LACTONE FORMATION OF CELLOBIONIC AND OF GLU- COARABONIC ACIDS AND ITS BEARING ON THE

STRUCTURE OF CELLOBIOSE.

BY P. A. LEVENE 9ND M. L. WOLFROM.*

(From the Laboratories of The Rockefeller Institute for Medical Research, Nem York.)

(Received for publication, March 10, 1928.)

A systematic study of the structure of disaccharides was under- taken under the incentive of the St. Andrews School, which, under the leadership of Purdie has developed the method of methyla- tion of polyhydric alcohols and of sugars. Over 25 years have passed since the method was introduced, and even to-day, there is no complete harmony between the views of individual workers on the structure of the common disaccharides.

In 1926 GBza Zemplen emphasized the necessity of introducing less drastic methods than those of the St. Andrews School for the study of the structure of disaccharides. Zemplen then sug- gested a way of progressive degradation of aldobioses until a sub- stance was obtained which no longer was capable of forming an osazone. It was then assumed by him that in the latter substance the hydrogen of the hydroxyl of carbon atom (2) was substituted by the second sugar radicle. The method undoubtedly is very important and ingenious. It has, however, one weak point; namely, that the final conclusion rests on a negative result, which sometimes may be accidental.

A still simpler method was suggested by Levene which is based on the observation of Levene and Simmsl that each non-substi- tuted sugar acid in solution passes into < 1, 4 > and < 1, 5 > lac- tones, and that the velocity of formation of each lactone is a function of the ring structure. With this information the structure

* National Research Fellow in Chemistry. 1 Levene, P. A., and Simms, H. S., J. Biol. Chem., 1925, Ixv, 31.

671

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672 Cellobionic and Glucoarabonic Acids

of a disaccharide can be determined from observations on the mutarotation of the bionic acid obtained on oxidation of the disac- charide and of the bionic acid obtained on removing carbon atom (1) from the original disaccharide. The argument may be best. illustrated by the following figures.

COOH----,---7, I I I / I

HCOR / I I

I 1 HCOH / ~

I I J&(-J+.1 I

H&OH--------J

I CHzOH

The hexonic acid gives two lactones. It cannot be de- graded to a pentose.

I.

COOH---,-P-T I

/ 1

HCOH ~ I COOH----,---

I I HCOR HCOR

,&OH-+- / I+ /

HCOH I

&OH--------; HioH ----I

I CHzOH &&OH------

Both the hexonic acid and the COP- responding pentonic acid give two la&ones.

II.

COOH -- l

HCOH I

HCOH I

HCOR I

HCOHm-pm I

CH,OH

7 / / I / I COOH---i / 1 / I / HCOH ~

J

The hexonic acid gives one la&one, < $5 >, and the corresponding pen- tonic acid two la&ones.

III.

1 COOHpm- I

HCOH I

HCOH I

HCOH----m I

HCOR I

CHzOH

I COOH----?;

/ I 1 HCOH i---f /

_I HCOH I

HCOR I

CH,OH---I

The hexonic acid gives one lactone, < 1, 4 >, and the cor- responding pentonic acid one la&one, < 1, 5 >.

IV.

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COOHp--T---l I / , /

HCOH j / COOH--- I

/ I I

HCOH I / I

HCOH I

HioH ----A -+ I

HCOH

I&OH ---.---p-i I

HCOHmpp-- I I

CH,OR CHzOR

The hexonic acid gives two lac- tones and the corresponding pentonic acid only one, < 1,4 >.

V.

The work on this pIan was begun by Levene and Sobotka2 in 1926 and was continued by Levene and Wintersteiner in 1927. The present investigation on the structure of cellobiose was begun in 1927 prior to the publication of the article of Haworth, Long, and Plant.4 From Tables I to IV and from Figs. 1 and 2 it is seen that cellobionic and the corresponding glucose pentonic acids behave like the substance of formula (III) and that therefore in cellobiose the reducing sugar is substituted in position (4). This conclusion is in harmony with those of Zempl&r,6 Irvine,‘j and Haworth, Long, and Plant.4

In the course of the present work an improvement was intro- duced in the purification of a-cellobiose octaacetate, a procedure was given for obtaining the oxime of cellobiose in crystalline form, an improvement was introduced in the method of thecon- version of the oxime into the nitrile octaacetate, a procedure was given for the preparation of the calcium salt of glucoarabonic acid in crystalline form, and also a procedure was developed for the preparation of basic lead acetate suitable for the precipitation of bionic acids and for some monocarboxylic sugar acids.

* Levene, P. A., and Sobotka, H., J. Biol. Chem., 1926-27, Ixxi, 471. 3 Levene, P. A., and Wintersteiner, O., J. Biol. Chem., 1927, lxxv, 315. 4 Haworth, W. N., Long, C. W., andPlant, J. H. G., J. Chem. Sot., 1927,

cxxxi, 2809. 3 ZemplBn, G., Ber. &em. Ges., 1926, lix, 1254. 6 Irvine, J. C., Chem. Rev., 1927, iv, 203.

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674 Cellobionic and Glucoarabonic Acids

EXPERIMENTAL.

Preparation of Calcium Cellobionate.

Crude a-cellobiose octaacetate, prepared from cellulose, was recrystallized several times from a mixture of 1 part of glacial acetic acid and 5 parts of 96 per cent alcohol. The addition of the acetic acid requires the use of less alcohol and gives about the same yield as when alcohol alone is used. The first recrystallization was easily effected by dissolving the material in 12 parts of the hot solvent mixture instead of the 30 parts required when alcohol alone is used. Further recrystallizations require the higher amount of solvent. The purified material showed a melting point of 222.5’ and the following specific rotation in chloroform.

Ialst = + 3.23” X 100 D 2 x 4.02

= + 40.2”.

Hudson and Johnson7 give the m.p. 229.5” and’ [arz = + 41” in chloroform for highly purified u-cellobiose octaacetate.

The octaacetate was deacetylated with sodium methylate ac- cording to the method of Zempl&P and the sugar recrystallized from alcohol and water. This material, dried in a vacuum oven, showed a specific rotation 13 minutes after solution of +22” and a final equilibrium value in wat’er as follows:

[a]‘,’ = + 1.55O x 100 = + 35,0” 1 x 4.43

Hudson and Yanovsky8 give the equilibrium value f35” for pure cellobiose and the value +26” 10 minutes after solution.

Cellobionic acid and a number of its salts were first prepared in amorphous condition by Maquenne and Goodwin.g We em- ployed the general method of Willstatter and Schudel as modified by GoebeP” in oxidizing cellobiose to the bionic acid. 18 gm. (I

7 Hudson, C. S., and Johnson, S. &I., J. Am. Chem. Sot., 1915, xxxvii, 1276.

8 Hudson, C. S., andyanovsky, E., J. Bm. Chem. SOL, 1917, xxxix, 1035. 9 Maquenne, L., and Goodwin, W., Bull. Sot. chim., 1904, xxxi, 854. lo Goebel, W. F., J. Biol. Chem., 1927, lxxii, 801.

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mol) of cellobiose were dissolved in 700 cc. of a solution containing 27 gm. (2 mols) of iodine and 50 gm. of barium iodide. To this was added over an interval of 5 to 10 minutes, with mechanical stirring, a solution of 50 gm. (3 mols) of barium hydrate in 800 cc. of water. The solution was allowed to stand 15 minutes, after which a solution of 18 cc. of concentrated sulfuric acid in 130 cc. of water was added under strong mechanical stirring. This was followed by the immediate addition of 220 gm. of washed basic lead carbonate. Under continual stirring the solution soon be- came neutral to Congo red. The precipitate was allowed to settle, filtered on a prepared bed of kieselguhr and charcoal, and the filtrate concentrated under reduced pressure to about 400 cc. The solution was filtered from the separated lead iodide; most of the lead was removed with sulfuric acid and the iodide ion with silver sulfate. After filtration, the remaining lead and silver were removed with hydrogen sulfide, the hydrogen sulfide by air, and the solution again filtered. In the filtrate the excess sulfate ion was removed quantitatively with barium hydroxide and the solution filtered. The filtrate was shaken with an excess of well washed, precipitated calcium carbonate for 15 minutes. Short boiling completed the formation of the calcium salt. The solution was cooled, filtered, and concentrated under reduced pressure to a thin syrup. This was poured into 12 to 15 volumes of anhydrous methyl alcohol. The salt precipitated as a dense, amorphous solid. This was taken up in about 3 times its weight of water and precipitated as a syrup by the addition of 1s volumes of 96 per cent alcohol. The supernatant liquid was decanted, the residue dissolved in a small amount of water, and poured into 10 to 15 volumes of absolute methyl alcohol. This procedure was repeated. Due to the high solubility of this salt, only 2.5 gm. of purified material were obtained. This, as well as the cadmium andbarium salts, resisted repeated attempts at crystallization. The salt analyzed as follows :

6.499 mg. substance: 11.65 mg. CaS04. 0.1456 gm. dried substance: 0.2028 gm. COz and 0.0720 gm. HZO. Mois-

ture, 7.30 per cent. Ca(C12H2101~)2. Calculated. C 38.18, H 5.61, Ca 5.31.

Found. ‘( 37.98, “ 5.53, “ 5.30.

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676 Cellobionic and Glucoarabonic Acids

The salt was kept in a weighed container over calcium chloride and subsequent weighings were corrected for a slow loss of mois- ture.

TABLE I.

Changes in Rotation of 0.090 N Cellobionic Acid Solution.

Experiment 1. 1 = 2 dm., t = 22”, X = 5892 b.

Time. a

min.

2 6 8

15 30 45 hrs.

1 2 4 6

20

-0.21 -3.3 -0.15 -2.3 -0.14 -2.2 -0.12 -1.9 -0.09 -1.4 -0.04 -0.6

SO.02 1-0.3 +0.05 $0.8 +0.05 $0.8 $0.05 $0.8 $0.06 $0.9

[aID

-i- I Experiment 2.

1 = 4 dm., t = 23”, X = 5892 A.

Time. a

min.

1

2 6 8

23 32 45 hrs.

1 2 4 6

24

degrees degrees -0.41 -3.2 -0.35 -2.7 -0.33 -2.5 -0.30 -2.3 -0.15 -1.2 -0.06 -0.5 -0.02 -0.2

+0.03 +0.09 +0.13 +0.13 +0.14

[aID

+0.2 +0.7 +1.0 +1.0 +1.1

TABLE II

Titration of 1.00 Cc. of 0.090 N Cellobionic Acid Solution with 0.1 a~’ Sodium Hydroxide.

Experiment 1. t = 22”

Time. 0.1 N NaOH.

hrs. cc.

1 0.90 2 0.89 5 0.89 8 0.90

20 0.89

Time.

hrs.

1

5

23

0.1 N NaOH.

cc.

0.90

0.90

0.91

Mutarotation and Titration Experiments.

A weight of 0.9020 gm. of calcium cellobionate containing 5.44 per cent of water, thus corresponding to 0.8529 gm. of dry sub- stance, was dissolved in about 15 cc. of water. The calculated

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amount, 4.52 f 0.01 cc. of 0.5 N hydrochloric acid was added and the solution quickly made up to 25.00 cc. The solution was thus 0.0905 N. The change in rotation was observed and samples of 1.00 cc., obtained with an accurate micro pipette, were titrated at suitable intervals with 0.1 N sodium hydroxide (phenolphthalein) delivered from a micro burette. Care was exercised that the water used in the experiments did not contain sufficient carbon dioxide to influence the end-point. The data are recorded in Tables I and II and the polarimetric change obtained is plotted in Fig. 1.

The interpolated initial value for the specific rotation of the free acid is -3.6”. At the time of the last reading the solution gave no reduction with Fehling’s solution, showing that no appar- ent hydrolysis had taken place.

FIG. 1. <1,5> lactone formation of cellobionic acid. Experiment 1.

Preparation of Calcium Glucoarabonate from Cellobiose.

I. Preparation of Crystalline Cellobiose Oxime.-For the prepa- ration of glucoarabinose the procedure of Zemp16n5 was followed with some modification. We were able to prepare cellobiose oxime in crystalline condition. The procedure of ZempMn was followed except that we used pure (99 per cent by Raschig’s reduction pro- cedure) hydroxylamine hydrochloride and removed the excess hy- droxylamine, after oxime formation, by distillation under reduced pressure, in the presence of solid calcium carbonate to neutralize any acidity. The quantities used by Zempldn in the decomposi- tion of the hydrochloride with sodium methylate leave theformer

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678 Cellobionic and Glucoarabonic Acids

in slight excess. The crude residue was dried by distillation with absolute alcohol. On nucleation the material crystallized. This material was then stirred with warm 96 per cent alcohol, cooled in an ice-salt mixture, filtered, washed with absolute alcohol, and dried. For the purposes of the next step this material is sufficiently pure. A product more nearly pure was obtained as follows: The crude evaporated product from 43 gm. of cellobiose was dissolved in 3 to 4 times its weight of water, decolorized at room temperature, and filtered. To the filt,rate ace- tone was added with stirring to opalescence. The solution was then nucleated and allowed to stand, first at room temperature and then for some time in the refrigerator. It was filtered, washed with 80 per cent acetone, and then with pure acetone. A weight of 24.2 gm., m.p. 122-123”, was so obtained. The mother liquor was concentrated to 50 cc. and the procedure repeated, 8.2 gm. of prod- uct melting at 119-120” being obtained. The filtrate was con- centrated to a thin syrup and treated with hot 96 per cent alcohol to slight turbidity, nucleated, and kept overnight in the refrigera- tor. A further quantity of 1.5 gm. of product melting at 119-120” was obtained, making a total yield of 76 per cent.

Cellobiose oxime crystallizes in plates, is very soluble in water, slightly soluble in cold methyl and ethyl alcohols, moderately soluble in warm methyl and ethyl alcohols, and insoluble in ace- tone, ether, benzene, ethyl acetate, and petroleum ether (40-60’). On one recrystallization from 80 per cent ethyl alcohol the material melted at 123-125”. A second recrystallization from the same sol- vent gave material melting at 123-125’ and having [a]: = -26.1” (1.0313 gm. of substance, 25 cc. of aqueous solution, Q: = -2.15”, 2 dm. tube) in aqueous solution, changing at a very slow rate in the dextro direction. A third recrystallization gave material melting at 123-125” and showing the initial [o(]r = -26.1” (1.1131 gm. of substance, 25 cc. of aqueous solution, 01 = -2.32’, 2 dm. tube). This purified material analyzed as follows :

5.310 mg. substance: 0.185 cc. Ng (760 mm., 24”). C12H23011N. Calculated. N 3.92.

Found. ‘( 4.00.

II. Preparation of Cellobionic Acid Nitrile Octaacetate.-The procedure of ZempEn calls for the acetylation of the syrupy oxime

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reaction mixture and in our experience always produced a violent reaction with loss of material and resin formation. With the crystalline oxime this reaction may be easily controlled by the following procedure. A mixture of 140 cc. of acet,ic anhydride and 21 gm. of fused sodium acetate was heated in an oil bath to 110”. The crystalline oxime (21 gm.) was then added in small portions. Reaction began immediately and the mixture was removed from the oil bath. The addition was continued, the rate being regulated so as to maintain the mixture at llO-115’, and the reaction’s own heat being utilized. The addition required about half an hour and was followed by 1 hour of heating at 110”. The preparation was finished according to the directions of ZemplBn, the recrystallized product melting at 132’ in agreement with ZemplBn. It is sug- gested for similar procedures with other sugars that, where the oxime does not crystallize, it be added gradually to the acetylating mixture in the form of an amorphous powder.

III. Preparation of Calcium Glucoarabonate from Clucoarabinose Ileptaacetate.-Glucoarabinose heptaacetate was prepared from cellobionic acid nitrile octaacetate according to the directions of ZemplBn. The recrystallized material melted at 194” and showed the following rotation in chloroform.

[a]:: = - 1.20” x 100 = _ 14”

2 X 4.42

ZernpEn records the melting point of 196” and [oL]~ = -16.95” in chloroform solution. 12.5 gm. of the acetylated sugar were deacetylated according to the directions of Zemplen and the resulting solution of sugar and sodium acetate concentrated to 40 cc. to remove methyl alcohol. This solution was oxidized to the aldobionic acid by exactly the same procedure as was previously described for the preparation of cellobionic acid. The amounts of oxidizing reagents used were 19 gm. of iodine, 37 gm. of barium iodide in 500 cc. of water, and 46 gm. of purified barium hydrate in 725 cc. of water, 12 minutes being taken for the addition of the alkali. After removal of the silver and lead ions, the solution containing the oxidation product together with a considerable amount of sodium acetate was neutralized with barium hydrox- ide solution until the solution was just acid to litmus. The

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680 Cellobionic and Glucoarabonic Acids

barium sulfate was removed by filtration and the filtrate concen- trated under reduced pressure at 40’ to a volume of 40 cc. A saturated solution of basic lead acetate was added to the solution until no further precipitation occurred. The voluminous precipi- tate so obtained was filtered and washed with a small amount of very cold water. To the filtrate barium hydroxide was added and a further quantity of precipitate obtained, which was combined with the first.

Fischer and Meye+ used basic lead acetate in the preparation of maltobionic acid. They stated that the common form of basic lead acetate did not precipitate the acid and described a method of preparing a basic compound suitable for this purpose. We were unable to obtain active material by repeating their procedure. The right form of basic lead acetate may easily be prepared in crystalline condition by adding barium hydroxide to a solution of ordinary basic lead acetate until a precipitate just begins to form. The solution is then concentrated until a separation of the active basic lead acetate in well formed needles takes place. These are removed by filtration.

The combined precipitates of the basic lead salt of the sugar acid were suspended in about 500 cc. of water and hydrogen sulfide passed into the solution. The solid material was filtered, ground in a mortar, and resuspended in the original solution, and again treated with hydrogen sulfide. This process was repeated once. The solution was filtered from the lead sulfide and the hydrogen sulfide removed from the filtrate with a current of air. A trace of barium was removed with sulfuric acid and the most of a small amount of acetic acid present removed by repeated extraction with a considerable volume of chloroform. The filtered solution was shaken with an excess of precipitated and washed calcium carbon- ate for 15 minutes, boiled 8 minutes, and allowed to stand over- night. After filtration, the solution was concentrated at 40” to a thin syrup and this poured into 10 to 15 volumes of absolute methyl alcohol. The amorphous material was dissolved in water and a mixture of methyl alcohol and acetone added to opalescence. On heating this mixture the material crystallized in radiating clusters of needles. The recrystallization process was repeated,

I1 Fischer, E., and Meyer, J., Ber. them. Ges., 1889, xxii, 1941.

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TABLE III.

Changes in Rotation of 0.112 N Glucoarabonic Acid Solution.

1 = 4 dm. x = 5892 ;i

Time.

min.

2.5 5 7.5

10 15 30 45

hrs.

1 1.5 2 2.5 3 4 5 6

10 23 29

-

- . a [aID

degrees degrees

+2.94 +20.0 2.87 19.5 2.84 19.3 2.82 19.2 2.78 1819 2.71 18.4 2.68 18.2

2.63 17.9 2.56 17.4 2.51 17.1 2.48 16.9 2.50 17.0 2.52 17.1 2.54 17.3 2.57 17.5 2.65 18.0 2.82 19.2 2.91 19.8

-

--

-

Time.

min. degrees degrees

6 10 15 30 45 hrs.

1 1.5 2

+2.75 +18.7 2.74 18.6 2.69 18.3 2.62 17.8 2.56 17.4

2.54 17.3 2.51 17.1 2.48 16.9

3 2.42 16.5

5 2.51 7 2.55

24 2.86

17.1 17.3

19.4

a

-

TABLE IV.

Titration of 1 .OO Cc. of O.lld N Glucoarabonic Acid Solution with 0.1 N

Sodium Hydroxide.

EH pteri_x& 1. Experiment 2. t = 23”

Time. 0.1 N NaOH. Time. 0.1 N N&OIL

hrs. cc. hrs. cc.

0.5 1.10 1 1.10 2 1.07 1.5 1.10 4 1.05 5.5 1.05

10 1.03 30 0.89 24 0.86

The permanent titer found by titrating the hot solution was 1.13 cc.

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682 Cellobionic and Glucoarabonic Acids

5 gm. of material being obtained. The analytical results of the material dried first at 100” and then at 110’ under reduced pressure over sulfuric acid indicated that it still contained 1 molecule of water. This behavior is similar to that of the crystalline calcium galactoarabonate obtained by Levene and Wintersteiner.

0.1078 gm. substance: 0.0088 gm. CaO. 0.1046 “ “ : 0.1424 “ CO, and 0.0514 gm. H,O.

Ca.(CIIR1~OI1)l.IlzO. Calculated. C 37.06, H 5.66, Ca 5.63. Found. “ 37.12, “ 5.49, “ 5.83.

FIG. 2. <1,5> and <1,4> la&one formation of glucoarabonic acid. Experiment 1.

The rotation of the calcium salt was as follows:

La1” = + 0.27” X 100 D 1 x 1.88

= + 14.4”.

Another preparation gave the following result.

[Jl = + 0.43” x 100 D 1 x 3.03

= + 14.2”.

Mutarotation and Titration Experiments.

A weight of 1.000 gm. of calcium glucoarabonate containing no water other than the molecule of water of crystallization, was dis- solved in about 15 cc. of water. The calculated amount, 5.61 f 0.01 cc., of 0.5 N hydrochloric acid was added and the solution

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quickly made up to 25.00 cc. The solution was thus 0.1123 N.

The change in rotation was observed and samples of 1.00 cc. ti- trated at suitable intervals with 0.1 N sodium hydroxide (phenol- phthalein) as described with cellobionic acid. The first end- point obtained was recorded. The data obtained are listed in Tables III and IV and the polarimetric change is plotted in Fig. 2.

The interpolated initial value for the specific rotation of the free acid is +21”. At the time of the last reading a sample of the solution containing 30 to 40 mg. of t,he sugar acid gave no re- duction with Fehling’s solution, showing that no apparent hydrol- ysis had taken place.

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P. A. Levene and M. L. WolfromCELLOBIOSE

BEARING ON THE STRUCTURE OF GLUCOARABONIC ACIDS AND ITS

CELLOBIONIC AND OF LACTONE FORMATION OF

1928, 77:671-683.J. Biol. Chem. 

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