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[CONTRIBUTION

ROM THE

CHEMICALABORATORY

F

THE

OHIO

STATE NIVERSITY

THE

ME C HANI S M O F CARBOHYDRATE OXIDATION. XVII I.*

T H E O X ID A T IO N O F C E R T AI N S UGAR S W I T H S I L VE R OX I DE

I N T H E P R E SE N CE O F P OTAS SIUM H YD R OX I DE

K .

G.

A. BUSCH,

J.

W.

CLARK , L. B. GENUNG, E .

F.

SCHROEDER,

AND

W.

L. EVANS

Received February 16 1986

A

clearer un ders tand ing of t he m olecular reaction m echan ism involved

in the oxidation of various carbohydrates may be obtained through an

experimental stu dy of th e behavior of these im por tant com pounds towards

reagents that will yield oxidation products differing respectively both in

kind an d in number. Glucose m ay be oxidized completely by alkaline

potassium permanganate solutions' to carbon dioxide, and oxalic and

trace s of acetic acids, w hile w ith silver oxide und er the sam e conditions,

carb on dioxide, an d oxalic, glycolic, and fo rmic acids are t he final reaction

products. I n th e acid medium of copper ace tate solutions containing an

excess of this s alt, glucose m ay be oxidized to glucosone, carbon d ioxide,

and formic, oxalic, and glyoxylic acids? A comparative study of the

d at a ob taine d throu gh th e use of reagents3 of differing oxidation po tentia l

on the sugars and their various theoretical degradation and oxidation

interme diates seems to offer

a

fruitful meth od of a ttac k on this imp orta nt

oxidation problem.

The reagent chosen for the studies reported in this paper was silver

oxide, both alone and in the presence of added alkali . K i l i a ~ ~ i , ~ef,6

Behrend and DreyerlBDenis' an d Witzem ann* are among those who hav e

studied t he action of th is reagen t on various sugars an d their inter-

mediate degradation com pounds. Th e use of silver oxide in the stu dy

of carb ohy drat e oxidation offers certa in unique adv anta ges. T he oxi-

datio n pro ducts formed a re carbon dioxide, and oxalic, formic and glycolic

* C o n t r i b u t i o n XVII of this series,

J.

Am. Chem. SOC. 7, 200 (1935). T h i s

ar t ic le was submit ted in response to t he inv i ta t ion of th e edi tors.

EVANS

ND COLLABORATORS, J . Am. Chem. Soc. 47,

3085, 3098, 3102 (1925).

EVANS, ICOLL,T R O U S E

ND

WARINC,

bid . ,

60, 2543 (1928).

a K A R R E R

ND

PFAEHLER,elv. Chim. Acta 17, 363, 766 (1935).

KILIANI,

er.

13,2 703 (1880);

Ann.

206, 187, 191 (1880).

NEF, Ann. 367, 287 (1907).

B E H R E N D

ND

DREYER,

nn.

416,

203

(1918).

D E N I S ,Am. Chem.

J.

38, 578 (1907).

* WITZEMAN,

h.D. d isser ta tion , Th e Ohio Sta te Univers i ty ,

(1912).

1

TEE

JOURNAL

OF O R G A N I C

CHDb IBTEY,

VOL 1,

NO.

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2 BUSCH, CLARK, GENUNG, SCHROEDER, AND EV A N S

acids, all of which can be determined qua ntitativ ely. Th e silver remain-

ing after oxidation may be easily separated from the unchanged silver

oxide, and from its weight the oxygen consumed may be calculated.

Th e reaction is com paratively rapid and hence does not necessitate a long

tim e for its completion. Th e main objective of these experim ents was to

stu dy the behavior of mannose, fructose, arabinose and compo unds re-

lated to these carbohydrates towards silver oxide in the presence and

absenc e of alkalies un der carefully controlled conditions for the purp ose

of obtaining accu rate qua ntitati ve d at a which might shed more light on

the mechanism involved in the oxidation reaction.

EXPERIMENTAL

Reagents.-All t h e reage nts used in these experime nts were examined for the ir

pur ity b y th e usual well-known lab ora tory procedures.

Carbohydrates.-The carbo hyd rates used were of th e highest ob tai na ble pu ri ty .

Th eir iden ti t ies were verified b y determinations of thei r specific rotations, and by

oth er means when necessary.

Sil ver Oxide.-A so lu tio n of 400 g. of AgNOa in 1200 cc. of d ist ill ed wa te r was

vigorously s t i r red and a solution of 150 g. of KOH in 800 cc. of water was added a t

the ra te of 100 cc. per minute. Th e size of t he bat ch was later increased to as much

as 2000 g. of AgNOa bu t th e sam e conc entratio ns were always ma intaine d. T he

brown Ag,O th us precipitated was washed with wate r by decantation unti l the wash

wa ter was free of Ag+ an d a 100-cc. sample req uired less tha n

0 . 3

cc. of 0.1N HC1

to neutra l ize the a lka l i present. Th is usua l ly required abo ut ten washings .

T h e

AgzO was th en dried

at

110C. under vacuum. When dried a t this temperature, i t

was changed from th e chocolate-brown color of t he fresh ly precip itated oxide to a

dark purplish-brown.

If

dried a t 85 C., th e original brown color w as retained.

After drying, t he oxide was passed through

a

100-mesh sieve, placed in brown bot-

t les and s tored in the dar k .

It

was believed a t first th a t different lo ts of silv er oxide

would give s l ightly different results , but i t was later shown that when the above

directio ns were carefully followed, uniform re sul ts were always obtain ed. AgnO

was analyzed before using for total s i lver, ammonia-insoluble matter and carbon

dioxide. Th e analysis of th ree typical batches of the AgzO th us prepared an d

labeled (a), (b), and (c) was as follows: Silver, (a) 92.6%; (b) 92.20%; (c) 92.95%;

the ore tica l 93.1%: COz, (a) 0.10%; (b) 0.05%; (c)

0.03%:

Ammonia-insoluble, (a)

0.07%; (b) 0.09%; (c) 0.12%.

Ap paratu s and Analytical Procedures.-Seven gram s of silver oxide was adde d t o

100 cc. of 1.ON KOH conta ined in a 150-cc. carbo n dioxide flask, fitted with a s to pp er

carry ing a thermometer and a smal l s topcock. Th e flask was then p laced in the

the rm os ta t ma in ta ined

at 50

and th e reagent was al lowed t o come to tem pera ture ,

after which one four-hundredth of a mole of the sugar (e.g., 0.45 g. of mannose,

glucose,

or

fructose, o r 0.375 g. of arabinose) was added, the s top per was inserted

in the flask and the s topcock closed. By closing the s topcock after insert ing th e

stopper, an y pressure effect caused by forcing in the lat t er was prevented. When

this was done, i t was found unnecessary to wire on the s toppers to prevent them

being blown out , an d no loss of c arbo n dioxide was experienced. In th e experiments

carried ou t in th e absence of added alkali , the only change made i n the above pro-

cedures waS t h a t of us ing

11

g. of AgzO instead of

7,

an d carbon-dioxide-free wate r

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MECHANISM

OF

CARBOHYDRATE OXIDATION 3

was used in place of t h e

1.ON

a lka li . T he reac t ion mixtures were then agi ta ted by

a mechanical shak er during the various periods of t im e indicated in Fig. 1.

Th is technique was a t tended by a quick r ise in temp era ture wi th

a

m axi m um of 5,

which rapidly subs ided to the th ermo s ta t ic temp era ture .

Determination of Consumed Oxygen.-After th e reaction flasks were removed from

the th erm os ta t the y were cooled and the reac t ion mixture was decanted th rough a

previously weighed and dried Gooch crucible. T he silver-silver oxide mix ture re-

maining in the flask was washed several t imes by decantation and the fi l trate and

washings were mad e up to a volume of 250 cc. Th is solutio n was used for th e deter-

min ation of oxalic, formic, and glycolic acids. T o th e residue in th e flask was add ed

50 cc. of dilu te NH aO H

1:3)

nd th e flask was shaken vigorously to h asten solution

of the A g20 . Th e undissolved s i lver was allowed to sett le and the l iquid was de-

canted through the Gooch cruc ible . Th e ammoniacal solu t ion , thus obta ined, was

immedia te ly added to a beaker containing an excess of HC1 to prevent explosions

of the kin d reported b y previous workers .

Th e s i lver residue was washed wit h two more 50-cc. portions of th e NHaOH, th e

final washing being tested with HC1 to make certain that al l the s i lver oxide was

removed. Th e Ag residue was now transferred to th e Gooch crucible, washed w ith

wa ter, dried in a vacuum oven a t llOC., and weighed. T he oxygen consumed in

th e oxidation was calculated from the weight of s i lver thus obtained afte r correcting

for ammonia-insoluble impurit ies .

Cai-bon Dioxide.-The determ ination of carbo n dioxide was made on a second

sample obta ined in exac t ly the same manner as the sample used for th e de terminat ion

of the acids. T he flask was cooled t o

a

tem pera ture below 40C. as soon

as

it was

removed from the th ermo s ta t and was then connected to a carbon dioxide appa ra tu s

aThich was esse ntially t he sam e as t h a t described b y F o ~ l k . ~

O d i c Acid. -Oxal ic ac id was de termined by prec ip i ta ting as ca lc ium oxa la te

wi th ca lc ium ac e ta te in th e presence of ace t ic ac id and th en t i t ra t in g th e ca lc ium

oxala te wi th potass ium permanganate in the usua l manner. Th e calc ium conten t

was verified by conversion of th e oxalate t o th e sulfate .

Foiemic Acid.-T wo-fifth s of the fi l trate from th e oxidrtt ion mixture was placed in a

500-co. roun d-bo ttom flask fitted wit h

a

dropping funnel, capil lary tub e, andKjeldah1

bu lb . Th e l a t t e r was connected , w i th an adap te r , t o a spi ral w ater condenser, thee nd

of which exte nded t o th e bo tto m of a 500-cc. suction flask. Sufficient6.OMphosphoric

ac id was added bo th to neutra lize the a lka li present in the oxida t ion mixture and to

form the monopotassium salt . Th e suction flask was the n connected to a water pump

and the mixture was dis t i l led under vacuum, the round-bottom flask being placed

in

n

wa ter bath k ep t a t 50C. Two successive 50-cc. portions of water were added

and dis t i l lation was carried to dryness each t ime t o insure the presence of al l the

formic ac id in the d is t i lla te. Th e d is t i l la te was t i t ra te d f i rs t wi th s tan dard a lka li ,

using thymol blue as th e indicator, an d th en by t he Jones10011 method. Usually,

th e two de terminat ions gave very near ly the same result , bu t when the tem pera ture

a t which th e distillatio n was carrie d ou t was allowed t o rise above 55, or when

large amounts of glycolic acid were present, the permang anate value was sometimes

higher than th a t g iven by the a lka li t i t ra t i on . Th is was found to be due to the d is ti l -

lat io n of sma ll am ou nts of glycolic acid wi th th e formic acid. Ap prop riate correc-

t ions were made in each case.

9

FOULKSNotes on Qua nt i ta t ive

Analysis,

McGraw-Rill Book Co.,

1938,

p. 220.

1 0

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4

BUSCH

CLARE, GENUNG, SCHROEDER, AND

EVANS

Glycolic Acid.-The residue rem aining aft er th e formic acid distillation was

ti tr ate d for glycolic acid by th e Jones method. Any glycolic acid th at had dis t i l led

over wi th th e formic ac id was ca lcula ted and add ed to th a t found in th e residue.

This method leaves much to be desired, s ince i t is essential ly

a

determinat ion by

difference, but

a

search thro ugh the l i te ra ture fa i led to offer

a

better procedure.

An a t tem pt was made t o prec ip ita te g lycolic ac id wi th bas ic lead ace ta te , but t he

prec ip ita t ion was found to be quan t i ta t ive only wi th in

a

very narrow range of condi-

t ions which could not be duplica ted when us ing th e oxida t ion mixture . T ha t the

compound measured by difference was glycolic acid was proved by using larger

qua nti t ie s of t he sugar a nd isolating the pure acid, which was identified by the

method of mixed melting points.

THEORETICAL PART

When aqueous solutions of mannose, glucose, fructose, arabinose,

eryt hrito l, glyceraldehyde, or glyco laldehyd e are oxidized with silver

oxide in t he presence a nd absenc e of 1.0

N

K O H a t 50C., th e final reaction

products in each case are carbon dioxide and oxalic, formic and glycolic

acids. T he experimental da ta presented in this paper will be inter prete d,

as

in ot he r papers of thi s series, from t he sta nd po int of Nef's12 enediolic

conception of the chem ical behavior of th e carboh ydrates. His views

were founded on and are an extension of those of F i ~ c h e r , ' ~nd Wohl and

Neuberg14 con cern ing th e presence of hexose enediolic forms of the carbo-

hy dr ate s in alkaline solutions. Some of the recent work in this field, and

points

of

view having

a

bearing on this theory are herewith summarized.

T h e existence of th e enediolic functiona l group , -C(OH)=C(OH)-, is

now a n accepted fact, as is evidenced by th e following instances. (a) Fen-

obtained dihydroxym aleic acid by t he oxidation of tartari c acid.

(b) M ore recently its presence has been established in th e molecular struc-

ture of Z-ascorbic acid (vita mi n C) and related substa nce s. (c) T h e isola-

t ion by Eu le r and M a r t i d B f

Redukton

C3H403,rom a n aqueous alkaline

solution of glucose which had been hea ted t o

90

unde r a stream of nitro-

gen is a discovery of th e first im por tanc e in thi s connection. The se in-

vestigators regarded

Redukton

(11) as a n enediol of tar tro nic aldehyd e ( I).

It

should be noted that

Redukton

may be considered as the enediol of

hyd rox ym ethy l glyoxal (111), discovered b y Ev an s and Waring.17

It

was

late r isolated by N orris h an d Griffiths'7 in th e photoche mical decom-

position of methy l glyoxal. These workers also pointed o ut t h a t th e

14 NEF,

Ann.,

336, 191

(1904);

367, 214 1907); 76, (1910); 403, 204 (1913).

l

FISCHER,

Ber., 28, 1145

(1895).

14 WOHL

ND

NEUBERG,bid . , 33, 3095 1900).

' ENTON,

J

Chem.

Soc.

87,

804 1905).

15EULERND MARTIUS,Ann., 606, 73 (1933).

17

EVANS

ND

WARINQ, Am .

Chem.

SOC., 8,2678

1926).

17.

NORRISH

ND

GRIFFITHS,.C.S.

928,

28-29.

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MECHANISM OF CARBOHYDRATE OXIDATION 5

aqueous solutions

of

hydroxy pyruvic aldehyde must contain its tauto-

meric form, the t ar tronic aldehyde enediol. T he interrelationship of these

three compounds may be shown as follows:

CH O H C-OH CHzOH

C H O H e C-OH 2-0

I I I

II

CHO

1)

With reference to the chemical character of the hydrogen atoms in the

enediolic functional group, Euler and Martius found that

Redukton

pos-

sessej th e acid ity of a n organic acid of average stre ng th K

= 1 X

;

i.e., it is a little weaker than acetic acid K 1.8

X

Th us it is seen

that the enediolic functional group imparts polar properties to the carbo-

hydrate in which it is present.

Winters1*glucic acid, obtained from a so lution of inv ert sugar in lime

water, and also isolated b y N elson a nd Brownelg from a similar solution

of glucose (cerelose), was assigned th e molecular form ula, C3H403,by the

latteir investigators. Like Redukton it possessed strong reducing proper-

ties, and also absorbed iodine. It was thought by Nelson and Browne

to be hydroxyacrylic acid, CHO H= CH COOH . T he properties of

glucic acid and

Redukton

compounds possessing the same empirical

formula, are strikingly similar.

Fro m the ir determ ination of t he alkali-fixing cap acity of t he m ost im-

po rta nt sugars, the conclusion reached by Hirsh and Schlags,20 would

indicate th at these compounds are dibasic. Th e da ta obtained by Urban

and 13haffeF appea r to indicate t h a t w ith glucose, fructose and sucrose a

third acidic group begins to function at high alkalinity; but because of

large errors in this region the existence of th e third gro up mus t be regarded

as uncertain.

Nef2 postulated furthermore th a t th e carbo hydrate enediols in alkaline

solutions would undergo scission

at

the double bond, thus yielding frag-

me nts containin g a bivalent c arbon atom , a typ e of compoun d whose exis-

tence was one of t h e cent ral ideas in his theo ry of o rganic chemical be-

havior.

If

these fragm ents were formed in th e presence of oxidizing agents,

they were oxidized to the corresponding acid, or to compounds having a

smaller num ber of carbon atoms tha n the fragments themselves. I n the

* a WINTER, 2.Ver . Rubenzucker Znd. 44 old series ,

1049 1894).

1s

NELSON

ND BROWNI E,.

Am. Chem.

SOC. , 1, 30 (1929).

20 HIRSH

AND SCHLAGS,.physik. Chem. 141, 387 1929).

2 1

URBAN N D SHAFFER, Biol. Chem. 94,697

1931-32).

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6 BUSCH, CLARK, GENUNG,

SCHROEDER, AND EVANS

abse nce of oxidizing agen ts, i.e. in the presence of alkalies,

a

scission of the

carboh ydrates would ta ke place, giving rise to fragm ents containing

a

smaller numb er of carbon atoms tha n th e original carboh ydrate.

This

has been am ply evidenced in the lite ratur e of this field.

Saccharinic acids

of th e original carb oh ydra te or of it s fragm ents m ay form , a reaction which

has be en experimentally investigated by Kiliani,*2NefI2, Shaffer an d Friede-

mann.23

T he postulate concerning the fragm entation of C=C has met with very

definite objections. (a) Such

a

reaction is not in accord with Schmidtsz4

rule which states that the fragmentation takes place at the C-C in the

CY-@

position to the

C=C.

It should be pointed out that the data upon

which this generalization rests were obtained under much more drastic

experimental conditions than have been employed in alkaline solutions of

the carboh ydrates. T he enediolic group is a polar one and is very reac-

tive even a t ordinary temp erature. (b) T he relative values of the well-

know n bond energies would fav or fragm entation

at

the

C-C

rather than

a t C=C.

Hirst25 an d his collaborators have shown t ha t with th e usual reaction

involving ozonization and subsequent hydrolysis, tetramethylascorbic

acid will give 3,4-dimethylthreonic an d oxalic acids. Fento n15 found

th at dihydroxymaleic dime thyl ester was decomposed in dilute am monium

hydroxide with the formation of oxam ide. H e suggested th at t he ester

had ruptured

at

the double bond.

If

it is assum ed th a t alkaline solutions of re ducing sugars con tain

enediolic forms, it w ould seem t ha t the mechanism involving

a

scission at

the double bond in the presence of alkalies and also th at takin g place in th e

presence of alkaline oxidizing agen ts would necessitate one of two poin ts

of view;

z.,

(a) either

a

scission of th e enediolic gro up followed b y ox ida-

tion, i. e. Ne fs view, or (b ) a different mec hanism of ru pt ur e in the presence

of alkalies alone, an d still ano ther m echan ism for alkaline oxidation in which

oxygen plays a pa rt, such as th e oxidation of ascorbic acid referred to above.

Among th e concepts which migh t offer a simple pictu re of th e me chanism

involved in the scission of

C=C

are those suggested by the electronic

theory,26 nd one outl ined by D r. C. L. Bernier of t his La bo rat ory which

involves

a

simple combination of th e enediol theory and a reverse aldol

condensation.

22 Cf. TOLLENS,Handbuch der Kohlenhydrate, 3 Auflage, 1914,

pp.

778-779.

23

SHAFFER

ND FRIEDEMANN,

.

Biol . Chem., 86,

345 (1930).

24 SCHMIDT,hem. Rev. 7, 137 (1935); Ber.,

68,

60, 795 (1935); Cf. Neuberg,

25

HIRST

ND COLLABORATORS, J SOC. hem. Ind. ,

62, 221, 1270 (1933).

20

Cf.

STIEGLITZ,

roc. Znst. Medic ine

of

Chicago,

1, 41 (1916);

Chem. Abstr., 17,

ibid,

505.

3878 (1923).

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MECHANISM O F CARBOHYDRATE OXIDATION 7

The following discussion is concerned primarily with the sources of the

four ireaction produc ts found i n these studies, an d th e ex ten t of fragm en-

tati on which occurs in mannose a nd fructose under th e experimental con-

ditions used.

Mtsnnose

an d Arabinose.-When a n aldohexose like manno se is acted

upon b y a n alkali it m ay undergo th e kind of chang e indicated in th e fol-

1owin.g reaction :

H O

H

CHO CHOH

\ /

I

II

I

C-OH

I

310-C-H HO-C-H

e

0-A-H H0-C-H

H-C-OH H-L-OH

(1)

HO-C-H?l

I

I

H- SOH

H-C-OH H-C-OH

CHzOH

I

I

H-C

I

CHZOH

HzOH

(d-man nose) (aldo-d-mannose) (d-mannose, 1,2-enediol)

A

fragmentation of the mannose 1,a-enediol between carbon atoms 1 a n d

2

would yield one molecule of arabinose a n d one of formaldehyde,

L e .

CHOH

I

-C-OH

II

C-OH

I

HO-C-H

4

t

I

I

I

H-C-OH

OH

\ /

/ \

C

H

H-C-OH H-C-OH

CHZOH

I

CHzOH

(d-mannose, 1,L- ened iol) (d-arabinose,)27 (formaldehyde,)*'

active f o r m ac t ive

form

T he form aldehyde molecule thu s formed would b e oxidized to formic a cid.

It is clear that the arabinose molecule in alkaline solution may in turn

undergo the same kind of fragmentation as does mannose, until finally

five molecules of formic acid would be formed from th e original pentose,

provided, however, that it reacts in only one direction.

As

set forth in

Fig. 1, th e results of o ur experiments show th a t hexoses, such as glucose

27 BALY,

Rice

Znst. Pamphlet,

12, 93

(1925).

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8

BUSCH, CLARK, OENUNO,

SCHROEDER,

AND E V A N S

an d galac tose, are oxidized b y silver oxide in th e absence of potassium

hydroxide to carbon dioxide, and oxalic, glycolic and formic acids, and

th at the ultimate fate of the o xidation produ cts formed is conversion to

carbon dioxide. I n the presence of added alkali, the salts thu s formed

tend to become stable tow ards silver oxide, a fact w hich confirms Witze-

manns observations in this respect with reference to potassium formate,

a substance which he found to undergo oxidation slowly when heated.

Ou r experimen tal da ta show th a t d-glucose in th e presence of silver

oxide and potassium hydroxide reached a maximum production of formic

acid a t the end of one hour

ie.,

1.5 moles per mole of sug ar), ma nnose

Percentage of Carbon Returned from d-Galactose

9ZW 9241 98.75

99.M

4:

ii

k 4

. y

t i j a g

ZG

; :

b o

2

s 20

3 ~

- - L

4 8 I2 2 24

H o u r s

A t o m s o f Oxygen p e r

Mole

of

d-Glucose

Atoms of

Oxygen

per Mo le

o f

d-Galactose

z4

Consumed 267564 11.75

Calculated

850 fl.10 12.0

Consumed

928 11.75

IbS I/.

Culculated d Y 11.22

Il

66 fm

FIG.

1

2.74

moles in

24

hours, galactose

2.42

moles in

12

hours and fructose

3.1

moles in 24 hours. Th e significance of these d at a lies in the fact th a t in

order t o p roduce yields of formic acid in excess of 1mole as demanded b y

equation (2)) either the original hexose must undergo a fragmentation

between carbon atoms 2 and 3 to give glycolaldehyde and erythrose, be-

tween carbon atoms 3 and 4 to give two molecules of glyceralde hyde, or

the pentose formed in the fragmentation between carbon atoms 1 and 2

mu st suffer a further degradation in the m anner just ou tlined for a hexose.

Tha t the

first

ste p in the frag men tation of the d-mannose molecule under

our exp erimen tal conditions would seem to be th e formation of arabinose

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MECHANISM OF CARBOHYDRATE OXIDATION 9

and formaldehyde [equation

(2)]

follows from an examination of the data

obtained a t th e end of four hou rs, at which time th e total carbon returned

in eac h case is

99.45

per cent for arabinose and 99.33 per ce nt for mannose.

If the mannose molecule, on fragmentation, yields one molecule of

form alde hyd e an d one of a rabinose per mole equivalen t of sug ar used,

TABLE I

DATA.XPRESSEDS MOLES ER GRAM-MOLEQUIVALENT

F

CARBOHYDRATESED

OXIDATION

PRODUCTS

Oxalic Acid

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Formic Acid . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Glycolic Acid. .

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

Carbon Dioxid e.

. . . . . . . . . . . . . . . . . . . . . . . . . . .

Atoms Oxygen Consumed, . . . . . . . . . . . . . . . . . .

MANNOSE

0.98

2.69

0 .56

0.19

6.69

A R A B IN O S E

0.89

1.64

0.71

0.13

5.46

DIFFERENCES

+0.09

+1.05

-0.15

+0.06

+1.20

Oxidation

of

Aqueous Solutions (a025

Malar of

d-Mannose

and I-Arabinose

wi th

S i l v e r

Oxide a n d 1.0N. Alkal i at 50C.

Percentage

o f

Carbon Returned from I-Arabinose

95zo 99.45 99.40 IOL80

Percentage of

Carbon Returned

from d-Mannose

U

.--Form/cAcid

Id-Maywse

f ,

97.67

99.33

97.17

96.63

B Y e

4 8 I2

: I6

20 24

Carbon

Dioxide

(d-Mannose)*

Ours

.Carbon Dioxide(L-Arabinw)

A t o m s of

Oxygen

per Mo le of & M a n n o s e

Consumed

630

669 6.95 7:N

Calculated

hJ4

656

6.66

k.

78

Atoms o f Oxygen

per Mole o f

Z-Arab inose

Consumed

528

X46

549 S 8

Calculated 509 529 5 5 6 9

FIQ.

the n it is clear th at the yields of formic acid from these two c arboh ydrates

should differ by

1.0

mole, and the yields

of

carbon dioxide, and glycolic

and oxalic acids should be the same from both sugars. Th e data in Table

I

show th at these assumptions are approximately tru e in this case. Hence,

it may be safely concluded that d-mannose (0.025 molar solution) is de-

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BUSCH, CL ARK, GE NUNG, SCHROEDER, AND E V A N S

O X I D A T I O N P R O D U C T S

Carbon

D i o x i d e . . . . . . . . . . . . . . . . . . . . . . . . . . .

Oxalic Acid. .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Formic Acid

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Glycolic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

graded approximately into formaldehyde and arabinose as the first step

in its oxidation with silver oxide in the presence of 1.0

N

alkali at 50C.

P. Fleury and J. Lang e2* btained one mole of fo rmald ehyd e by the oxida-

tio n of glucose with periodic acid , which K arr er an d Pfaehler, loc. c i t . )

ascribe to

a

rupture of glucopyranoside between carbon atoms 5 and 6.

T he complete da ta over a period of 24 hours are shown in Fig. 2.

T he fa te of th e arabinose molecule from mannose can be fairly well

understood b y a comparative stu dy of it s oxidation da ta obtain ed after

24 hours with those from erythritol, a compound which we had to use

instead of t he ra re suga r erythrose, a t this writing know n only as a sirup.

These are shown in the following table.

A R A B I N O S E

0.180

1.010

1.690

0 . 6 0

TABLE

I1

DATA XPRESSED

S

MOLES

ER

GRAM-MOLEQUIVALENTSED

O X I D A T I O N P R O D U C T S

Carbon

Dioxide. . . . . . . . . . . . . . . . . . . . . . . . . . . .

Oxalic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Formic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Glycol ic Ac id . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

T W O

MOLES

E ~ ~ ~ H ~ ~ ~ c

LYCOLALDE- DIFFERENCES

H Y D E

0 .124 0 .092

f O ,032

0 .594 0 .648 -0 .054

1.045 1.174 -0.035

0 .803 0 .720

0.08

E R Y T R R I T O L

0 .124

0 .594

1 .045

0.803

D I F F E R E N C E S

0.064

$0.416

0.645

-0 .203

From the differences in the yields of formic and oxalic acids it is clear

that the arabinose is not being fragmented into formaldehyde and ery-

throse,

a

condition which would cause a difference in th e yields of fo rmic

acid of one mole per mole

of

arabinose used. Previous work has shown

t h a t

a

tetr os e sugar ten ds to give two molecules of glycolaldehyde instead

of one each of glyceraldehyde and formaldehyde. If this is so, it is con-

ceivable th at th e erythrose formed from a fragm entation of arabin ose

might yield oxidation data approximately the same as that for two mole-

cules of glycol aldehy de. Since we do no t know th e conce ntration of th e

erythrose in this reaction, th e d at a used can be assumed to show merely

~ ~ F L E V R Y

ND

LANGE, . pharm. chim.,

8 ]

17, 1 (1933); Chem. Abstr .

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MECHANISM OF CARBOHYDRATE OXIDATION

11

th e tendency of th e reaction. Such da ta are given for eryt hrito l an d

glycolaldehyde after twenty-four hou rs in Tab le

111.

T he d at a for glycol-

aldehyde are two-thirds of those obtaine d from a thr ee molar solution, an d

are used only as an approximation.

Glucose.-Under ou r experim ental conditions glucose an d arabinose do

not show the same simple relationship with reference to formic acid yields

which seems to exist between mannose and arabinose, as is evidenced by

the fact that at the end of one hour glucose yielded 1.5 moles of formic

O x i d a t i o n o f Aqueous

Solutions

of

d-Glucose (0.025 Molar) and dl-Glyceric Aldehyde (0.050 Molar)

with Silver

Oxide a n d 1.0

N. Alkal i

a t

50C.

Percentage

of

Carbon Returned f r om dZ-GlycericAldehyde

96.36

96.72

97.11

OJ Percentage o f Carbon

Returned f rom d-Glucose

r 98.00 9650 9833 96.50

-5

5 2

0 .u_

u l i

= J 3

E 8

G g

% I

=2

._

z k

0

k

w e

2

0

1

9

Hours

-

4

8

/Z

/6

20 24

Atoms of

Oxygen per

Mole o f d-Glucose

C o n s u m e d 718

iT40 253

i If

Caliculated

6.7/

6.70

728

232

C o T s u m e d

552

566

24

Calculated 562 584 AM

FIQ.

A t o m s

o f Oxygen

per

TWO

Moles of dl Glyceric

Aldehyde

acid, and arabinose 1.55 moles per mole equiva lent of c arb oh yd rate used.

Fig. 3

is

a graphic comparison of the data obtained from glucose with

those which would have been obtained had this hexose molecule undergone

cleavage in to tw o molecules of glyceraldehyde. It is evident from these

experimental results that glucose reacts in more than one direction.

T h e formation of oxalic acid from glucose, manno se, galactose, arabinose,

fructose, glyceraldehyde and similar compounds may arise from the for-

mation of an a-keto-acid

of

th e hexose as well as

of

the other theoretically

possible keto-acids of fewer carbon atoms. T he keto-acid m ay enolize an d

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12

BUSCH, CLARK, GENUNG, SCHROEDER, AND EVANS

then undergo a fragm entation, th us yielding glyoxylic acid, which in tu rn

is oxidized to oxalic acid, in accordance with the following reaction.

R

R

R

CH O H --&-OH >(O H) .corn

C-OH

I

(3)

co

- -OH

I I

+

l + o

COOH

COOH

COOH

OOH

Fructose:

Glycolic

Acid.-The most plausible source of glycolic acid is

glycolaldehyde. Fro m our studies we are led to believe th at t he primary

alcohol group in the sugars studied is only slowly attacked under our

conditions and it appears in the oxidation products chiefly as glycolic

acid. On th e oth er han d the secondary alcohol groups are rapidly oxidized.

Our experiments show that ethylene glycol is stable towards silver oxide

and alkalies while glycerol is most readily attac ked . Ta ble

I V

shows the

T A B L E

I V

M OLESOF GLYCOLIC CID FORMEDER M OLE O F COM POUNDXIDIZED

&Glucose . . . . . . . . . . 0.70 &Mannose. . . . . . . . . ..0.75 &Galac tose . . . . . . . . ..0.76

d-Fruc tose . .

. . . . . . . . 1.09 d-Arabinose. . . . . . . . .

0.81

&Xylose . , . . . . . . . . . . 0 . 4 5

Ery th r i to l .

. . . . . . . . .

.1.18 dl-Glyceraldehyde.. . 0 . 5 7 Glycolaldehyde.

. . . .

O . 52

nu mbe r of moles of glycolic acid form ed per mole of com pou nd oxidized

after th e reaction has proceede d for one hour a t 50 . Since the glycolic

acid obtained from the aldo-sugars tends to approach, but never exceed,

one mole per mole of com poun d used, and t h at from fructose an d ery-

thritol does exceed one mole, the glycolic acid can be easily accounted for

on th e basis of glycolaldehyde form ation arising in each case from t he

primary alcohol group and its neighboring carbon atom, formed by the

fragmentation of the sugar at th at point. T he forma tion of

2 ,3 -

and

3

,

-enediols would furnish additional primary alcohol groups in the aldo-

sugars a nd hen ce should furn ish more th an one mole of glycolic acid per

mole of sugar used. Fro m the da ta tabu lated it

is

seen that this does not

take place under o ur experimental conditions. Dr. Charles L. Bernier of

this laboratory has shown that d-glucose in aqueous potassium hydroxide

solutions a t 50 will yield 51.19% of lactic acid , th e for eru nn er of which

m u st be g l y ~e r a l d e h y d e .~ ~

I n view of th e da ta obtaine d with fructose and er ythr itol, and m ore

especially th e probab le fac ts with reference to glycolic acid form ation giv en

20 WEISENHEIMER, er., 41, 1009 (1908).

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MECHANISM OF CARBOHYDRATE OXIDATION 13

QLYCOL-

A L D E H Y D E

0.046

0 .324

0.590

0 . 3 6

above, it may be assumed that it is th e keto-hexose which will undergo

a

fragmentation to give erythrose and glycolaldehyde according to the fol-

lowing equation :

HO CHzOH CHzOH CHzOH

C-OH

C-OH CHzOH -C-OH

H-C-OH

H-C-OH

CHzOH

2,a-enediol) hyde, activ e activ e form)

I

I

I

I

bo

H--C:-oH o H - L H H-C-OH A

\ /

I

I

c

HO-( &-H HO-CH

I - + I

C-OH+ I

H-( --OH H- c -OH H-C-OH

CHzOH

I

CHZOHHZ

(d-f ructose) (d-keto-fructose) (d-fructose (glycolalde- (erythrose,

form)

That this fragmentation

of

th e fructose molecule probably does take place

under these experimental conditions seems to be borne out by the data

obtained from erythritol and th at obtained by using one-third of the values

BUM FRUCTOSE

. 1 7 0 0 . 1 8

0 . 9 1 8 0 . 8 5

1 .635 1 .77

1 . 1 6 3 1 . 1 7

TABLE

V

Carbon

Dioxide.

. . . . . . . . . . . . . . . . .

Oxal ic Ac id . . . . . . . . . . . . . . . . . . . . . .

Formic Acid . . . . . . . . . . . . . . . . . . . . .

Glycolic Ac id . . . . . . . . . . . . . . . . . . . .

OXIDATIONRODUCTSN MOLESPER

O X I D A T I O N P R O D U C T 8 E R Y T H R I T O I

0 .124

0 , 5 9 4

1 .045

0 .803

I

MOLE O F SUBSTANCE XIDIZED

DIFFEREWCB

-0 .010

+0.068

-0 .135

-0.007

TABLE

VI

OXYGENCONSUMPTION-MOLES

E R

MOLEOF SUBSTANCE XIDIZED

I

Oxygen, Used

. . . . . . . . . . . . . . . . . . . . .

5 .07

2 . 0 5 7 . 1 2

6 . 1 2 6 . 1 0

Oxygen, Calc'd . . . . . . . . . . . . . . . . . . . 4 . 8 8

2 . 0 1 6 . 8 9

5 . 8 9 5 . 8 6

from glycolaldehyde oxidation as a n app roximation to th e da ta which we

would ha ve obta ined had we had sufficient glycolald ehyde for all our ex-

perim ental purposes. If the keto-hexose undergoes fragmentation in this

manner the sum

of

the products obtained from the tetra-hydric alcohol

and .the glycolaldehyde should be approximately equal in value to those

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BUSCH, CLARK, GENUNG, SCHROEDER, AND EVANS

0.18

0.85

1 . 7 7

1 .17

6.10

obtained with fructose. T ha t this is approximately

so

under the experi-

mental conditions used

is

seen from Table

V.

Th e tetrose formed in this

reaction is believed t o fra gm en t into two m ore m olecules of glycolaldehyde.

0.04

o. 12

0.00

-0.09

f 0 . 0 5

TABLE VI 1

COMPARISON

F

OXIDATIONATA

OR

FRUCTOSEONE

MOLE)AND

GLYCOL-

A L D E H Y D E

(THREE

OLES)

O X I D A T I O N P R O D U C T S

Carbon

Dioxide.

. . . . . . . . . . . . . . . . . . . . . . . . . . .

Oxalic Acid.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Formic Ac id . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Glycolic Acid. .

............................

Oxygen Consumed. .

. . . . . . . . . . . . . . . . . . . . . . . .

0.14

0 . 9 7

1 . 7 7

1 . 0 8

6 . 1 5

Oxidaf ion

o f

Aqueous

Solutions

o f

d -

Fructrose (0.025 Molar)

and Glycol

Aldehyde (0.075 Molar)

with

Si lver Oxide

and

1.0N Alkali

a t

5 0 O C .

P e r c e n t a g e o f Carbon Returned from Glycol Aldehyde

4g7l

/&2W

Im07

d

2 r I

P e r c e n t a g e

o f

Carbon Returned f rom

d-Fructrose

= 9967 96100 D O a9

99B

- t /E

I I I I

-

2

6 8

I2 16

20

24

s

Hours

Atoms

of

Oxygen p e r Mole

of

d-Fructrose

Consumed 107 5.55 5 1 6.ID

Calculated 485 419 548 5: 6

Calculated

4.98

5.49 603

FIQ.4

Atoms of

O x y g e n

pe r Three Moles

of Glycol

A l d e h y d e

Consumed

4.98

549

6.G

(See Table

IV.)

The oxygen relations at this point (Table VI) are of

equal interest in th is connection.

W hen the da ta obtained with fructose at the end of 24 hours are com-

pared with those obtained from

a

three mole equivalent of glycolaldehyde

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MECHANISM O F CARBOHYDRATE OXIDATION 15

under the same conditions, the differences are sufficiently small to justify

th e belief th a t fructose is oxidized to i ts final products indicated in T able

YII , by way of a n erythrose

+

glycolaldehyde stage, the erythrose thus

formed th en yielding two mo re molecules of glycolaldehyde-the to ta l

oxidation being, in effect, equivalent to oxidation of three molecules of

glyco [aldehyde. T h e complete experimental d at a for these oxidations

are giraphically shown in Fig. 4.

Carbon

Dioxide.-An exam ination of the graphica l da ta will show t h a t

th e yields of carb on dioxide were uniform ly low thr ou gh ou t th is series of

experiments, much to our surprise. Among th e possible sources of carbon

dioxide are potassium formate, glycolaldehyde, and any possible keto-

acids formed as oxidation pro ducts in th e course of t he reaction.

I n a sep arate experiment w ith glycolic acid

(0.57

g.) only 0.29% of the

carbon was conv erted to carbon dioxide. At the end of twenty-four hou rs,

a

three molar solution of glycolaldehyde 0.75 molar) gave

2.3%

of its

carbon a s carb on dioxide. Since we had no available keto-acid we oxi-

dized galactonic lactone because, as has been show n, the seco ndary alcohol

TABLE

VI11

OXIDATION RODUCTS

F

GALACTOSEN D GALACTONICACTONE

QLYCOLIC

ACID

Galactose.

.

.

.

. .

.

. .

.

. . .

.

. . . .

. .

. . . . .

Galac tonic L actone , . .

.

. . . .

.

. .

.

.

.

.

CARBON

DIOXIDE

0 . 3 5

0 . 3 4

OXALIC

ACID

1 . 4 4

1 . 6 6

FORM IC

ACID

2 . 1 5

1 . 79

0 . 3 2

0 . 2 3

group is readily at ta ck ed with silver oxide in the presence of potassium

hydroxide. I ts da ta are compared a t this point with those of galactose

a t t he end of

24

hours, the results in each case being in moles per mole of

compound used (Table VIII). It would seem that the chief sources of

th e carbon dioxide in these experiments are th e possible keto-acids formed

as intermediate oxidation produc ts, a n d the slow oxidation of glycolic and

formic acids.

SUMMARY

1. Glucose, mannose, galactose, and fructose have been oxidized in

.025 molar solutions with silver oxide in the presence of

1.0 N

potassium

hydroxide. T he ultim ate products in each case were carbon dioxide a nd

oxalic, formic and glycolic acids.

2 . Through a similar study of a number of the available theoretically

possible intermediate degradation compounds light has been shed upon

th e oxidation mechanisms of th e abo ve hexoses. T he following intermedi-

ates have been studied in this connection: arabinose, erythritol (for

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16

BUSCH,

CLARK, GENUNG, SCHROEDER, AND EVANS

erythrose), glyceraldehyde, glycolaldehyde, galactonic lactone (for a keto-

acid). I n all these cases the ultim ate products were the same as those

obtained with the hexoses.

3.

Th e first stage in th e oxidation m echanism of mannose seems to be

th e fragm enta tion of this hexose into on e molecule each of fo rmald ehyde

and arabinose, since its oxidation d at a differ from those of th e pentose by

appro xima tely one mole of form ic acid.

4.

Th e & st stage in the oxidation m echanism of fructose seems to be

th e fra gm enta tion of thi s hexose int o eithe r one molecule each of glycol-

aldehy de an d eryth rose , or int o thre e molecules of glycolaldehyde, since

the quantitative data obtained from the fragments in the two cases are

practically the same as that obtained from the keto-hexose.

5 .

Th e behavior of glucose is best understood on t he assum ption th at

it undergoes oxidation in more t ha n one direction.

6 . Th e formaldehyde obtained in the fragm entation of these sugars and

related compounds is th e source of th e oxidation p rodu ct form ic acid.

We have indicated that oxalic acid and the chief portion of the carbon

dioxide is derived from the fragmentation and subsequent oxidation of

keto-acids. Evidence is given for th e general stabil ity of th e primary

alcohol group towards th e oxidation m edium ; hence th e presence of this

group as glycolic acid in the final reaction products.