THE PREPARATION OF BORNEOL GLYCURONIC ACID AND ... - … · from this study a new test for liver function. The preparation of glycuronic acid from borne01 glycuronic acid offers no

THE PREPARATION OF BORNEOL GLYCURONIC ACID AND GLYCURONIC ACID.

BY ARMAND J. QUICK.

(From the Department of Surgical Research, Cornell University Medical College, New York City.)

(Received for publication, June 14, 1927.)

The significance of glycuronic acid in the economy of the organism is not fully understood, but it seems rather certain that its biological importance is underestimated. There can be no doubt that it plays a prominent part in the mechanism of de- toxication in the body. Like hippuric acid and the ethereal sul- fates, the conjugated glycuronic acids appear to be constant con- stituents of normal human urine as indicated by the slight levo- rotation which is nearly always observed and by the fact that glycuronic acid has been isolated from urine in the form of the p-bromophenylhydrazine derivative (1, 2). Glycuronic acid is conjugated both with phenolic types of compounds and with various aromatic acids such as benzoic acid and phenylacetic acid, thus complementing on the one hand the action of the ethereal sulfates and that of glycocoll on the other. A quantitative study of the curious interrelationship of these detoxication mechanisms may possibly offer a means for obtaining further information concerning the synthetic power of the various organs, especially of the liver. Little emphasis has been given to the marked ability of the organism to produce relatively large amounts of glycuronic acid without any perceptible embarrassment. As an example of this power it might be cited that a dog after eating 2 pounds of prunes excreted 11 gm. of glycuronic acid in the form of glycuronic acid monobenzoate in the course of 24 hours. Even in the human organism the amount of glycuronic acid produced for conjugation with phenolic compounds is much greater than the ethereal sul- fate output. The fact that glycuronic acid is produced in response to benzoic acid, phenylacetic acid, and other phenyl substituted

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332 Glycuronic Acid

aliphatic acids which resist oxidation within the body, suggests the interesting probability that a similar conjugation may occur between the fatty acids and a carbohydrate molecule in normal metabolism, but that the compounds thus formed are so readily oxidized that their existence has thus far escaped detection.

Before these various problems just mentioned can be success- fully studied, it stands to reason that more ought to be known about glycuronic acid itself, especially of its metabolism. Before such a study can be made, however, it is essential to secure rela- tively large amounts of the pure acid, and therefore the first step in the proposed research consisted in developing a satisfactory method for preparing the acid and in finding a suitable source of material. Glycuronic acid has been isolated by a number of the earlier investigators who studied this compound, but always as the acid lactone, and it was not until 1925 that the acid itself was prepared by Ehrlich and Rehorst (3). Prior to the appearance of their publication, the author, on hydrolyzing menthol glycuronic acid obtained a small amount of substance, the crystalline struc- ture and chemical behavior of which corresponded to the glycuro- nit acid described by Ehrlich and Rehorst, but further work on the compound was postponed at that time on account of other experiments which were then being carried out. The method employed by the author is in principle essentially the same as the method of Ehrlich and Rehorst, but it is considerably simpler, more time-saving, and better adapted for the preparation of large quantities of glycuronic acid. In short the process consists in hydrolyzing borne01 glycuronic acid with dilute sulfuric acid, subsequent removal of the mineral acid and the borneol, and finally, direct isolation of the acid from the concentrated filtrate. It may be remarked, however, that the real difficulty encountered was not in the actual preparation of the acid, but in finding a suitable source from which it might be obtained, or in other words, the primary task in hand was to find a satisfactory gly- curonogenic drug. Any substance suitable for this purpose must be relatively non-toxic, thus permitting the administration of relatively large doses over long periods of time. It should further- more give rise to a large quantity of a conjugated glycuronic acid which can readily and fairly quantitatively be isolated from urine. Although the number of conjugated glycuronic acids which have

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been qualitatively determined is very large, few are suitable for the preparation of glycuronic acid. Thierfelder (4) and other pioneer investigators of glycuronic acid employed euxanthic acid, which is a conjugated glycuronic acid obtained from the natural dyestuff, purree. Since this substance is now difficult to obtain and offers no particular advantages, it was not further considered. Menthol appears to be the only substance which has given satis- factory results. It is relatively non-toxic and its conjugated glycuronic acid can readily be separated from urine by means of ammonium sulfate. When one attempts, however, to prepare large quantities of the acid, one finds menthol not altogether ideal. Of the common laboratory animals, the rabbit seems to be the only one suited for producing menthol glycuronic acid; dogs apparently can destroy menthol and also the conjugated menthol, for on feeding either form no free menthol and only a very small amount of the conjugated compound can be recovered in the excreta. When using rabbits one is compelled to give the drug by stomach tube, and since the maximum daily production of men- thol glycuronic acid per animal is not very much over 2 gm., it can readily be seen that the preparation of several hundred gm. of material, the amount desired for the present series of experi- ments, would become a tedious and laborious task.

In order to find a drug which was more satisfactory than men- thol, a number of compounds especially of the terpene series was investigated, and it was found that borne01 was by far themost suitable for the purpose at hand. It is practically an innocuous drug which can be fed to dogs in 5 gm. doses per day for a period of weeks without causing any apparent deleterious effect, and without causing the slightest loss of appetite. It can be mixed directly with the food, which is a great convenience. About 50 per cent of the borne01 is excreted in the urine as borne01 gly- curonic acid. Attempts to increase this amount have so far yielded rather unsatisfactory results, but it is hoped that further studies will determine the fate of the unaccounted for borne01 as well as throw some light on the other factors concerned in the mechanism of this conjugation. The isolation of borne01 gly- curonic acid from urine is exceedingly easy and simple, since it can be directly and almost quantitatively precipitated as the zinc salt. Although both Hildebrandt (5) and Fromm and Clemens

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Glycuronic Acid

(6) prepared this salt, they apparently overlooked the possi- bility of utilizing it as a means of isolating borne01 glycuronic acid from urine and other body fluids. Since the precipitation is carried out in acidified urine, no other insoluble salt of zinc is formed unless the urine contains uric acid which is also precipi- tated under these conditions. By treating the urine with lead acetate uric acid as well as pigments and other interfering sub- stances are removed without precipitating any of the desired compound. From the clarified filtrate the zinc salt of borne01 glycuronic acid can be precipitated in practically chemically pure condition. On dissolving this salt in hot 3.5 N sulfuric acid, borne01 glycuronic acid with a yield of about 95 per cent is obtained. It is nearly pure, and can readily be further purified by recrystalli- zation from hot water. In this respect it offers an advantage over menthol glycuronic acid since the purification of the latter is more difficult due to the fact that the last trace of ammonium sulfate is removed with difficulty and entails a certain loss of material.

Borneo1 glycuronic acid belongs to the glucoside type of con- jugated glycuronic acids and therefore does not reduce Fehling’s solution. Its solubility in water is about 2 parts per 100, but even slight impurities will greatly increase its solubility. Fromm and Clemens (6) state that it crystallizes with 1.5 molecules of water of crystallization, but it was found that in the carefully purified air-dried product obtained in this study the water content was somewhat variable, but approximated more nearly 1 molecule of water of crystallization. The specific rotation of the compound was [a]“,” = -48, whereas the value for pure d-borne01 glycuronic acid is [a]:’ = -37, and for the Z-borne01 glycuronic acid [cx]~’ = -69. It is quite evident that the preparation was a mixture of the dextro and levo forms and this can be explained by the fact that the borne01 used, although labelled d-borneol, was really a mixture of both isomers, for the rotation of the borne01 employed was [c&’ = +5 instead of [&,’ = +37, the value for the pure dextro modification. This is however of minor importance since it has no bearing on the results reported in this paper. Like other glycuronic acids, borne01 glycuronic acid is a relatively strong acid although it is somewhat weaker than either the free glycuronic acid or glycuronic acid monobenzoate. Like the corresponding menthol compound, the acid is precipitated by a half saturated

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solution of ammonium sulfate. It appears that this is a common property of several conjugated glycuronic acids, for purified thy- mol glycuronic acid is likewise precipitated, and it seems likely that other compounds of this type will be found. As previously stated, the zinc salt is insoluble even in fairly acid solution. Zinc in a dilution of 1 to 5000 can readily be detected by the rather characteristic crystalline precipitate which forms on adding an excess of borne01 glycuronic acid. Since the salts of the other common metals except cadmium are soluble in either acid or neutral solution, borne01 glycuronic acid is an excellent reagent for the qualitative detection of zinc and for its separation from other metals. Since the glycuronic acid in zinc borne01 glycuron- ate can readily be determined by any of the common quantitative sugar methods, it is hoped that the insolubility of the zinc salt will form the basis for a new quantitative method for zinc. As for the determination of borne01 glycuronic acid, the method developed for menthol glycuronic acid (7) is entirely satisfactory.

While borne01 glycuronic acid is especially suited for preparing glycuronic acid, it also seems promising as a satisfactory drug for the study of the synthesis of glycuronic acid in the human or- ganism. Preliminary work on this problem indicates that a dose of several gm. can be taken with impunity and that the response of the body to conjugate it with glycuronic acid is prompt and the output of the conjugated acid larger than in the case of dogs and rabbits. Since it seems probable that the liver participates in the synthesis of glycuronic acid, it may be possible to develop from this study a new test for liver function.

The preparation of glycuronic acid from borne01 glycuronic acid offers no technical difficulties. The conjugated acid is hydrolyzed by means of 0.2 N sulfuric acid. The liberated borne01 is insoluble in water and can be filtered off, while the sulfuric acid can be quantitatively removed by means of barium hydroxide. On con- centrating the filtrate which now contains only the desired product, glycuronic acid can be obtained as a white crystalline powder. It is not pure acid, however, but contains about 30 per cent of the acid lactone. This conversion of the acid to the la&one occurs during the hydrolysis, for the solution immediately after the hy- drolysis contains practically the same ratio of acid to lactone as the crystalline product. When the hydrolysis is conducted on a

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336 Glycuronic Acid

water bath, instead of the solution being boiled over a free flame, the amount of la&one formed is slightly less, but no scheme of hydrolysis was found which would yield only the pure acid. The separation of the acid from the lactone offers considerable difficulty because their solubility in the various solvents is not great enough to effect a clean cut separation. Fortunately, the lactone is slightly more soluble in alcohol than the acid, and by extracting the mixture with 95 per cent ethyl alcohol one is able to obtain a product containing over 99 per cent of the free acid. Another successful though somewhat more laborious method consists in converting the acid and the lactone to a salt, reliberating the acid in the cold, removing the inorganic salt, and finally concentrating the filtrate at a low temperature by distilling under reduced pressure. This was the principle followed by Ehrlich and R.ehorst (3), for they formed the barium salt as an intermediate step in their preparation of pure glycuronic acid. For most purposes it is rarely necessary to employ the pure acid; especially is this true in physiological experiments as in these the sodium salt is apt to be used almost entirely. Even for purely chemical studies it will hardly be found essential to use the pure acid unless one is specifi- cally studying the physical or chemical properties of pure glycuronic acid.

The determination of lactone and free acid in any given sample can be readily determined by direct titration with standard alkali when phenolphthalein is used as the indicator. The complete neutralization of the acid is indicated by the first temporary per- sistence of the pink color. For the titration of the la&one, the solution is heated to boiling and titrated to a permanent end-point.

The lactone, or glucuron, as it is commonly called, is the more stable form, and it can be readily prepared from the acid. When glycuronic acid is merely exposed to warm dry air, it slowly loses water and becomes converted into the lactone. On dissolving the acid in hot glacial acetic acid, the lactone crystallizes out from the cooled solution. This offers a convenient method for preparing the lactone. The compound is best purified by recrystallization from hot water. On dissolving the purified product in water, one obtains a solution which is neutral to litmus and which even on standing several days shows no sign of free acid. On refluxing a solution of the lactone about 50 per cent of the acid is regenerated,

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but at the same time a certain amount of disintegration occurs as indicated by the fact that the solution becomes brown and the total titratable acidity is increased. The la&one shows a specific rotation of [01]2,’ = +19, which agrees with the value found in the literature. Theoretically there ought to be at least two modifica- tions of the lactone, namely the w and fl-butylene oxide forms, and it seems probable that this product having [cy]:’ = +19 may be one of these two.forms. The crystals of the lactone are very characteristic. They consist of thick monoclinic plates with bevelled edges, which present a marked contrast to the needle crystals of glycuronic acid.

Glycuronic acid is a strong organic acid (K< 1 X 1 OW3). It is readily soluble in water and also slightly soluble in alcohol. In cold water it is stable, but on boiling it is partly converted to the lactone. As in the case of the la&one some decomposition also takes place. An equilibrium between the acid and lactone, as observed by Ehrlich and Rehorst, occurs on refluxing a solution of either the lactone or the acid. In further accordance with the findings of these two authors the acid showed mutarotation. An initial rotation of [cx]~’ = +16 and a maximum rotation of [a]“,” = +36 obtained in 3 hours agree fairly well with the values obtained by Ehrlich and Rehorst, namely +12 and +36 re- spectively. At this point it might be interesting to recall that glycuronic acid monobenzoate (8) does not show mutarotation until it is neutralized. It is rather difficult to explain why the esterification of one of the hydroxyl groups should definitely hinder mutarotation. Glycuronic acid ought to be a type of com- pound which should lend itself well to further study of the rela- tionship between optical rotation and the chemical structure of the carbohydrate molecule. In fact glycuronic acid with its two strongly positive groups should prove to be a useful substance for the study of carbohydrate chemistry in general, especially from a synthetic point of view, now that it can be prepared in large quantities.

EXPERIMENTAL.

Preparation of Borneo1 Glycuronic Acid.-5 gm. of pulverized borne01 were fed daily to each of several dogs. The drug was incorporated directly with the food, or, which proved somewhat,

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338 Glycuronic Acid

more satisfactory, first suspended in a small volume of, a gelatin- glucose mixture in order to insure better mixing with the food. The urine was collected, slightly acidified with acetic acid, and treated with lead acetate. The precipitate which carried down with it most of the coloring matter was filtered off, the clear light yellow filtrate heated to boiling, and an excess of zinc acetate added, whereupon a voluminous crystalline precipitate of zinc borne01 glycuronate filled the container. The product was filtered off immediately and washed with hot water until no more color- ing matter was extracted. In this way a pure white crystalline product of practically chemically pure zinc borne01 glycuronate was obtained. The purity of this compound is emphasized be- cause on it depends the success of preparing pure borne01 gly- curonic acid. The yield was about 1 gm. of the zinc salt for every gm. of borne01 fed.

Glycuronic acid was prepared by dissolving the finely pulverized zinc salt in hot 3.5 N sulfuric acid, about 140 cc. of the acid for every 100 gm. of zinc borne01 glycuronate being used. When the salt was completely dissolved, the solution was rapidly cooled and then placed in the ice box for several hours, whereupon the crystallization of the acid was complete. The crystalline mass was filtered off, washed with a small amount of cold water, and dried in the air. The product thus obtained was pure white and when redissolved gave a nearly colorless though somewhat turbid solution. The yield obtained was often as high as 87 gm. per 100 gm. of the zinc salt. On recrystallizing the prcduct once from hot water and using a little decolorizing charcoal, one obtains a pure white material which when dissolved gave a water-clear solution.

Analysis. Titration with 0.1 N sodium hydroxide. Sample 0.5 gm. Required 14.25 cc. Calculated for C16H280,+1H20,

14.4 cc. Glycuronic acid. Sample 0.1 gm. Found 0.0560 gm. Calculated for C,&L~O~~lH~O,

0.0558 gm.

Preparation of Glycuronic Acid.- 100 gm. of borne01 glycuronic acid were dissolved in 1500 cc. of 0.2 N, sulfuric acid, and the solution boiled for 3 hours under a reflux condenser. The borne01

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was filtered off, and the hot filtrate treated with a sufficient amount of barium hydroxide to remove quantitatively the sulfuric acid. The complete removal of the mineral acid is essential since even traces of the acid will seriously interfere with the iso- lation of glycuronic acid. For the sake of convenience the barium sulfate was allowed to settle, the supernatant liquid siphoned off, and the final separation of the precipiate from the liquid effected by centrifuging. The colorless solution was concentrated under reduced pressure to a syrupy consistency. Standing caused crystallization to set in, and in the course of several hours the solution became almost a solid mass. The product was filtered of3 and washed with small amounts of alcohol until all traces of coloring matter were removed. The yield was 45 gm., or over 80 per cent of the theoretical.

Analysis. Titration with 0.1 N sodium hydroxide. Sample 0.2 gm. Titration to transitory end-point, 7.5 cc.

“ “ permanent “ 10.6 “ Composition of mixture calculated from titration: glycuronic acid 70.8

per cent, lactone 29.2 per cent. Apparent molecular weight of mixture.

Calculated from total titration, 188.7. “ on basis of 70.8 per cent acid and 29.2 per cent lactone, 188.8.

Glycuronic acid. Sample 0.06 gm. Found 0.063 gm. Calculated on basis of 70.8 per cent

acid and 29.2 per cent lactone, 0.062 gm.

Separation of Glycuronic Acid from Its Lactone.-The prepara- tion of pure glycuronic acid from the mixture of acid and lactone, which is obtained by hydrolyzing borne01 glycuronic acid, is most conveniently accomplished by extraction with alcohol. In this solvent the acid is less soluble than the lactone, and by extracting a sample several times practically all of the lactone is removed. The method entails no loss of material since the portion which is dissolved in the alcohol can easily be recovered. The exact pro- cedure is best presented by citing an actual specific experiment.

4 gm. of the product containing 70.8 per cent acid and 29.2 per cent lactone were covered with 200 cc. of 95 per cent alcohol and set aside for 12 hours. The undissolved portion was fil- tered off and dried.

Yield 2.6 gm. Analysis: acid 77.5 per cent, lactone 22.5 per cent.

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The remainder of the product (2.5 gm.) was again covered with 100 cc. of alcohol, and after 12 hours the product was again filtered off and dried.

Yield 2.2 gm. Analysis: acid 91 per cent, lactone 9 per cent.

Again the remaining portion was covered with 100 cc. of alcohol and allowed to stand for 24 hours. The temperature throughout the experiment was about 25°C.

Final yield 1.5 gm. Titration with 0.1 N sodium hydroxide. Sample 0.2 gm. Titration to transitory end-point, 10.15.

“ “ permanent “ 10.25. Purity of glycuronic acid, 99 per cent.

Optical Rotatio?.-0.5 gm. of glycuronic acid was dissolved in 25 cc. of water. The initial rotation observed in 3 minutes after the substance was dissolved was [ar]:’ = +16.5. The maxi- mum rotation obtained in 3 hours and which remained constant for several days was [cy]2d) = +36.

Preparation of Lactone of Glycuronic Acid.-5 gm. of the mixture of acid and lactone were dissolved in 25 cc. of hot glacial acetic acid. On cooling, the lactone alone crystallized out. The yield was 3 gm. of a slightly brown crystalline solid which when dis- solved in water gave a solution which was neutral to litmus. A pure colorless preparation of the lactone was obtained by one recrystallization from hot water.

Analysis. Titration with 0.1 N sodium hydroxide. Sample 0.2 gm. Required 11.4 cc. Found 11.4 cc. Glycuronic acid. Sample 0.05 gm. Found 0.0558 gm. Calculated 0.0551 gm.

Optical Rotation.-0.5 gm. was dissolved in 25 cc. of water. The specific rotation observed was [oL]~’ = +19.

Borneo1 glycuronic acid, because of the ease with which it can be obtained in large quantities, offers one of the best sources for the preparation of glycuronic acid. The procedure developed

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for preparing borne01 glycuronic acid consists in feeding borne01 to dogs, isolating the conjugated glycuronic acid from the urine as the zinc salt, and finally preparing from this salt the free acid.

The hydrolysis of borne01 glycuronic acid by means of dilute sulfuric acid yields a colorless crystalline product containing approximately 70 per cent of glycuronic acid and 30 per cent of the lactone. From this mixture pure glycuronic acid can be prepared by extraction with alcohol since the la&one is more soluble in that solvent than the acid. A convenient method for preparing the lactone consists in dissolving the mixture in hot glacial acetic acid from which the lactone alone crystallizes on cooling. The properties of the acid and the lactone correspond to those described in the literature.

BIBLIOGRAPHY.

1. Hervieux, C., Bull. sot. chim., 1908, iii, 349. 2. Mayer, P., 2. physiol. Chem., 1901, xxxii, 518. 3. Ehrlich, F., and Rehorst, K., Ber. them. Ges., 1925, Iviii, 1989. 4. Thierfelder, H., 2. physiol. Chem., 1887, xi, 388. 5. Hildebrandt, H., Biochem. Z., 1909, xxi, 1. 6. Fromm, E., and Clemens, P., 2. physiol. Chem., 1902, xxxiv, 385. 7. Quick, A. J., J. Biol. Chem., 1924, lxi, 667. 8. Quick, A. J., J. Biol. Chem., 1926, Ixix, 549.

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Armand J. QuickGLYCURONIC ACID

GLYCURONIC ACID AND THE PREPARATION OF BORNEOL

1927, 74:331-341.J. Biol. Chem. 

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