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ON CONDENSATION OF MONOSACCHARIDES BYMEANS OF DILUTE MINERAL ACID.

BY P. A. LEVENE AND R. ULPTS.(From the Labora tories of Th e Rocke feller Institute for Medica l Research.)

(Received for publication , April 30, 1925.)

The results to be reported in this communication had theirorigin in an unexpected observation made in the course of thepreparation of chitose. It was stated in a previous paper that the2,5-anhydrosugars are capable of existing in only one form;namely, the aldehydic. Only one 2,banhydrohexose has beenprepared in cryst,alline form; namely, epichitose. The preparationof this sugar is very laborious and therefore an attempt was madeto obtain it either in a crystalline or at least in a sufficiently purecondition for the preparation of some mportant derivatives. Anunexpected result was obtained at this phase of the work. In-stead of an alcohol-soluble product which was expected, a productpractically insoluble in all common reagents was obtained. It wasinsoluble in dilute aqueous alkalies and acids. It could be washedfree of adhering mineral acids by means of alkali. On the otherhand, the substance was very readily hydrolyzed by heating withvery dilute mineral acids. The hydrolysis by boiling with 2 percent sulfuric acid is completed in approximately 90 minutes. Infact, the substance is 80 per cent hydrolyzed as soon as ts solutionis accomplished in boiling 2 per cent sulfuric acid. The specificrotation of the completely hydrolyzed and partially hydrolyzedsubstance is practically unchanged and is the same as that ofuncondensed chitose; namely, in the neighborhood of [(YE =+33.0 (&l.O). It thus became evident that, under the condi-tions above described, a condensation of chitose was effected. It isnot certain whether the condensation product was a di- or polysac-charide in view of the fact that the insolubility of the product didnot 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 Monosaccharidesentertained that this sugar would be obtained crystalline. Chond-rose (2,5-anhydrotalose) has not as yet been prepared. Thedeamination of chondrosamine was accomplished by means ofnitrous acid. The reducing power of the substance was equal to55 per cent of that of glucose and its specific rotation was foundto be [a]: = +20.0, calculated on the basis of the quantity ofdeaminized chondrosamine. When the solution was concentratedunder the same condition as that of chitosamine, a solid residuewas obtained which was still soluble in water, but only partiallysoluble in glacial acetic acid. The product which remained in-soluble in glacial acetic was no longer soluble in water. When theoriginal substance was redissolved in water and reprecipitated bymeans of alcohol, the resulting product still possessed some reduc-ing power, but equivalent only to about 22 per cent of the original.On hydrolysis, an increase in reducing power was observed. Thespecific rotation of the substance was [a]?,= 20.5. The same valuewas obtained foruncondensedchondrose. The molecular weight ofthe soluble product was 351. Thus, it became evident t,hat2,5-anhydrohexoses condense quite readily. The question natur-ally arose whether this property was a peculiarity of the aldehydicforms of sugars in contrast with the saccharidic.r It thereforebecame desirable to test the behavior of simple monosaccharideswhen treated under the conditions which brought about the con-densation of the 2,5-anhydrosugars. In the past, glucose hasbeen condensed to di- and polysaccharides, but always throughthe action of concentrated acids. Experiments here reported dealwith glucose, mannose, and lyxose.

Glucose.-A solution containing 10 per cent of glucose and 1per cent hydrochloric acid, evaporated under a pressure of about10 to 12 mm. in a water bath, temperature of 45-5OC., andfurther treated as in the experiment of chitose, formed a solidresidue which was soluble in water, but insoluble in alcohol andother organic solvents. It was only faintly colored, was nolonger sweet, had only a slight reducing power, and a specificrotation of [a], = +95. The reducing power of the substancewas about 12 per cent of that of glucose. It was not possibleto prepare a phenylosazone of this product. From the hydrolyzed

1 Saccharidic is proposed to be used in place of the confusing terms g luco sidic or oxidir .

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P. A. Levene and R. Ulpts 477material, the osazone was readily obtained in very good yield-approximately 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 thefollowing peculiarities. It was more soluble in water but showedgreater resistance towards hydrolytic agents. Apparently it isa disaccharide. The product was completely hydrolyzed by 2per cent sulfuric acid in a sealed tube at the temperature ofthe boiling water bath only after 420 minutes, whereas the water-insoluble product of chitose was practically completely hydrolyzedin 90 minutes.

Mannose and Lyxose.-These sugars behaved in exactly thesame manner as glucose.

In the experiments thus far reported, alcohol was used to bringtlhe product to a state approaching complete dryness and, there-fore, there was a possibility that the condensation product wasa mixture of the CY-and P-ethyl saccharides. True, the solubili-ties of the condensation products differed from simple ethylsaccharides. Furthermore, it was found that the condensationproduct was formed even when the step of the operation whichinvolved t,he use of alcohol was omitted.

As to the structure of the condensation product, little can besaid. It may be suggested that in the main, the condensationproducts are built upon the plan of trehalose. Thus the productformed in the absence of alcohol which was quite impure showed areducing power equal to 25 per cent of glucose, whereas a disac-charide of the type of maltose should possess the reducing powerequal to 50 per cent of that of glucose. However, since the sub-stances are al l amorphous, and since the products dried by means ofalcohol still contain some alcohol, the possibility is not excludedthat admixtures were present of either l-ethyl disaccharides or ofl-ethyl monosaccharides.

The information obtained from the observations on the reactionswith enzymes is not very conclusive. Thus the products were nothydrolyzed by invertase, diastase, and maltase. Emulsin hydro-lyzed the condensation products to some extent. Thus, com-pared with /3-methyglucoside, the condensation product of glucosewas hydrolyzed only to the extent varying from 35 to 50 per centand that of chitose to the extent of 20 to 30 per cent.

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478 Condensation of MonosaccharidesFrom the view-point of the peculiarities of 2,5-anhydrosugars

two very important points were brought out. First, the specificrotations of the condensation products of the simple sugars werehigher than those of the equilibrium forms of the sugars whereasthose of the 2,5-anhydrosugars had practically the same specificrotations as the original sugars. Such behavior is to be expected inview of the fact that 2,6anhydrosugars are capable of existingonly in the aldehydic form. Second, the condensation products ofthe 2,5-anhydrosugars seem less stable than those of the simplesugars and this peculiarity also may be attributed to the differ-ences in the functions of carbon atom (1) in the simple and in the2,5-anhydrosugars.

EXPERIMENTAL PART.Chitose.

Chitosamine hydrochloride recrystallized until free from mineralimpurities was deaminized in the cold by means of silver nitriteand hydrochloric acid until the amino nitrogen could no longerbe detected in the solution. At the end of the operation it wasfound that 0.1637 gm. of chitose had the reducing power of 0.1000gm. 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 chitosam-

ine hydrochloride was evaporated in a 1 liter distilling flaskunder pressure of 11 to 14 mm., from a bath of 4045. Theremaining syrup was evaporated three times with 75 cc. of 95 percent 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 15minutes. The residue weighed 13 gm. and was it britt le, slightlycolored, amorphous substance, insoluble in the usual solvents,glycerol and dilute alkali; dilute mineral acids dissolved it onlyafter hydrolysis. The product was powdered, stirred with 2per cent ammonia water, filtered, and washed with hot water,alcohol, and ether. The yield was 9 gm. The substance wasdried at 60C. under reduced pressure. The substance turned

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P. A. Levene and R. Ulpts 479faintly yellow at 15OC., and at 222C. remained solid. Thereducing power of the substance suspended in water was equalto 6 per cent of that of glucose. The substance contained about14 per cent of alcohol when dried at 80C.

For analysis, the substance was dried at 150C. under reducedpressure.

0.0956 gm. substan ce: 0.1760 gm. COz and 0.0530 gm. HzO.GJLsOo. Calculated. C 47.2, H 5.89.CHO 8 26 13. 48.0, 5.78.

Found. 50.20, 6.2.0.1085 gm. substan ce: 0.0806 gm. Ag I.

Found. OCzHs 14.23.

Hydrolysis of the Condensed Chitose.The reducing power of chitose measured by the Maquenne-

Lehman method was found to be equal to 60.7 per cent of that ofglucose. Since chitose was available only in solution obtained ondeamination of chitosamine, it was desirable to determine thereducing power of crystalline epichitose.

It was found that 0.1000 gm. of glucose had the same reducingpower as 0.1640 gm. of epichitose. Hence, the reducing power ofepichitose is the same as that found for chitose; namely, 60.7 percent of that of glucose.

3 gm. of condensed chitose in 150 cc. of 2 per cent sulfuric acidwere heated in a sealed test-tube to 100C. for 30 minutes. Thetube was rapidly cooled to 20C. and the solution distributed intofour tubes which were then sealed and heated in a steam bath.

Time heated.

min.306090120

150

Reducing power of 0.100 g m .as glucose.

0.04150.04250.04930.05000.0515

Calculated as chitose .

0.06810.09680.08100.08200.0845

The optical rotation was unchanged throughout the experimentand found to be Ial2o = fo.64 X 100D 1x2 = + 32

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480 Condensation of MonosaccharidesIn another experiment, 0.5000 gm. was heated in a steam bath

with 25 cc. of 2 per cent sulfuric acid until solution was com-pleted (10 minutes after placing in bath) ; the solution was rapidlycooled 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 0.0415gm. 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 250cc. of solution with a specific rotation of [cr12i= +20. Itsreducing 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 chondrossminehydrochloride. The substance had a bitter taste, was partlysoluble 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 crudeproduct with thirteen times its volume of 98 per cent alcohol, asubstance was obtained which had the following specific rotation.

[J = + 0.41" x 100D 1x2 = + 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, therotation did not change and the reducing power of 0.1000 gm. waslowered to the value of 0.0125 gm. of glucose. The molecularweight of the substance determined cryoscopically was 351.

Condensation of Glucose.10 gm. of glucose were dissolved in 80 cc. of water. 5 cc. of10 per cent hydrochloric acid were added and the solution was

evaporated under diminished pressure in a 1 liter flask from a bathof 40-45. The residue was three times evaporated with 75 cc. of95 per cent alcohol and then four times with 75 cc. of 98 per centalcohol, allowing the temperature to rise gradually to 95-100 andkeeping it at that temperature for 15 minutes. The brittle res-

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P. A. Levene and R. Ulpts 481idue was readily soluble in cold water, reduced Fehlings solution,and had the following optical rotation.

Ial = + 1.90 x 100D 2x1 = + 9.s

0.1000 gm. had a reducing power equal to that of 0.0093 gm. ofglucose.Rate of Hydrolysis of Condensed Glucose.-3.0000 gm. of the sub-stance were dissolved in 150 cc. of 2 per cent sulfuric acid andheated in seven separate sealed tubes in a steam bath.

NO. Time.

min.00306090120

180300420

-

-

Reducing power of0.1000 gm . equalsgm. glucose.

0.00930.02750.03760.04700.05200.06550.07670.0865

Temper&m.

C.95.086.581.078.573.565.565.557.0

There remained 74 cc. of the solution containing 1.3 gm. ofglucose. The free sulfuric acid was neutralized with sodiumacetate and to this solution 2.2 cc. of phenylhydrazine and 2 cc.of glacial acetic acid were added and the solution was allowed tostand on the water bath for 1% hours. The yield of glucophenyl-osazone was 1 gm. Once recrystallized from methyl alcohol, itmelted at 204-205. The osazone in pyridine-alcohol (2:l) at21 in a 1 dm. tube gave OL, = - 0.72 (c = 1). After 24 hours:o!D = - 0.28.

CJBHS~O~N,. Calculated. N 15.47.Found. 15.43.

Molecular weight determination by the cryoscopic methodgave a value of 309.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 dissolvedin 125 cc. of water and 10 cc. of 10 per cent hydrochloric acid.

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482 Condensation of MonosaccharidesThis solution was treated in the same manner as the solution

of glucose. The yield was 10 gm. of a bitter tasting, britt le,amorphous substance, soluble in water. It was redissolved in 10cc. 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= + 5g 51x2The substance did not reduce Fehlings solution. The reducingpower of the substance after hydrolysis for 16 hours in a sealedtube placed in a steam bath was equal to 80 per cent of its weightof glucose and the specific rotation of the product of hydrolysiscalculated on this basis was [ar]2: = +13.

From this solution a levorotatory phenylosazone was obtainedwhich had a melting point of 203-205C. and had the following.composition.0.1000 gm. substance: 13.7 cc. nitrogen gas at 26C. and 757.4 mm.CI&~O~N~. Calculated. N 15.47.Found. 15.59.

Condensation of Mannose in the Absence of Alcohol.10 gm. of mannose were dissolved in 125 cc. of water and 10cc. of 10 per cent hydrochloric acid. This solution was evaporatedunder reduced pressure from a bath of 45C. (1 liter flask, widecapillary, pump of large capacity). After 20 minutes the contents.of the flask had a syrupy consistency. The temperature of thebath was slowly raised to 65 when the syrup began to solidify.10 minutes later the temperature was raised to 73. The reactionwas interrupted and the solid, somewhat colored product was re-moved 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 100D 1x1 = +33"The substance was dissolved in 5 cc. of water and reprecipitatedwith absolute alcohol. The optical rotation of the substance thenrose slightly to

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P. A. Levene and R. Ulpts 483Ial = + 0.74 x 100D 1x2 = + 37

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

Condensation of Lyxose.10 gm. of lyxose ([aID = - 13) were dissolved in 90 cc. of water

and 10 cc. of hydrochloric acid and the solution was concentratedin the same manner as described in previous experiments. Theyield was 10.0 gm. of a dry substance easily pulverized and verysoluble in water. The molecular weight of the substance wasfound to be 338.

The optical rotation of the substance was the following.falz, = + 0.83 X 100D 1x2 = + 41.5

The substance had the following composition.0.1020 gm. substance : 0.1728 gm. CO, and 0.0618 gm. H,O.0.0975 ( : 0.0538 AgI.

The composition of the substance approached that of a dipen-tose with 1 mol of alcohol.CloHlaOg + C2H60H. Calc ulated . C 43.9, H 7.3, OCzHs 13.7.

Found. * 45.75, 6.71, 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 hydrol-ysate was equivalent to 85 per cent of glucose. From thehydrolysate a phenylosazone was obtained which melted at 162C.,was levorotatory, and had the following composition.

0.0965 gm. substanc e: 15.00 cc . nitrogen gas at 24 C. and 749.4. mm.C,&oO~N~. Calculated. N 17.07.

Found. 17.60.

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