48 JAMES COLQUHOUN I R V I N E

yield the stable or butylene-oxide variety of tetramethyl glucose.This result may be translated into the statement that although

glucose can and does exist in a highly reactive form, this varietydoes not appear to play a permanent part in the formation ofglucosides in Nature. Reference will be made later to artificialglucosides derived from an unstable modification of glucose, butat this stage it may be stated that such compounds show littletendency, in the absence of hydrolytic conditions, t o revert to themore stable isomerides by alteration of the oxygen ring. Thischange seems in fact to be dependent on the presence of thereducing group, and the combined evidence indicates that it is

the stable form of glucose which is utilized by plants in theformation of natural glucosides. Some indications exist tha t theglucosidic components of plant pigments are not invariablyderived from a single variety of glucose, but our work in thisdirection is not sufficiently advanced t o permit of a final decisionbeing made.

CONSTITUTION OF DISACCHARIDES

Five methylated hexoses, functioning in the capacity of refer-

ence compounds, have served t o determine the essential con-stitution of the most important disaccharides and polysaccharides.

The hexoses in question are : tetramethyl glucose, two isomericforms of trimethyl glucose, tetramethyl yfructose, and tetra-methyl galactose.

+HOH rCHOH +HOH CHlOMe

01 01 0 1 f l z M e

0 CHOMeCHO M e 17CH LCH

I &OMe I LHOMe I AHOMe I

IY O M e

II kMeHOMe

CHOH

I

ICHOH

CH20Me

I

ICHOMe

CH20Me

2:3:5:6-Tetra- 2: 3 :B-Trimethyl- 2:3 : -Trimethyl Tetramethyl-

methyl glucose glucose glucose y-fructose

or galactose

IX X XI XI1

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Of these, 2 : : : -tetramethyl glucose has proved of greatest

service, and it is fortunate that the compound is the most readilyidentified of all methylated sugars.

In the experimental treatment of reducing disaccharides theprocedure adopted with monosaccharides is followed in that aglucosidic methyl group is introduced before the methylation ofthe sugar chain is carried out. By a simple but extremely

effective met,hod, these consecutive reactions are conducted inone operation and in this way a number of fully methylateddisaccharides have been obtained. The hydrolysis of thesecompounds has given results which, as in the examples already

quoted, are decisive in displaying the manner in which the hexosecomponents are united. As the principles involved have already

been explained the structural studies of typical disaccharides canbe readily synopsized.

Maltose

The essential steps required in this particular investigation areshown below :

2 :3:5 :6-Tetramethyl glucose. ............ X

2:3:5-Trimethyl glucose ................ XI

Heptamethy 1methylmaltoside

altose +

tilieing the structures for IX and XI, the coupling of the

Uucose residues in maltose is shown to be:

ICHOH

r - O iCHQ-O-CH*CHOH*CHOH*CH*CHOH.CH~OH

The formula thus established finds application, as will be de-scribed later, in deducing the constitution of the starch molecule.

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50 J A M E S C O L Q U E O U K IRVISE

Cellobiose

An entirely different node of combining two hexose residuesis displayed by cellobiose, as one glucose molecule is attachedthrough its reducing group to the 5 position of the second glucosemolecule. This is shown from the sequence of changes:

.. 1 f2 :3: 5:g-Tetrarnethvl elucose. ....... 1X

From these results it follows that cellobiose should be repre-sented by the structure:

F H O H

I /

I CHOH

01I CHOH XI11I 1

I r - O - 7

LCH

CH- 0-CH*CHOH*CHOH.CH.CHOH.CH20II

ICHzOH

Apparently the attachment of the hexose molecules depicted inthe above formula is characteristic only of natural carbohydrates,as the same coupling is present in cellulose, in starch, and inglycogen but not in synthetic dextrins. Meanwhile it is ofinterest to find that precisely the same condition exists in thedisaccharide lactose.

Lactose

The mutual relationships of the glucose and galactose residuesin lactose are at once evident from the experimental scheme:

2:3:5:6-Tetramethyl galactose.. ......... 1XHeptamethyl

................actose

methyllactoside 2:3:6-Trimethyl glucose.. .X

Considering the origin of lactose, it is important to find that thegalactose component belongs t o the stable type and that the

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reactive, or y-form, is definitely absent. This point was readily

established, as although tetramethyl galactose is a liquid, it wasidentified by conversion into the well-defined crystalline anilidein yields which show that no other isomeride was present.

Taking into consideration the mutual relationships of thecornpound sugars, the constitution of other reducing disaccharidesmay be deduced from the results already obtained experimentally,but meanwhile the complicated case presented by sucrose maybe discussed.

Sucrose

So far as two factors are concerned, research on sucrose provedto be comparatively simple. In the absence of a reducinggroup the actual methylation presented few difficulties andte trane thyl glucose, which is one of the scission products ob-

tained from octamethyl sucrose, crystallizes readily. Closerinspection of the practical details will, however, reveal some of theobstacles encountered.

2:3 :6:6-Tetramethyl glucose. ................. X

a Tetramethyl fructose.. ................... XI1

Octamethyl ~

Sucrose -+

sucrose

The fructose derivative was a liquid and its separation by dis-tillation proved to be tedious and imperfect. This was overcomeby replacing octamethyl sucrose by the corresponding hepta-methyl derivative, but another difficulty was then encountered,as the constitution of the tetramethyl fructose was unknown.The compound was moreover abnormal, being dextro- in placeof laevo-rotatory, and although earlier investigations had alreadyrevealed this irregularity, prolonged research has been necessary

in order to ascribe a forlzlula to the reference sugar.It is not known that the compound belongs to the type generally

known as “7-sugars,” and I hope to refer to these curious iso-merides at a later stage. Meanwhile, it may be stated that the

normal laevo-rotatory form of fructose has been definitely provedto be a butylene-oxide, and that in the tetramethyl fructose now

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52 JAIVES COLQUHOUN IRVINE

under consideration the internal oxygen ring is displaced to oneof the alternative positions. The first suggestion that anethylene-oxide ring is present, is no longer tenable, and a formercolleague provides experimental evidence in favor of an amylene-

oxide structure. According to this view the formula for sucrosebecomes :

CHiOH

H-O-C----q

I

ICHOH CHOH 1CHOH 0

r;:0 1

I CHOH

LCH I XIVCHOH

CHa II L ACHOH

CHzOH

It may be mentioned that both the dimethyl and the trimethylfructose obtained from inulin are likewise y-sugars, and containthe same internal ring which is present in the ketose fragment of

sucrose. The examination of these sugars, which is in progress,will throw further light on the structure of sucrose.

From what has been said, it will be admitted that a new

complication has been introduced into studies on the inversionof sucrose, as the initial condition of one of the hydrolysis productsis transient. The disaccharide thus furnishes an interesting

case where methylation reveals a feature of molecular structurewhich other processes fail to detect.

POLYSACCHARIDES

Although doubtless any reducing sugar is capable of yieldinga corresponding polysaccharide, authentic examples of the latterclass of compound are not numerous and, up to the present time,our attention has been restricted to the four most importantrepresentatives : cellulose, starch, glycogen and inulin. It is

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COKSTITUTIOK OF POLYSACCHARIDES 53

perhaps advisable to express the opinion that we are scarcely

justified in claiming that the names used above refer to individualcompounds and, strictly speaking, we should meanwhile regardthem as applicable to groups of close-related compounds.

Systematic study of the polysaccharides on constitutionallines is naturally more difficult than that of the related sugars.The absence of crystalline structure, the inconvenient solu-bilities involved, and the uncertainty regarding the uniformityof the material under examination are disadvantages, the fullmeasure of which quickly becomes apparent in the experimentaltreatment of the compounds. These obstacles have not been

allowed by chemists to stand in the way of investigating poly-saccharides, which have been examined with commendablepatience, yet it must be admitted that the reactions availablefor elucidating constitution are few in number and are difficultto interpret. Above all, the lack of precise information leadingeven approximately to the molecular weight of members of thegroup has proved a serious disadvantage. Two fundamentalpoints, however, have long been recognized. The polysaccharidesas a class possess the formula (C6HIO0,), nd are apparently

polymerides of definite molecular units. Although little infor-mation can be gleaned from an empirical formula of this nature,the situation becomes clearer when it is remembered that, oncomplete hydrolysis, each polysaccharide is converted into a

monosaccharide. Familiar examples are :

Cellulose ---+ Glucose

Starch - lucose

Glycogen- lucose

Inulinv

ructose

The evidence afforded by hydrolysis is important but, on occa-sions, may be utterly misleading, and consideration of oneexample will give an indication of the pit-falls which await theinvestigator in this field. The fact that inulin is converted bythe action of dilute acids into the ordinary laevo-rotatory variety

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of fructose led very naturally to the idea that the polysaccharideis derived from this form of the sugar. Such is not the case,

and it is now certain that the fructose molecule liberated wheninulin is decomposed is a dextro-rotatory ketose, which revertsat once to the stable laevo-rotatory variety. An extreme case,parallel with that of sucrose, has been quoted, but i t serves toillustrate the caution which must be exercised in work of thisdescription. Failure to appreciate the complex isomerism ofwhich the monosaccharides are capable has led in other cases tohasty speculation.

Although, from the structural point of view, inulin is the most

unique and, in many respects the most interesting of the poly-saccharides, it is perhaps advisable to deal, in the first instance,with members of the class which are related t o glucose. I turn,therefore, to the case presented by cellulose.

Cellulose

In a paper recently presented before the Chemical Society ofLondon, results are described from which the constitution of themolecular unit of cotton cellulose may be deduced. To avoid

repetition, I shall omit a detailed discussion and confine myselfstrictly to a synopsis of the essential evidence. Cotton cellulosehas been converted into a-methylglucoside in yields which arepractically quantitative, so that there can be little reason todoubt that polysaccharide is composed entirely of glucose units.This result, of course, gives no clue as to the linkage of theseglucose residues, but the information has been supplied by aparallel research on fully methylated cellulose. The alkylation

proceeds readily up to the dimethyl stage, but thereafter theniethoxyl content rises very slowly, and i t has been a matter ofsome difficulty to obtain a trimethyl cellulose and to prove thatthis product represents the experimental limit of the substitution.Kow, it has been known for some years that when dimethylcellulose is hydrolyzed there results a complex mixture of meth-ylated sugars, one component being 2 : :6-trimethyl glucose.This result, significant in itself, justified only one step being taken

’

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in ascribing a formula to cellulose which, in consequence, might

be represented by the expression:FCH-0-X

1 CHOHI 1

01I cI-roI-r

CH-0-Y

CHIOHI

Beyond the fact that they must consist of some form of glucoseresidues, the nature of the groups X and Y remained quiteunknown until the fully methylated cellulose I have mentioned

was available for hydrolysis. In addition a further complicationwas introduced (and the point is somewhat elusive) through lackof any information as to how the hydroxyl groups in celluloseare distributed between the different Cs units. Applying theconsiderations already discussed, reflection will show that accord-

ing to the manner in which the hydroxyl groups are shared byeach C6 omponent, a trimethyl cellulose may undergo hydrolysisin four different ways and that, as a maximum, 49 meth-ylated sugars may then be produced. These formidable possi-bilities are dealt with in the paper referred to, but the resultactually obtained was the simplest of all the alternatives.Trimethyl cellulose was converted quantitatively into 2 : :6-

trimethyl methylglucoside and this, in turn, yielded thecorresponding sugar, unmixed with any isomeride or withany other derivative of glucose. -4 triking simplicity is thus

established fo r the cellulose molecule, as the unknown fractionsX and Y are shown to be identical with each other and with thenucleus glucose residue. In order to expand this result into a

molecular structure, it is necessary to know how many of theseglucose residues go to form the unit. The question is largelysolved when the constitution of cellobiose is taken into account,as good grounds exist for claiming that this sugar is a definite

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CHOH

01

IIT O HLCH

fraction of the cellulose complex. The simplest expression of

the cellulose molecule therefore consists of two anhydroglucoseresidues connected as shown in formula XVI:

0 XVII

CHOH

La, XVI

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ylation process fail to complete the architectural scheme of the

cellulose complex. No indication is given of the a- or @-con-figuration of the glucose components, and no idea is conveyedof the mechanism or extent of the polymerization undergone bythe simple molecule. We realize, however, tha t our work is not

at an end and it may be stated that, by the degradation ofesparto cellulose, a compound corresponding to the molecularunit postulated above has already been isolated. Now that theoutlines of the cellulose molecule have been defined, many furtherdevelopments will be apparent and we are engaged in extending

our researches to various forms of modified celluloses.

Starch

Turning to the problem of the constitution of starch, verymuch the same difficulties emerge as have been referred to in thecase of cellulose and, viewed superficially, the two polysac-charides have much in common. Like cellulose, starch is com-posed entirely of glucose residues and contains three hydroxylgroups for each Cs unit present in the molecule. It does not

follow, although this is almost invariably assumed, that eachglucose residue contains three substituted hydroxyl groups, ortha t the distribution of the three groups is uniform. Theprimary reaction of starch which, so far as we can tell, must beaccommodated in a structural formula is the production of thedisaccharide maltose by the action of diastase. Starch is alsodistinguished sharply from cellulose in being more readilyhydrolyzed by acids and this at once points either t o

1. A different type of polymerization

2. A different mode of attachment between the individual sugar

3 . A variation in the type of th e glucose units

residues.

In other words, part of the starch molecule might conceivablybe related to y-glucose, but this possibility can now be definitelyexcluded from the results furnished by methylation. Theseresults are in themselves simple, but few reactions impose greaterdemands on the experimenter than the alkylation of starch.

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As a rule, the procedure in methylating compounds of this

nature is to use, in the first instance, methyl sulphate as thereagent and, when the methylation has proceeded far enough torender the product soluble in organic solvents, to complete the

substitution by means of the silver oxide reaction. In the caseof starch, there is clear evidence that these alternative reagentsact in a different manner, owing to the preferential attack ofsilver oxide and methyl iodide on one particular hydroxyl group.The methylation can, in fact, be arrested definitely when a

dimethyl starch is the sole product (OMe = 32 percent), butby varying the procedure, the methoxyl content can be graduallyincreased until it reaches the value 37 per cent. It should be

stated that this is not an absolute maximum, but represents asecond definite stage beyond which further methylation becomes

a matter of increasing difficulty. The lower value for methoxyl(32 per cent) corresponds, as stated, to a dimethyl starch, andno definite structural evidence is forthcoming from the hydrolysisof this compound. On the other hand, the higher methoxyl

content (37 per cent) agrees with the theoretical amount requiredfor an anhydrotrisaccharide in which three methyl groups are

attached to one glucose residue, while four such groups areshared by the two remaining glucose residues. The same resulthas been obtained consistently in different preparations, andultimate analysis has confirmed the composition. Hydrolysis

of this methylated starch shows that this is not a coincidence.The sugars thus produced were isolated in the form of the cor-responding methylglucosides, and separated by vacuum dis-tillation. Two products were in this way obtained, one being atrimethyl methylglucoside, and the other a dimethyl methyl-

glucoside. It is significant that these products were formed inthe molecular ratio of 1:2. On isolating the respective sugarsfrom the compounds, a totally unexpected result was obtained,as 2 : :6-trimethyl glucose was produced from the trimethylmethylglucoside. As will be seen from inspection of formula X

we are forced to the conclusion that in starch one glucose com-ponent is linked in a manner which is duplicated in cellulose,

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I1 CHOH01

I

cellobiose, and in lactose but is absent in maltose. Combining

these factors, it follows that, as a minimum, the starch moleculemust contain three glucose units arranged in such a manner that

one pair displays the coupling present in maltose while anotherpair reproduces the linkage characteristic of cellulose. Theresults summarized in the scheme given below indicate the con-ditions which must be satisfied, and these are fulfilled by anyony of four formulae.

2:3:5:6-Tetramethyl glucose., ......... 1mol.

(2:3:5-Trimethyl glucose.. ............. 1 mol.

2:3:6-Trimethyl glucose.. ............. 1mol.

Dimethyl glucose.. ................... 2 mols.

Maltose-/

IStarch

r- O-1

iethylated starch -+

rCH-0 - H CHOH.CH CHOH*HOH CHI

0

T o - 1,-CH- 0-CH CHOH. HOH CH. CHO H*C H~

I

0 1

I CHOHI CHOH

id,

0I

XVIII

XIX

I IIL-

CH-0 -CHs*CHOH.CH*CHOH*CHOH*CH

CH3OH

I

0

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60

1 dHOH

0

01

rH

J A M E S COLQUHOUN IRVINE

r- O-1

rCH-0

-CH *CHOH CHOH * CH *CHOH* H*

0

I I ILCH

I

XXI

In reviewing the four possibilities, there is no diagnostic reactionwhich will enable a final decision to be made between thembut the formulae differ in one important respect. Maltosemay be produced in one way only from either formulae XIX,XX, and XXI and in two ways from the remaining alternativewhich is thus preferred until the completion of work now inprogress. The objections which may be offered against theadoption of such a formula have been discussed elsewhere, and

the view now expressed receives strong support from the factthat very similar results have been obtained with glycogen.

Glycogen

In general it may be said that the methylation of glycogendisplays precisely the same features as were encountered in thecase of starch. The reaction shows a distinct tendency to cease

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COKSTITUTIOK O F POLYSACCHARIDES 61

at a stage where a dimethyl glycogen is the essential product,

but frequent repetition of the alkylation occasions a slow increasein the methoxyl content, which has now been raised to a valueclose to that required for a trimethyl derivative. In a numberof respects, however, minor differences are apparent betweenmethylated starch and methylated glycogen and these suggestthat although a close parallel exists between the respectivemolecular units, they are unlikely to prove identical.

One highly important factor has already been established withcertainty, and that is the formation of crystalline 2: 3: 6-trimethylglucose by the hydrolysis of a methylated glycogen containing37 per cent of methoxyl. The reaction which gave this resultproceeds in exactly the same way as the hydrolysis of methylatedstarch and points to the idea that an anhydro-trisacchariderepresents the glycogen molecule. Consequently both starchand glycogen resemble each other and also cellulose in possessingthe unit shown in formula X. Beyond this, it is impossible tospeak with certainty at the present time as the constitution ofthe other hydrolysis products obtained from methylated glycogenhas not been fully developed, but the evidence so far accumulated

indicates that one of the formulae XVIII , XIX, XX, and XXI,represents the molecular unit of glycogen.

Taking a general view of the three polysaccharides related toglucose, a number of interesting points emerge. The first un-doubtedly is their surprising simplicity. In the production of

these compounds in Nature complications of structure have beenavoided, and although in the end there are wide differencesbetween the polymerized aggregates to which we apply thenames cellulose, starch and glycogen, the inner molecular

structure is remarkably alike. Apparently the contrasting prop-erties of these polysaccharides are dependent chiefly on the extentand, more particularly, on the mechanism of the polymerizationundergone by the molecules, and we know little or nothing con-cerning these factors. Analogy is dangerous, but as we aredealing with molecular architecture, I may remind you that manydifferent buildings, varying in design and function, may beerected with bricks of identical material and mould. Theanalogy may serve to reveal my opinion of the vast problems

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62 JAMES COLQUHOUN I R V I N E

which have still to be solved before we can trace how, and why,

an anhydro-trisaccharide molecule can take up its place as part

of a: cellulose fibre or of a starch grain.A second generalization is that polysaccharides are not compIex

glucosides in which the groups of one glucose residue are sub-stituted by chains of other glucose residues. Two syntheticdextrins have been shown to conform t o this model, but noevidence is forthcoming that any natural polysaccharide is con-stitut.ed in such a way. But the most remarkable feature incellulose, starch and glycogen is the absence of any sugar residuepossessing the structure of y-glucose, and on first inspection,

this may be regarded as somewhat damaging to the theory thatthe reactive type of this sugar plays a part in natural syntheses.On the other hand, a greater importance must hereafter beattached to position 5 in the glucose molecule, as it functions inthe coupling of the hexose residues in five highly importantsaccharides. In submitting these formulae, it is perhaps neces-sary to sound a note of caution, as the double assumption is madethat maltose is an integral part of starch, and similarly that thecellobiose unit is present in cellulose. There seems no reason

to doubt these conclusions and they are generally accepted, butthey have not been proved.Issues which are of special interest to the physiologist are

raised in the relationship now established between starch andcelIulose on the one hand, and between starch and lactose on the

other, as one molecular fragment is common to all the com-pounds. A similar statement may be made regarding sucroseand inulin, as will be evident from the consideration of thispolysaccharide.

Inulin

A common view is that inulin is the direct analogue of starchfrom which i t differs in being based on fructose in place of glucose.The distinction is, however, more deep-seated than the abovestatement implies, and inulin occupies a position which is uniqueamong natural carbohydrates. It is the only compound, so faras known, which is built up of hexose units belonging exclusivelyt o the type known as “7-sugars.’’ This feature of the poly-

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COSSTITUTION OF POLYSACCHARIDES 63

saccharide has already been referred to, and a diagranmatic

scheme will show the succession of reactions which lead to thisconclusion.

Inu l in _I) Fructose ---+ 1:3:4:&Tetramethyl fructose.. . . . , ... A(laevo) (laevo)

(dextro) (dextro)

Irimethyl +Trimethyl f r uc to s s ---f Tetramethyl y-fructose. . . . .. .. . . .. . I3inulin

The ultimate products A and B are definitely isomeric, and afundamental change in structure must therefore take place duringone of the reactions involved. On the basis of evidence which,

if complicated, is nevertheless conclusive, it has been shown thatit is the conversion of inulin into laevo-rotatory fructose which isat fault. During this change the internal ring in the sugarassumes the stable butylene-oxide position. This is not presentin the original polysaccharide, but no such intramolecular changefrom the unstable to the stable type is possible in the parallelhydrolysis of trimethyl inulin, as the hydroxyl group necessaryfor the alternation is substituted.

Our earlier work on inulin established several points of primaryimportance. All the fructose residues were proved to conformthe 7-type, and moreover, each C 6 unit was shown to containthree hydroxyl groups. One important piece of structural evi-dence remained undetermined but this can now be supplied.

If the hydroxyl groups in inulin are similarly distributed amongall the C e chains then the trimethyl 7-fructose obtained fromtrimethyl inulin must be a single chemical individual. Con-

versely, in the event of the hydroxyl groups being unsymmetri-cally arranged, then a mixture of the four possible trimethyl

7-fructoses would result. T o obtain the necessary evidence has

been difficult, and has demanded the preparation of large quan-tities of trimethyl 7-fructose which, unfortunately, is a viscousuncrystallizable syrup. The liquid has been shown to be homo-geneous in every respect, and the complete symmetry of theinulin molecule is thus established. It is, on paper, a simplematter to utilize these results so as to construct a molecularformula for inulin, but some doubt still remains as to the con-stitution of trimethyl 7-fructose. The compound has the same

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internal oxygen ring which is present in the ketose component ofsucrose, but the position of the unsubstituted hydroxyl group isnot yet determined with certainty. Until this has been done,it is premature to ascribe a complete structural formula to inulinbut the most probable structure is

CHiOHI

0 CHOH

k-o-yXXII

the same unit being represented at XY or at X and Y.It is my belief that the key to the mechanism of Nature’s

method of building up polysaccharides from primitive moleculeslies in the comparative study of starch and inulin. Thesenatural processes, must a t some stage, involve the formation of a

compound which by simple intramolecular change gives rise,

according to the conditions, to either glucose or fructose. Theliberation of one of these sugars leads ultimately to starch or

inulin respectively, while the simultaneous formation of thehexoses would result in the formation of sucrose. This suggestion

is not at variance with the order in which sucrose and starchmake their appearance in the plant, provided it is established thatthe ketose molecule can be rapidly formed and speedily removedby condensation. Such a condition is not met by the reactionsof the ordinary variety of fructose, but is fulfilled by the highly

reactive modification of the sugar which is included under theexpression “ fructose.” This brings me to the considerationof a novel aspect of carbohydrate chemistry.

*/-SUGARS

In the preceding sections, I have been under the necessity fromtime to time of referring in passing to r-sugars and it seemsexpedient, in closing, to summarize our knowledge regarding

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these important variations in sugar molecules. This is desirable,as

hitherto no attempt has been made to compile a general surveyof what is known regarding y-sugars and, as considerable interestis taken in these compounds, I begin with the statement tha t thename applied to them is not descriptive but is, in a sense,accidental.

As is well-known, the best-defined reducing sugars can generallybe obtained in two isomeric forms which display muta-rotationin opposite directions, and this is attributed to the rearrange-ment of the groups in the reducing position. The expressionsa- and 8- were applied to designate these isomerides, and the two

muta-rotatory forms of glucose serve as standard examples inillustration. Now this isomerism has been proved t o be in no

way dependent on the position of the internal oxygen ring in thesugar since both a- and @-glucosehave been correlated with thebutylene-oxide form of tetramethyl glucose. The discovery thatanother variety of glucose exists, in which the oxygen ring is

differently attached, could not be accommodated by the nomen-clature then in use and the expression “y-glucose” was appliedto it. The name “7-sugar” has therefore a very general sig-

nificance, and is used to include all forms in which the oxygenring is displaced from the normal stable position. This is notentirely satisfactory, but a more exact nomenclature cannot beapplied until the complete structure of each of these sugars hasbeen established. Meanwhile it may prevent confusion if it ispointed out that y-sugars, retaining as they do a reducing group,can exist in OC- and p-modifications, and the same remark appliesto their glucosides.

Up to the present it has been ascertained that glucose, galac-tose, mannose, rhamnose and fructose are all capable of existingin y-forms and there seems to reason to doubt that theproperty is general. In no case, however, has an unsubstituted7-sugar been isolated, although we recognize their transientexistence, and can in some measure study their properties by

examining their methylated derivatives. The presence of themethyl groups in these derivatives prevents any alteration in theoxygen ring, and thus, the constitutional type is preserved.

CH~X ICAL EVIEWS, VOL. I, NO. 1

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66 J A M E S COLQUHOUN I R V I N E

Before summarizing the chemistry of y-sugars, it may be men-tioned tha t their discovery did not originate in any single dramaticobservation, but that the evidence accumulated gradually. Oneof my laboratory note-books, describing work carried out exactlytwenty years ago, gives an account of the methylation of sucrose,and the separation of the sugars thereafter formed on hydrolysis.Crystalline tetramethyl glucose was isolated and, in addition,the corresponding fructose derivative was obtained by means ofcondensation in the cold with methyl alcohol, followed by theusual fractional distillation and hydrolysis. The completeanalysis of the product was carried out, its composition was

ascertained to be that of a tetramethyl fructose, and its specificrotation was shown to be of the order + 27” . The entry in thebook states that the work was at this stage suspended, therebeing no adequate explanation as to why a methylated fructoseshould be dextro-rotatory. The compound was, in fact, puretetramethyl y-fructose, but many consecutive researches werenecessary before the discovery could be interpreted. A sub-sequent paper on the methylation of fructose added materiallyto the evidence that the ketose can react in a dextro-rotatory

form, and about the same time, a result of equal significancewasforthcoming. A laevo-rotatory trimethyl glucose was obtainedfrom glucose monoacetone and, in investigating the origin of thiscompound, a new laevo-rotatory form of tetramethyl methyl-glucoside was isolated, thus showing that not only can d-fructose

react in a dextro-form but d-glucose can exist in a laevo-rotatory

variety.

At this stage Fischer published his paper on y-methylglucosidewhich was quickly followed by our account of the isolation andproperties of tetramethyl y-glucose. In view of this theoretical

development, the way was now clear to resume the study ofsucrose and to expand the scope of our work.

For reasons already stated, the properties of y-sugars have tobe inferred from those of their methylated derivatives and, so

far, the following examples together with the corresponding“methylglucosides” are available for study:

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CONSTITUTION OF POLYSACCHARIDES 67

Tetramethyl 7-glucose (laevo)

Trimethyl ?-glucose (leavo)Tetramethyl 7-galactose (laevo)

Tetramethyl 7-mannose (laevo)Tetramethy1 7-fructose (dextro)Trimethyl 7-fructose (dextro)

In reviewing their reactions, one is struck by the close resem-blance shown to natural processes. For example, the com-pounds undergo oxidation with extreme ease, being affected bymild oxidizing agents, and in illustration i t may be mentioned tha ttrimethyl 7-glucose reduces Fehling's solution instantaneously

in the cold. At ordinary temperatures, also, they are convertedinto glucosides, at a speed which is remarkable, especially whenit is remembered tha t the formation of a-methylglucoside requiresa treatment of approximately sixty hours at looo. In a t least one

case (tetramethyl y-galactose) a reducing sugar of the 7-type isknown to pass spontaneously by auto-condensation into a non-reducing disaccharide. These striking properties are far removed

from those of a normal sugar.Great difficulties have been encountered in attempts to solve

the constitution of y-sugars and experimental work has been

largely confined to tetramethyl y-glucose, trimethyl 7-fructoseand tetramethyl 7-fructose. I n the case of the glucose deriva-tive, the first opinion formed was that an ethylene oxide ring waspresent, but this view has been modified in favor of the propylene-oxide structure. Similarly, an amylene-oxide constitution is atpresent supported for tetramethyl 7-fructose so that , meanwhile,

the parent sugars may be represented as:

A H O H CHnOH CHiOH1 10

CHOH

QH

I

I I,-COH

IrYoHI CHOH or OCHOH

I I0 CHOH

I

II

CHOH

CHOHI

ICHOH

CHnOHHiOH

Y-GIUCOM y-Fructoae

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It is already evident that the butylene-oxide structure cannot

be assumed for any sugar even in its most stable form. Thuscrystalline xylose, which displays none of the properties of a7-sugar, has recently been shown to be an amylene-oxide andthis respect at least resembles 7-fructose.I admit that speculation in the absence of experiment is best

avoided in the carbohydrates, but in the course of investigatingmany varieties of y-compounds, I found it impossible to rejectthe idea that the ordinary, crystalline, comparatively stablesugars of the laboratory are not the forms which are primarilyelaborated by the plant or disrupted by the animal. The fruc-

tose which we store in the chemical museum, and produce as a,

lecture specimen, is not fructose as it exists in combination insucrose or in inulin.

The idea conveyed above is that the natural processes leadingto the formation of sugars and their derivatives, as also theultimate utilization of carbohydrates by the animal organisminvolve as intermediate stages the transient formation of 7-forms.The evidence may not be particularly convincing save to thosewho have handled all types of carbohydrates at the working

bench, but it is certainly the case that we have fleeting glimpsesof extraordinarily reactive sugars which, in the process of artificial

isolation from either plant or animal sources, undergo rearrange-ment to the sugars as we know them. This is not inconsistent

with the results obtained in either hydrolysis or synthesis effectedby enzymes. If this suggestion is substantiated, i t may have far-reaching effects in studying physiological processes, and in thetreatment of conditions such as diabetes in which the metabolismof sugars is abnormal. Research designed to test this idea was

commenced three years ago, and the first tentative steps havebeen taken to ascertain if living tissue promotes the formation of7-glucose. The evidence obtained is encouraging, but furtherprogress is hampered by the inaccessibility of y-sugars, and thelack of trustworthy methods of identifying them. When it is

remembered that y-glucose and y-fructose revert instantaneouslyto their respective stable forms in the presence of 0.1 per cent of

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hydrochloric acid, it will be agreed tha t there is little prospect ofdetecting these fugitive isomerides by ordinary processes. Ana-lytical methods are of no avail, and polarimetric evidence aloneis insufficient and often misleading. No doubt these difficulties

will be surmounted in time, but only when the detailed chemistryof 7-sugars has been advanced on systematic lines, and whenknowledge exceeds impatience.

Having in a sense created ?-sugars for you, it may appearcontradictory if I say tha t I have some doubts as to whether thesesubstances are, after all, chemical individuals. They may only

represent a condition of the sugar molecule which originates inthe mechanism whereby a sugar forms certain of its derivatives.To make the point clear by means of an example, we need notassume that in the formation of sucrose a molecule of normalglucose condenses with a molecule of 7-fructose in such a mannerthat each loses a hydroxyl group.

CHiOH............................fjlH OIH HOi I1

I CHOH...........................

CHOHI II

+ CHOH 0

LCH CHOH I1I

CHOH

CHoOH

I suggest tha t 7-fructose may have no real existence, and that thecondensation may be equally well explained on the assumption

t,hat fructose functions as a ketone. In such a case, two hydroxylgroups become involved, and although one of these must be thereducing group of the aldose molecule, there is no reason, oncethe reaction is initiated, why the ketose itself should not furnishthe second group:

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CHzOH, . . .

.. .. ... ..

I

01

'. ,.. :.I. :. ,

.. :.. :1

CHzOH

An amylene-oxide ring is thus formed in the ketose componentand here we see the production of a y-residue without the previous

existence of a y-sugar. Similar considerations apply to the forma-tion of 7-glucose derivatives which may be formed in condensationreactions in which glucose reacts as an aldehyde. These sug-gestions are not without the support afforded by numerousanalysis, and meanwhile the whole subject is a maze of possi-bilities, and enlightenment is still remote.

For example, if the question is raised as to why sucrose is so

remarkably unstable towards hydrolysts the existence of anamylene-oxide ring in the fructose component provides noanswer. Methyl xyloside is likewise an amylene-oxide, yet thecompound resists the effect of dilute acids while p-glucosan whichcontains a hexylene-oxide ring is notably stable. Again,fructose mono- and diacetones both undergo hydrolysis with afacility which resembles that displayed by sucrose but never-theless both of these compounds are based on the stable type of

fructose in which a butylene-oxide ring is present. These appar-

ently contradictory results reveal that the special instability of?-derivative does not depend primarily on the presence of anypirticular internal ring in the sugar.

On the other hand, when aldehydes or ketones condense withhydroxy-compounds (including sugars) the products resemblesucrose in their ease of hydrolysis. Numerous isopropylideneand benzylidene derivatives may be quoted in illustration, and

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the reactions in which such compounds are formed have been

shown to follow the course indicated below:

R HO *Rz+R><:C):>o + -i HO*Ra Ri

Applying this to the special case of carbohydrates, 7-sugars maybe regarded in a new light; 7-fructose may, in fact, be nothingmore than the ketonic form of the sugar and 7-glucose the cor-responding aldehyde. It may well be that the aldoses and ketosesfunctioning in their primary capacity as aldehyde and ketone

are the reactive sugars in Nature, but this much seems clear.No naturally-occurring compound of glucose, whether glucoside,disaccharide or polysaccharide, has been found to contain theyglucose structure and the reactive type of the sugar has hithertobeen obtained only under artificial conditions. In sharp con-trast, the two most important nature derivatives of fructose areeach based on 7-fructose which may possibly prove to be theconnecting link in natural processes whereby the interconversionof ketoses and aldoses is effected.

The structural studies which I have laid before you are ad-mittedly incomplete and the various formulae now suggestedrepresent only stages in knowledge and thus have no claim tofinality. Much reliance has been placed on the validity of themethylation process as a means of determining constitution, butit has to be remembered that in the carbohydrates most specula-tion of the kind is now based on results obtained by this particularmethod. If I may add one personal note, it is tha t I have sub-mitted these results not as an individual contribution but as thework of a loyal research school. The colleagues, collaborators

and students who have played their part in the laborious workinvolved, if unnamed now, are always gratefully rememberedby me; but memory dwells specially on the one who carried thespirit of chemical research to St. Andrews and who provided thefacilities and the enthusiasm which it has been my good fortunet o share. I refer to Thomas Purdie.

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