ON CONDENSATION OF MONOSACCHARIDES MEANS OF DILUTE MINERAL ACID.

BY P. A. LEVENE
(From the Laboratories (Received of The Rockefeller for publication,

BY

AND R. ULPTS.
Institute April for Medical 30, 1925.) Research.)

The results to be reported in this communication had their origin in an unexpected observation made in the course of the preparation of chitose. It was stated in a previous paper that the 2,5-anhydrosugars are capable of existing in only one form; namely, the aldehydic. Only one 2,banhydrohexose has been prepared in cryst,alline form; namely, epichitose. The preparation of this sugar is very laborious and therefore an attempt was made to obtain it either in a crystalline or at least in a sufficiently pure condition for the preparation of some important derivatives. An unexpected result was obtained at this phase of the work. Instead of an alcohol-soluble product which was expected, a product practically insoluble in all common reagents was obtained. It was insoluble in dilute aqueous alkalies and acids. It could be washed free of adhering mineral acids by means of alkali. On the other hand, the substance was very readily hydrolyzed by heating with very dilute mineral acids. The hydrolysis by boiling with 2 per cent sulfuric acid is completed in approximately 90 minutes. In fact, the substance is 80 per cent hydrolyzed as soon as its solution is accomplished in boiling 2 per cent sulfuric acid. The specific rotation of the completely hydrolyzed and partially hydrolyzed substance is practically unchanged and is the same as that of uncondensed chitose; namely, in the neighborhood of [(YE = +33.0 (&l.O). It thus became evident that, under the conditions above described, a condensation of chitose was effected. It is not certain whether the condensation product was a di- or polysaccharide in view of the fact that the insolubility of the product did not permit the measurement of its molecular weight. An attempt was then made to prepare chondrose. Hope was
475

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476

Condensation

of Monosaccharides

entertained that this sugar would be obtained crystalline. Chondrose (2,5-anhydrotalose) has not as yet been prepared. The deamination of chondrosamine was accomplished by means of nitrous acid. The reducing power of the substance was equal to 55 per cent of that of glucose and its specific rotation was found to be [a]: = +20.0, calculated on the basis of the quantity of deaminized chondrosamine. When the solution was concentrated under the same condition as that of chitosamine, a solid residue was obtained which was still soluble in water, but only partially soluble in glacial acetic acid. The product which remained insoluble in glacial acetic was no longer soluble in water. When the original substance was redissolved in water and reprecipitated by means of alcohol, the resulting product still possessed some reducing power, but equivalent only to about 22 per cent of the original. On hydrolysis, an increase in reducing power was observed. The specific rotation of the substance was [a]?,= 20.5. The same value was obtained foruncondensedchondrose. The molecular weight of the soluble product was 351. Thus, it became evident t,hat 2,5-anhydrohexoses condense quite readily. The question naturally arose whether this property was a peculiarity of the aldehydic forms of sugars in contrast with the saccharidic.r It therefore became desirable to test the behavior of simple monosaccharides when treated under the conditions which brought about the condensation of the 2,5-anhydrosugars. In the past, glucose has been condensed to di- and polysaccharides, but always through the action of concentrated acids. Experiments here reported deal with glucose, mannose, and lyxose. Glucose.-A solution containing 10 per cent of glucose and 1 per cent hydrochloric acid, evaporated under a pressure of about 10 to 12 mm. in a water bath, temperature of 45-5OC., and further treated as in the experiment of chitose, formed a solid residue which was soluble in water, but insoluble in alcohol and other organic solvents. It was only faintly colored, was no longer sweet, had only a slight reducing power, and a specific rotation of [a], = +95. The reducing power of the substance was about 12 per cent of that of glucose. It was not possible to prepare a phenylosazone of this product. From the hydrolyzed
1 Saccharidic is proposed g lucosidic or oxidir I. to be used in place of the confusing terms

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P. A. Levene and R. Ulpts

477

material, the osazone was readily obtained in very good yieldapproximately 1.0 gm. of osazone from 1 gm. of the product. The molecular weight of the substance was 342. As compared with condensed chitose this substance had the following peculiarities. It was more soluble in water but showed greater resistance towards hydrolytic agents. Apparently it is a disaccharide. The product was completely hydrolyzed by 2 per cent sulfuric acid in a sealed tube at the temperature of the boiling water bath only after 420 minutes, whereas the waterinsoluble product of chitose was practically completely hydrolyzed in 90 minutes. Mannose and Lyxose.-These sugars behaved in exactly the same manner as glucose. In the experiments thus far reported, alcohol was used to bring tlhe product to a state approaching complete dryness and, therefore, there was a possibility that the condensation product was a mixture of the CY-and P-ethyl saccharides. True, the solubilities of the condensation products differed from simple ethyl saccharides. Furthermore, it was found that the condensation product was formed even when the step of the operation which involved t,he use of alcohol was omitted. As to the structure of the condensation product, little can be said. It may be suggested that in the main, the condensation products are built upon the plan of trehalose. Thus the product formed in the absence of alcohol which was quite impure showed a reducing power equal to 25 per cent of glucose, whereas a disaccharide of the type of maltose should possess the reducing power equal to 50 per cent of that of glucose. However, since the substances are all amorphous, and since the products dried by means of alcohol still contain some alcohol, the possibility is not excluded that admixtures were present of either l-ethyl disaccharides or of l-ethyl monosaccharides. The information obtained from the observations on the reactions with enzymes is not very conclusive. Thus the products were not hydrolyzed by invertase, diastase, and maltase. Emulsin hydrolyzed the condensation products to some extent. Thus, compared with /3-methyglucoside, the condensation product of glucose was hydrolyzed only to the extent varying from 35 to 50 per cent and that of chitose to the extent of 20 to 30 per cent.

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478

Condensation

of Monosaccharides

From the view-point of the peculiarities of 2,5-anhydrosugars two very important points were brought out. First, the specific rotations of the condensation products of the simple sugars were higher than those of the equilibrium forms of the sugars whereas those of the 2,5-anhydrosugars had practically the same specific rotations as the original sugars. Such behavior is to be expected in view of the fact that 2,6anhydrosugars are capable of existing Second, the condensation products of only in the aldehydic form. the 2,5-anhydrosugars seem less stable than those of the simple sugars and this peculiarity also may be attributed to the differences in the functions of carbon atom (1) in the simple and in the 2,5-anhydrosugars.
EXPERIMENTAL PART.

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Chitose. Chitosamine hydrochloride recrystallized until free from mineral impurities was deaminized in the cold by means of silver nitrite and hydrochloric acid until the amino nitrogen could no longer be detected in the solution. At the end of the operation it was found that 0.1637 gm. of chitose had the reducing power of 0.1000 gm. of glucose. The specific rotation of chitose was found to be [& = $33.2. Condensation of Chitose.

The solution obtained on deamination of 20.0 gm. of chitosamine hydrochloride was evaporated in a 1 liter distilling flask under pressure of 11 to 14 mm., from a bath of 4045. The remaining syrup was evaporated three times with 75 cc. of 95 per cent alcohol and then four times with 75 cc. of 98 per cent alcohol. The temperature of the bath was allowed to rise to 95-100. At the final temperature the distillation was continued for 15 minutes. The residue weighed 13 gm. and was it brittle, slightly colored, amorphous substance, insoluble in the usual solvents, glycerol and dilute alkali; dilute mineral acids dissolved it only after hydrolysis. The product was powdered, stirred with 2 per cent ammonia water, filtered, and washed with hot water, alcohol, and ether. The yield was 9 gm. The substance was dried at 60C. under reduced pressure. The substance turned

P. A. Levene and R. Ulpts

479

faintly yellow at 15OC., and at 222C. remained solid. The reducing power of the substance suspended in water was equal to 6 per cent of that of glucose. The substance contained about 14 per cent of alcohol when dried at 80C. For analysis, the substance was dried at 150C. under reduced pressure.
0.0956 gm. substance: 0.1760 gm. COz Calculated. GJLsOo. CHO 26 13. 18 Found. gm. substance: 0.0806 Found. and C 0.0530 gm. HzO. 47.2, H 5.89. 48.0, 5.78. 50.20, 6.2.

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0.1085

gm. Ag I. OCzHs 14.23.

Hydrolysis

of the Condensed Chitose.

The reducing power of chitose measured by the MaquenneLehman method was found to be equal to 60.7 per cent of that of glucose. Since chitose was available only in solution obtained on deamination of chitosamine, it was desirable to determine the reducing power of crystalline epichitose. It was found that 0.1000 gm. of glucose had the same reducing power as 0.1640 gm. of epichitose. Hence, the reducing power of epichitose is the same as that found for chitose; namely, 60.7 per cent of that of glucose. 3 gm. of condensed chitose in 150 cc. of 2 per cent sulfuric acid were heated in a sealed test-tube to 100C. for 30 minutes. The tube was rapidly cooled to 20C. and the solution distributed into four tubes which were then sealed and heated in a steam bath.
Time heated. Reducing power of 0.100 gm. as glucose. Calculated as chitose.

min.
30 60 90 120 150 0.0415 0.0425 0.0493 0.0500 0.0515 0.0681 0.0968 0.0810 0.0820 0.0845

The optical rotation and found to be

was unchanged throughout

the experiment

Ial2o = fo.64 X 100 = + 32 D 1x2

480

Condensation

of Monosaccharides

In another experiment, 0.5000 gm. was heated in a steam bath with 25 cc. of 2 per cent sulfuric acid until solution was completed (10 minutes after placing in bath) ; the solution was rapidly cooled to 25 and the rotation taken in a 1 dm. tube. [cr12i= f34.5. The reducing power of 0.1000 gm. is at that time equal to 0.0415 gm. of glucose. Condensation of Chondrose.

When chondrosamine hydrochloride was treated in the same way as glucosamine hydrochloride for the preparation of chitose, a solution was obtained containing 14.5 gm. of chondrose in 250 cc. of solution with a specific rotation of [cr12i= +20. Its reducing power was equal to 54.64 per cent of that of glucose. The solution, evaporated under the same conditions as chitose, gave a light yellow dry residue which was readily pulverized. The yield was from 9 to 10 gm. from 20 gm. of chondrossmine hydrochloride. The substance had a bitter taste, was partly soluble in glacial acetic acid, and on cooling, partly settled out. This precipitate could not be completely redissolved in water. After reprecipitating a concentrated aqueous solution of the crude product with thirteen times its volume of 98 per cent alcohol, a substance was obtained which had the following specific rotation.

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[J

= +

0.41" 1x2

x 100 = +

20.5"

The reducing power of 0.1000 gm. equals that of 0.0140 gm. of glucose. When once more purified in the same manner, the rotation did not change and the reducing power of 0.1000 gm. was lowered to the value of 0.0125 gm. of glucose. The molecular weight of the substance determined cryoscopically was 351. Condensation of Glucose.

10 gm. of glucose were dissolved in 80 cc. of water. 5 cc. of 10 per cent hydrochloric acid were added and the solution was evaporated under diminished pressure in a 1 liter flask from a bath of 40-45. The residue was three times evaporated with 75 cc. of 95 per cent alcohol and then four times with 75 cc. of 98 per cent alcohol, allowing the temperature to rise gradually to 95-100 and keeping it at that temperature for 15 minutes. The brittle res-

P. A. Levene and R. Ulpts

idue was readily soluble in cold water, reduced Fehlings and had the following optical rotation.
Ial = + 1.90 x D 2x1 100 = + 9.s

481
solution,

0.1000 gm. had a reducing power equal to that of 0.0093 gm. of glucose. Rate of Hydrolysis of Condensed Glucose.-3.0000 gm. of the substance were dissolved in 150 cc. of 2 per cent sulfuric acid and heated in seven separate sealed tubes in a steam bath. NO. Time.
min. 00

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Reducing power of 0.1000 gm. equals gm. glucose.

Temper&m. C.

30 60 90 120 180 300 420

0.0093 0.0275 0.0376 0.0470 0.0520 0.0655 0.0767 0.0865

95.0 86.5 81.0 78.5 73.5 65.5 65.5 57.0

There remained 74 cc. of the solution containing 1.3 gm. of glucose. The free sulfuric acid was neutralized with sodium acetate and to this solution 2.2 cc. of phenylhydrazine and 2 cc. of glacial acetic acid were added and the solution was allowed to stand on the water bath for 1% hours. The yield of glucophenylosazone was 1 gm. Once recrystallized from methyl alcohol, it melted at 204-205. The osazone in pyridine-alcohol (2:l) at 21 in a 1 dm. tube gave OL, = - 0.72 (c = 1). After 24 hours: o!D = - 0.28.
CJBHS~O~N,. Calculated. Found. N 15.47. 15.43.

Molecular weight determination gave a value of 309.

by

the cryoscopic

method

Condensalion of Mannose. 10 gm. of mannose with the specific rotation of [LY]~~= +14.5 (having the reducing power of 0.095 gm. of glucose) were dissolved in 125 cc. of water and 10 cc. of 10 per cent hydrochloric acid.

482

Condensation

of Monosaccharides

This solution was treated in the same manner as the solution of glucose. The yield was 10 gm. of a bitter tasting, brittle, amorphous substance, soluble in water. It was redissolved in 10 cc. of warm water and precipitated with 240 cc. of absolute alcohol. The operation was repeated twice and the final yield was 6.5 gm. The molecular weight was 378. The optical rotation of the substance was as follows:

[a],= + 1.19x 100= + 5g5 1x2

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The substance did not reduce Fehlings solution. The reducing power of the substance after hydrolysis for 16 hours in a sealed tube placed in a steam bath was equal to 80 per cent of its weight of glucose and the specific rotation of the product of hydrolysis calculated on this basis was [ar]2: = +13. From this solution a levorotatory phenylosazone was obtained which had a melting point of 203-205C. and had the following. composition.
0.1000 gm. substance: 13.7 cc. nitrogen Found.

CI&~O~N~. Calculated.

gas at 26C. and 757.4 mm. N 15.47. 15.59.

Condensation of Mannose in the Absence of Alcohol. 10 gm. of mannose were dissolved in 125 cc. of water and 10 cc. of 10 per cent hydrochloric acid. This solution was evaporated under reduced pressure from a bath of 45C. (1 liter flask, wide capillary, pump of large capacity). After 20 minutes the contents. of the flask had a syrupy consistency. The temperature of the bath was slowly raised to 65 when the syrup began to solidify. 10 minutes later the temperature was raised to 73. The reaction was interrupted and the solid, somewhat colored product was removed from the flask. It still contained a little hydrochloric acid.. The yield was about 8 gm. The optical rotation of the substance was

[/

= + 0.33" x 100 = +33"

1x1

The substance was dissolved in 5 cc. of water and reprecipitated with absolute alcohol. The optical rotation of the substance then rose slightly to

P. A. Levene and R. Ulpts

Ial D = + 0.74 x 1x2 100 = + 37

483

The reducing power of this substance was equal to 20.5 per of its weight of glucose. The reducing power rose to 82.5 per of glucose after an 8 per cent solution of the substance in 2 per sulfuric acid was heated for 6 hours in a sealed tube placed steam bath. The specific rotation of the mannose in the solution was [a]: = +13.75. Condensation of Lyxose.

cent cent cent in a final

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10 gm. of lyxose ([aID = - 13) were dissolved in 90 cc. of water and 10 cc. of hydrochloric acid and the solution was concentrated in the same manner as described in previous experiments. The yield was 10.0 gm. of a dry substance easily pulverized and very soluble in water. The molecular weight of the substance was found to be 338. The optical rotation of the substance was the following.
falz, = + 0.83 X 100 = + 41.5 D 1x2

The substance
0.1020 0.0975 gm.

had the following

0.1728 : 0.0538 gm.

composition.
CO, AgI. and 0.0618 gm. H,O.

substance: (

The composition of the substance tose with 1 mol of alcohol.

CloHlaOg + C2H60H. Calculated. Found.

approached
C 43.9, * 45.75, H 7.3, 6.71,

that of a dipenOCzHs 13.7. 10.57.

The substance was hydrolyzed by means of a 2 per cent solution of sulfuric acid. After 14 hours the reducing power of the hydrolysate was equivalent to 85 per cent of glucose. From the hydrolysate a phenylosazone was obtained which melted at 162C., was levorotatory, and had the following composition.
0.0965 gm. substance: 15.00 C,&oO~N~. cc. nitrogen Calculated. Found. gas at 24 C. and 749.4. N 17.07. 17.60. mm.