DERIVATIVES OF GLUCURONIC ACID

V. THE SYNTHESIS OF GLUCURONIDES

BY WALTHER F. GOEBEL AND FRANK H. BABERS

(From the Hospital of The Rockefeller Institute for Medical Research, New York)

(Received for publication, May 28, 1935)

This series of studies on the chemistry of glucuronic acid was instigated primarily by the desire to prepare synthetically aldo- bionic acids similar to those found in the hydrolytic products of numerous naturally occurring complex bacterial polysaccharides. These derivatives of uranic acids were first described when it was shown that the specific polysaccharide elaborated by the Type III pneumococcus during its growth in liquid medium is composed of units of a glucose-glucuronide (1). The striking immunological properties exhibited by the bacterial polysaccharide derived from encapsulated microorganisms are not entirely lost when the carbo- hydrates undergo hydrolysis, for it has been shown that the products of partial hydrolysis of Type III pneumococcus specific polysaccharide retain the capacity to precipitate specifically in homologous antipneumococcus horse serum (2).

The first step in the synthesis of aldobionic acids involves the preparation of the appropriate acetohalogen derivative of the hexose uranic acid. The syntheses of diacetylchloroglucuron and of triacetylchloroglucuronic acid methyl ester have been described in earlier communications of this series (3). The present report describes the synthesis and properties of several simple glucuron- ides prepared from these two acetohalogen derivatives of glucu- ronic acid.

When methyl alcohol is condensed with diacetylchloroglucuron, or with triacetylchloroglucuronic acid methyl ester in the presence of silver carbonate, the corresponding acetylated methylglycosides are formed. Both these derivatives, however, exhibit properties

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708 Derivatives of Glucuronic Acid. V

which distinguish them sharply from true glycosides. When aque- ous acetone solutions of the two derivatives are titrated with a dilute base, in each instance one acetyl group remains firmly attached to the glycosidic molecule. Furthermore, it has been observed that both compounds, when dissolved in 0.005 N HCl in 95 per cent dioxane, undergo a rapid change in specific rotation with a corresponding loss of the glycosidic group. These proper- ties are characteristic of methylglycosides having the orthoacetate structure (4). The glycosides must be regarded therefore as having the structure represented in figures (I) and (II), and not as true methylglycosides.

OCHs OCHs

/ / H O----

AcO

/

I I \ 0 CHa

OAc

Hy 7 H

COOCH,

I II

Diacetylmethylglycoside of glucuron Triacetylmethylglycoside of glucuronic acid methyl ester

It has been suggested that the formation of glycosides having the orthoacetate structure occurs whenever adjacent hydroxyl groups congregate in clusters on the same side of the 6 atom ring, as in the mannose, rhamnose, and lyxose series (5). Although it is obvious that a congregation of this nature does not exist either in the glucuron or glucuronic acid molecule, yet the acetohalogen derivatives of both these substances yield methylglycosides having the structures of orthoacetates. This question has also been dis- cussed by Pacsu (6), who has pointed out that the third aceto- chloromaltose of Freudenberg (7) fails to have a clustering of adjacent hydroxyl groups, yet the derivative possesses the struc- ture of an orthoacetate.

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W. F. Goebel and F. H. Ea,bers 709

If p-nitrobenzyl alcohol is condensed with diacetylchloroglucu- ron in the presence of silver oxide, or with triacetylchloroglucuronic acid methyl ester in the presence of silver carbonate, derivat.ives are formed which have the structure of true glycosides and not those of orthoacetat,es. That these derivatives are indeed true glycosides is evident from the fact that the diacetyl-p-nitrobenzyl- glycoside of glucuron neutralizes 3 equivalents of ammonium hy- droxide when the glycoside is dissolved in a 0.1 N solution of the base, and that the triacetyl-p-nitrobenzylglycoside of glucuronic acid methyl ester utilizes 4 equivalents of 0.1 N sodium hydroxide. Furthermore these derivatives, unlike the corresponding methyl- glycosides having the orthoacetate structure, do not undergo hydrolysis in solutions of 0.005 N HCl dissolved in 95 per cent dioxane.

The reducing properties exhibited by both the methyl- and p-nitrobenzylglycosides of diacetylglucuron may be attributed to the lactone ring, for it has been observed that this property is lost when the ring is opened by allowing the glycosides to stand in an alcoholic ammonia solution. It is well known that certain lac- tones, such as the lactone of mannosaccharic acid, have the prop- erty of reducing Fehling’s solut,ion.

The fact that both diacetylchloroglucuron and triacetylchloro- glucuronic acid methyl ester may, under slightly different experi- mental conditions, yield glycosides having either the orthoacetate structure, or the structure of a true glycoside, raises a question concerning the constitution of the parent halogen derivatives them- selves. It is hoped that this question may be further investigated.

EXPERIMENTAL

Preparation and Properties of Diacetylmethylglycoside of Glucuron -5.0 gm. of diacetylchloroglucuron were mixed with 1.5 moles of freshly prepared dry silver carbonate. The mixture was added to 100 cc. of cold anhydrous methyl alcohol and shaken at 0” until the alcoholic soIution gave no test for chloride. The contents of the flask were then filtered at O’, and the residue of silver salt washed with successive portions of anhydrous chloroform. The alcohol and chloroform filtrate was concentrated to small volume under diminished pressure. The flask was set aside in the ice box until crystallization of the glycoside rea,ched completion.

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710 Derivatives of Glucuronic Acid. V

2.5 gm. of glistening prismatic needles, the diacetylmethylglycoside of glucuron, were filtered from the colorless mother liquors.

Several recrystallizations from anhydrous ether gave 2.0 gm. of glycoside having a melting point of 110-111” (uncorrected).

[a]: = +112.5” in CHC18 (c = 0.6 per cent). AnaZ~sis-CJ%Os (COCH& OCHI

Calculated. C 48.2, H 5.2, COCH3 31.4, OCH, 11.3 Found. “ 48.5, “ 5.3, “ 31.1, “ 11.3

The diacetylmethylglycoside of glucuron is extremely soluble in chloroform, dioxane, acetone, and methyl alcohol, and considerably less soluble in ethyl alcohol and ether. It was observed that a solution of the glycoside in methyl alcohol underwent a rapid change in specific rotation, although solutions of the same con- centration in chloroform and dioxane exhibited no such change. It was found that the change in rotation of the methyl alcoholic solution of the glycoside was due to a spontaneous opening of the lactone ring with the subsequent formation of the corresponding methyl ester of the glycoside. Thus a 1 per cent solution of glyco- side in methyl alcohol was allowed to stand until the rotation became constant and the solvent removed in vacua. The oily residue, dried to constant weight in vacua over sulfuric acid, was found to contain 17.8 per cent methoxyl. Approximately 70 per cent of the glycoside of glucuron had thus been converted to the corresponding methyl ester. The reaction between the glyco- side and methyl alcohol apparently reaches an equilibrium in which 30 per cent of unchanged methylglycoside of diacetylglucuron is still present.

A second unusual property which the diacetylmethylglycoside of glucuron exhibits is its capacity to reduce Fehling’s solution. This property may be attributed to the presence of the lactone ring, for it has been found that when the ring is opened by dis- solving the glycoside in methyl alcohol saturated with anhydrous ammonia, the reaction mixture no longer reduces alkaline copper solutions.

Conductometric Titration of Acetyl Groups of Diacetylmethylgly- coside of Glucuronl-The acetyl group of the diacetylmethylgly-

* The authors wish to express their thanks to Dr. Torsten Teorell who ha.s kindly assisted them in carrying out the conductometric titrations described in this report.

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W. F. Goebel and F. H. Babers 711

coside of glucuron cannot be titrated directly with dilute sodium or barium hydroxide, since the derivative undergoes a severe de- composition in the presence of fixed alkali. Aqueous acetone solutions of the glycoside turn a brilliant yellow, even upon the cautious addition of a few drops of 0.01 N NaOH at 0”. It was observed, however, that in the presence of dilute ammonium hy- droxide the decomposition of the glycoside was greatly minimized. In order to establish the number of equivalents of base neutralized, 0.0812 gm. of glycoside was dissolved in 5 cc. of acetone, 5 cc. of N NHdOH were added, and the mixture diluted to 50 cc. in a volumetric flask. 4 cc. samples of the reaction mixture were removed from time to time and titrated conductometrically (8) with 0.05 N acetic acid. At the end of 2 hours it wa,s found that 0.47 cc. of 0.1 N NH,OH, or exactly 2 equivalents, had been neutralized by the glycoside in the sample titrated. On further standing (24 hours or more) a slight increase in the consumption of base occurred, indicating a very gradual hydrolysis of the re- maining acetyl group. From the results of the experiment it may be concluded that one of the acetyl groups of the glycoside is firmly attached to the glycosidic molecule.

Triacetylmethylglycoside of Glucuronic Acid Methyl Ester-This derivative was prepared from triacetylchloroglucuronic acid methyl ester exactly as was the corresponding diacetylmethyl- glycoside of glucuron. From 6.0 gm. of the halogen derivative 2.8 gm. of crystalline glycoside were recovered. By repeated recrystallization from ether, 2.0 gm. of material were recovered which melted sharply at 118” (uncorrected). Further crystalliza- tion failed to change either the melting point or the specific rota- tion of the derivative.

[cx]: = +54.0” in chloroform (c = 0.6 per cent). Analysis-CsH~O,(COCH,),(OCH,),

Calculated. C 48.3, H 5.8, COCH, 37.1, OCH, 17.8 FouIld. “ 48.5, “ 5.8, “ 36.7, “ 17.9

The triacetylmethylglycoside of glucuronic acid methyl ester sepa- rates as beautiful cubic crystals from anhydrous ether. The sub- stance is very soluble in chloroform, acetone, and dioxane, and considerably less soluble in ethyl alcohol and ether. The gly- coside does not reduce Fehling’s solution. When an aqueous

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712 Derivatives of Glucuronic Acid. V

acetone solution of the glycoside is treated with an excess of 0.1 N

NaOH, warmed to loo”, and the excess alkali titrated with 0.1 N

HCl, it was found that only 3 equivalents of base were neutralized. 0.0642 gm. of glycoside neutralized 5.54 cc. of 0.1 N NaOH (calcu- lated for 3 equivalents, 5.53 cc.). Similarly, when a weighed sample of the glycoside was dissolved in aqueous acetone contain- ing a known amount of 0.1 N NHIOH, and titrated after several hours conductometrically with 0.05 N acetic acid, again it was found that exactly 3 equivalents of base had been neutralized by the glycoside. From these experiments it may be concluded that this derivative retains one acetyl group, firmly bound in the mole- cule, which cannot be removed by alkaline hydrolysis. It appears, therefore, that both the diacetylmethylglycoside of glucuron, and the triacetylmethylglycoside of glucuronic acid methyl ester must be regarded as derivatives of orthoacetic acid having the struc- tures given in figures (I) and (II), respectively.

Repeated attempts have been made to isolate the monoacetyl derivatives of both glycosides in a crystalline state, but without success. The diacetylmethylglycoside of glucuron cannot be hy- drolyzed either with barium hydroxide, or with barium methylate, without undergoing severe decomposition. Attempts to prepare the corresponding monoacetylmethylglycoside amide by dissolving the acetylated methylglycoside of glucuron in alcoholic ammonia yielded an amorphous material, so sensitive to hydrogen ions that aqueous solutions left in open vessels are rapidly hydrolyzed by the carbon dioxide of the air. On account of its instability the mono- acetylmethylglycoside of glucuronic acid, prepared by hydrolyzing the triacetylmethylglycoside methyl ester with barium hydroxide, has likewise not been isolated as a crystalline derivative.

Hydrolysis of Diacetylmethylglycoside of Glucuron, and Triacetyl- methylglycoside of Glucuronic Acid Methyl Ester by Hydrochloric Acid Dissolved in 95 Per Cent Dioxane--One of the most charac- teristic properties of the methylglycosides of sugars having an orthoacetate structure is the extreme lability of the methoxyl group in the presence of very dilute acid (9). The rate of hydroly- sis of the diacetylmethylglycoside of glucuron, and of the triacetyl- methylglycoside of the methyl ester of glucuronic acid has been measured in 95 per cent dioxane solution containing 0.005 N HCl. The change in optical rotation was observed and the velocity

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W. F. Goebel and F. H. Babers 713

constant was calculated. The results of these experiments are given in Table I. From the results given in Table I it may be seen that both the acetylated methylglycosides of glucuron and of the methyl ester of glucuronic acid are rapidly hydrolyzed by very dilute hydrochloric acid in aqueous dioxane. From the values of the constant, k, it may be seen that the reaction is in each

TABLE I

Hydrolysis of Diacetylnethylglycoside of Glucuron and Triacetylmethylglyco- side of Glucuronic Acid Methyl Ester by Hydrochloric Acid Dissoloed in

95 Per Cent Dioxane

Substance observed

Diacetylmethyl- glycoside of glu- curon

Triacetylmethyl- glycoside of glu- curonic acid methyl ester

Concentration

3.0696 gm. in 10 cc. Of 0.005 N SOlU-

tion of HCl in 95 per vent dioxane

3.1009 gm. in 10 cc. Of 0.005 N SOlU-

tion of HCl in 95 per cent dioxane

____ min. degrees

0 +1.47 3 +1.57 5 +1.62 8 +I.68

12 +1.73 17 +1.7C, 27 f1.79 57 +1.80

240 +1.80 0 f1.29 3 +1.46 6 +1.59 8 +1.69

11 +1.77 15 f1.89 21 f2.01 28 f2.10 38 +2.20 52 +2.26 75 +2.31

320 +2.31

0.052 0.052 0.055 0.056 0 ,054

0.026 0.025 0.027 0.025 0.025 0.025 0.024 0.025

instance apparently unimolecular. Although the kinetics of hy- drolysis of the two glycosides proceeds in accordance with a unimolecular reaction, it does not follow that only one product is, in each instance, formed during the course of the reaction. It has been found that after the hydrolysis of the methylglycoside of glucuron reaches completion, there are two substances present

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714 Derivatives of Glucuronic Acid. V

in the reaction mixture in approximately equimolecular propor- tions. The first is a crystalline hydroxydiacetylglucuron having a specific rotation of + 115” (in 95 per cent dioxane) and identical wit)h the diacetylglucuron previously described (4). The second product is an oil analyzing correctly for hydroxydiacetylglucuron, and having a specific rotation of + 143” (in 95 per cent dioxane). E’rom the hydrolysis of 1 gm. of glycoside in aqueous dioxane con- taining 0.005 N HC1 approximately equal quantit.ies of the amor- phous and crystalline hydroxy derivatives were obtained. The two substances were separated from the reaction mixture by evapo- rating the solvent in vacua, dissolving the residue in 5 cc. of ether, and separating the resulting crystalline derivative by filtration. The oily residue was dried in vacua over sulfuric acid to constant weight.

On examination of the reaction product of the methylglycoside of triacetylglucuronic acid methyl ester in 0.005 N HCI dissolved in 95 per cent dioxane, two substances are likewise obtained. The first is a crystalline hydroxytriacetylglucuronic acid methyl ester melting at 126” and having a specific rotation of 146.8” (in 95 per cent dioxane). The second product of reaction is an oil having a specific rotation of +76” (in 95 per cent dioxane) and analyzing approximately for hydroxytriacetylglucuronic acid methyl ester. These two substances are likewise formed apparently in equi- molecular proportions.

The structural relationship between the crystalline and amor- phous forms of hydroxydiacetylglucuron, and between the two forms of hydroxytriacetylglucuronic acid methyl ester is at present not understood. It is hoped, however, that this question may eventually be more thoroughly investigated.

Diacetyl-p-Nitrobemylg2ycoside of Glucuron-6.0 gm. of diacetyl- chloroglucuron and 4.0 gm. of p-nitrobenzyl alcohol were dissolved in 50 cc. of pure anhydrous chloroform and 5 gm. of freshly pre- pared dry silver oxide were added. The mixture was shaken for 48 hours at room temperature and then filtered. The dark brown chloroform solution was concentrated to a syrup in vacua, and 75 cc. of ethyl alcohol were added. Within a few minutes crystal- lization of the p-nitrobenzylglycoside of diacetylglucuron com- menced. 2.2 gm. of glycoside were separated by filtration. The

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W. F. Goebel and F. H. Babers 715

substance was recrystallized from ethyl alcohol, yielding 2.0 gm. of pure glycosidc melting at 133-134” (uncorrected).

[a]: = +39.9” in chloroform (c = 1 per cent). Analysis

C,8H,,0sN(COCH3)2. Calculated. C 51.6, H 4.3, COCH, 21.8, N 3.5 Found. “ 51.9, ” 4.2, ‘I 21.2, (( 3.4

The diacetyl-p-nitrobenzylglycoside of glucuron is di&ultly soluble in ether and in cold ethyl alcohol, but is readily soluble in ethyl acetate, dioxane, chloroform, and acetone. Like the cor- responding methylglycoside of diacetylglucuron, the p-nitrobenzyl derivative reduces Fehling’s solution, and in aqueous acetone solu- tion is very sensitive to fixed alkali. The hydrolysis of the acetyl groups of the glycoside with dilute barium hydroxide at 0” yields a brownish tarry residue from which nothing could be crystallized. It has likewise not been possible to obtain in crystalline form the deacetylated amide by treating the glycoside with alcoholic am- monia. By conductometric titrations of aqueous acetone solu- tions of the glycoside in 0.1 N NH,OH, it was found that 3 equiva- lents of base were neutralized, indicating that two acetyl groups had been removed and that the la&one ring had been opened. When solutions of the glycoside in 95 per cent dioxane containing 0.005 N HCI were observed in a polarimeter, no change in rotation occurred. Indeed, when the concentration of acid was increased IO-fold the specific rotation of the solution remained constant for many hours, and the value was identical with that of a solution of the glycoside in 95 per cent dioxane containing no HCl. The diacetyl-p-nitrobenzylglycoside of glucuron must be regarded, therefore, as a true glycoside and not as a derivative of ortho- acetic acid.

Triacetyl-p-Nitrobenzylglycoside of Glucuronic Acid Methyl Ester- 3.0 gm. of triacetylchloroglucuronic acid methyl ester and 1.4 gm. of p-nitrobenzyl alcohol were dissolved in 25 cc. of pure anhydrous chloroform and the mixture shaken for 7 days with 3.0 gm. of silver carbonate. At the end of this time the chloroform solution still showed the presence of the halogen derivative, so the mixture was heated and stirred at 60” in a mercury-sealed apparatus. The mixture was filtered and the chloroform solution was concentrated

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716 Derivatives of Glucuronic Acid. V

to a syrup in vacua. The syrup was dissolved in ethyl alcohol and the solution placed in the ice box for 2 weeks. Crystalliza- tion of the glycoside was very slow, and only 0.4 gm. of substance was recovered. The derivative was recrystallized from ethyl alcohol, yielding 0.3 gm. of the triacetyl-p-nitrobenzylglycoside of glucuronic acid methyl ester.

[a]: = -57.8”in CHCl, (c = 0.6 per cent). Analysis

C,,H,,O,N(COCH,),(OcH,). Calculated. C 51.2, H 5.0, N 3.0, OCH, 6.6 Found. “51.3,” 5.1,“2.8, “ 6.3

The glycoside crystallized as pale yellow silky needles, difficultly soluble in cold, but soluble in hot ethyl alcohol. The glycoside does not reduce Fehling’s solution. The substance melts at 175- 176” (uncorrected). 15.211 mg. of glycoside were dissolved in 3 cc. of acetone, and 14.00 cc. of ~/70 NaOH were added. After standing overnight the excess alkali required 4.98 cc. of N/70 HCl for neutralization. Thus 9.02 cc. of ~/70 NaOH were utilized by the acetyl groups and carboxyl groups of the glycoside. (Calcu- lated for 4 equivalents, 9.09 cc. of ~/70 NaOH.) From this experi- ment it may be seen that all of the acetyl groups are removed by alkaline hydrolysis, and at the same time the methyl ester grouping is hydrolyzed. The derivative must therefore be regarded as a true glycoside, and not as a derivative of orthoacetic acid. On account of its levorotation, the glycoside probably has the fi con- figuration.

In conclusion the authors wish to express their thanks to Dr. Duncan MacInnes and to Dr. I’. A. Levene for their generous advice.

SUMMARY

1. The synthesis of the diacetylmethylglycoside of glucuron, and of the triacetylmethylglycoside of glucuronic acid methyl ester has been described. Both these derivatives have the struc- tures of orthoacetates.

2. The synthesis of the diacetyl-p-nitrobenzylglycoside of glucuron, and the triacetyl-p-nitrobenzylglycoside of glucuronic

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W. F. Goebel and F. H. Babers 717

acid methyl ester has been described. Both these derivatives have the structures of true glycosides.

BIBLIOGRAPHY

1. Heidelberger, M., and Goebel, W. F., J. Biol. Chem., 74,613 (1927). 2. Heidelberger, M., and Kendall, F., J. Ezp. Med., 67, 373 (1933). 3. Goebel, W. F., and Babers, F. H., J. Biol. Chem., 101, 173 (1933); 106,63

(1934). 4. Freudenberg, K., and Scholy, H., Ber. them. Ges., 63, 1969 (1930).

Braun, E., Ber. them. Ges., 63, 1972 (1930). Bott, H. G., Haworth, W. N., and Hirst, E., J. Chem. Sot., 1396 (1930). Haworth, W. N., Hirst, E., and Samuels, H., J. Chem. Sot., 2861 (1931).

5. Haworth, W. N., Dixieme conference de I’union internationale de chimie, Liege, 38 (1930).

6. Pacsu, E., J. Am. Chem. Sot., 67,537 (1935). 7. Freudenberg, K., Naturwissenschuften, 18, 393 (1930). 8. Kolthoff, J. M., Konduktometrische Titrationen, Dresden and Leipsic

(1923). 9. Pacsu, E., J. Am. Chem. Sot., 66, 2451 (1933).

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Walther F. Goebel and Frank H. BabersGLUCURONIDES

V. THE SYNTHESIS OF DERIVATIVES OF GLUCURONIC ACID:

1935, 110:707-717.J. Biol. Chem. 

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