ADIPIC ACID AS AN OXIDATION PRODUCT OF THE DIAMINO ...

ADIPIC

ACID AS AN OXIDATION PRODUCT OF THE DIAMINOCARBOXYLIC ACID DERIVED FROM BIOTIN*

BY KLAUS (From

the

HOFMANN, Department

DONALD B. MELVILLE, DU VIGNEAUD

of Biochemistry, New York

Cornell City)

(Received for publication,

University

AND

Medical

VINCENT College,

May 7, 1942)

* Preliminary reports of this work have been published (1, 2). The authors wish to express their appreciation to the S. M. A. Corporation for a research grant which has aided greatly in this work. They also wish to thank Mr. W. 0. Frohring and the Research Staff of the S. M. A. Corporation and Dr. R. Major and the Research Staff of Merck and Company, Inc., for supplies of biotin. 513

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In recent communications we have established the molecular formula as well as the nature of the functional groups of biotin (3-5). We now wish to report experiments which give information regarding the carbon skeleton of this compound. The diaminocarboxylic acid, C9H1802N2S,which we had obtained by the treatment of biotin with Ba(OH)z at 140” served as the starting material. This compound upon oxidation with alkaline potassium permanganate at room temperature was found to yield an ether-soluble acidic oxidation product which crystallized from ether in small prisms and melted at 152-153”. This product was identified as adipic acid by the neutral equivalent, by a mixed melting point, and by the preparation of the diamide and the di-/3-naphthylamide. The quantity of adipic acid that we were able to isolate in pure form from the oxidation products amounted to about 20 per cent of that theoretically possible. Adipic acid was also obtained in a somewhat higher yield by oxidation of the diaminocarboxylic acid with nitric acid. The isolation of the same compound under both these oxidizing conditions minimizes to a great extent the possibility of a rearrangement to an intermediate which could have yielded the adipic acid. Thus the consistent formation of adipic acid as an oxidation product of biotin may be interpreted in one of two possible ways. Either biotin contains an aliphatic side chain which is capable of yielding adipic acid or the latter has its origin in a cyclic structure which is cleaved by the oxidation. In the first case one of the carboxyl groups of the adipic acid must be the carboxyl group originally present in biotin, and it should therefore be possible, by the oxidation of a derivative of the diaminocarboxylic acid in which the carboxyl group has been eliminated, to decide between the two alternatives. After several attempts by other methods the objective was achieved by a Curtius degradation. In this way the carboxyl group was replaced by an amino group. Biotin methyl ester was

514

ADIPIC

ACID

FROM

RIOTIN


converted into the hydrazidc, CloIIl&~N$. When the calculated amount of nitrous acid was added to this in dilute HCl, a rather stable azide was formed which, when dissolved in absolute ethanol and boiled for 2 hours, was transformed into the corresponding ethyl urethane, C&HZ103N3S. The further breakdown of the urethane was performed in two ways. The first route was the complete hydrolysis with strong Ba(OH)2 which led to the triamine, C8H19N$, characterized as the sulfate, the tripicrolonate, and the tribenzoyl derivative. The second route was a stepwise degradation first with strong HCl to the monoamine, GH170N3S, followed by the drastic Ba(OH)2 treatment to the triamine, CsH19N&S, which was identical with the triamine above. Evidently the alkaline hydrolysis opens the urea ring and cleaves the urethane group to yield the triamine; the acid treatment, however, splits the urethane selectively, without affecting the urea ring, to give the monoamine. The latter corresponds to biotin with the, carboxyl group replaced by an amino group. Subjection of this compound to alkaline treatment opens the urea ring to yield the triamine. The triamine was subjected to the same oxidation procedures which we employed for the oxidation of the diaminocarboxylic acid. In the first experiment 10 mg. of the triamine were oxidized with nitric acid. No indication of the presence of adipic acid among the ether-soluble oxidation products was observed. A repetition of the experiment on the ‘same amount of material led to a similar result. Under the same conditions adipic acid had been readily isolated after the oxidation of similar amounts of the diaminocarboxylic acid. Finally when more material became available, 50 mg. of the triamine sulfate were oxidized with potassium permanganate under the same conditions employed in the oxidation of the diaminocarboxylic acid. Here again no trace of adipic acid could be detected in the ether-soluble oxidation products, although the amount of adipic acid which might have been formed from the relatively large amount of triamine used would have made its isolation and identification comparatively easy. The absence of adipic acid in isolable amounts among the oxidation products of the triamine therefore affords substantial evidence that one of the carboxyl groups of the adipic acid. formed by oxidation of the diaminocarboxylic acid is identical with the original carboxyl group of biotin. This means in effect that the 6-carbon moiety giving rise to adipic acid upon oxidation is not present in biotin as a cyclic structure, but indicates the presence of an aliphatic acid side chain in biotin which is capable of yielding adipic acid on oxidation. The possible interpretation of these findings and other data previously published (1,3-7) with respect to the structure of biotin has been discussed in the preliminary note (2) and will not be repeated here.

HOFMANN,

MELVILLE,

AND

DU

VIGNEAUD

515

EXPERIMENTAL


Oxidation of Diaminocarboxylic Acid with Nitric Acid-10 mg. of the diaminocarboxylic acid sulfate were dissolved in 1 cc. of nitric acid (sp. gr. 1.42). After the first vigorous reaction the solution was heated on the steam bath for 1 hour and was then evaporated to dryness in vacua. The residue was taken up in 1 cc. of water and was evaporated to dryness again, this process being repeated twice to free the products from excess nitric acid. The residue was then continuously extracted with ether for several hours. The ether extracts were washed with a small amount of water, were dried over sodium sulfate, and were evaporated. The resulting 3 mg. of acids were washed with a few drops of ether and were purified by sublimaThe sublimate was recrystallized from tion in vacua (loo’, 0.01 mm.). ether and yielded 1 mg. of short prisms melting at 152-153”. No depression of the melting point was observed when this material was mixed with a sample of pure adipic acid. Oxidation oj Diaminocarboxylic Acid with Barium Permanganate-20 mg. of t,he diaminocarboxylic acid sulfate were dissolved in 2 cc. of water and 1.2 cc. of 0.1 N Ba(OH)2 and were oxidized at room temperature by adding The solution was kept dropwise 1.8 cc. of 0.1 M barium permanganate. at room temperature overnight and the MnOz was removed by filtration and was washed repeatedly with hot water. The clear filtrate was concentrated to a small volume in vacua, was acidified to Congo red with N HzS04, and was extracted with ether for several hours. 3 mg. of crude acids, melting at 120-130”, were obtained from the ether extract. The acids were purified by sublimation in vacua and the fraction subliming at 80-100” (0.01 mm.) was further purified by crystallization from ether. 1 mg. of short prisms melting at 152-153” was obtained. The substance gave no depression of the melting point when mixed with a sample of pure adipic acid. Biotin-l mg. of adipic acid from biotin Diamide of Adipic Acid from was transformed into its acid chloride by treatment with 5 drops of thionyl chloride. The acid chloride was distilled in vacua and the distillate was mixed with a solution of ammonia in water. After standing for 10 minutes the solution was evaporated to dryness and the residue was washed with ice water. The latter was then sublimed at 150-160” (0.01 mm.). The purified material melted at 224-226’, with a change at 210’ from short prisms to long needles. A sample of adipic acid amide prepared in the same manner from authentic adipic acid behaved exactly as described for the product from biotin and a mixture of the two compounds showed no depression of the melting point. Di-P-naphthylamide of Adipic Acid from Biotin-1 mg. of the acid was transformed into the acid chloride as described and this was coupled with

516

ADIPIC

ACID

FROM

BIOTIN

CzeH2402N2. (396.45)

Calculated. Found.

C 78.76, “ 78.88,

H 6.10 “ 6.03

Biotin Hydra&de-20 mg. of biotin methyl ester and 0.5 cc. of hydrazine hydrate were heated in a sealed tube at 130” for 3 hours. The clear solution was then evaporated to dryness in vacua and the crystalline residue was washed with methanol and ether. 19 mg. of material melting at 237-239” were obtained which on recrystallization from water gave clusters of prisms which melted at 238-240’. CIoH1802NaS. (258.3)

Calculated. Found.

C 46.50, “ 46.82,

H 7.02, “ 7.09,

iX 21.68 “ 21.88

Ethyl Urethane, C~zH,103N&--52 mg. of biotin hydrazide were dissolved in 0.5 cc. of N HCI and the solution was cooled with ice. To this was added 0.4 cc. of a solution containing 200 mg. of NaNOz in 5 cc. of water and the solid precipitate of the azide was collected, was washed with ice water, and The dried azide (39 mg.) was was dried over PZOS at room temperature. boiled for 2 hours with 5 cc. of absolute ethanol and the solution was then evaporated to dryness in vacua. 40 mg. of crude urethane were obtained which sintered at 165’ and melted at 171-176”. 10 mg. of the compound


purified fl-naphthylamine in ether. The ether was evaporated and the crystalline residue was transferred to a filter with N HCl and was washed The material was further purified by crystallizawith N HCl and water. tion from glacial acetic acid, followed by sublimation in vacua at 255’ (OS01 mm.). The pure material melted at 267-268” and likewise showed a transformation from prisms to needles at 240-250”. No depression of the melting point was observed when this compound was mixed with a sample of authentic adipic acid di-p-naphthylamide, which melted at 267-268” and showed the change from prisms to needles at 250”. Oxidation of Diaminocarboxylic Acid with Alkaline Potassium Permanganate-50 mg. of the diaminocarboxylic acid sulfate were dissolved in 2 cc. of N NaOH and to this solution was added dropwise with stirring a 5 per cent solution of potassium permanganate until the violet color remained for 10 minutes. The reaction mixture was then heated on the steam bath for 10 minutes to destroy the excess permanganate, the manganese dioxide was removed by filtration, was washed with hot water, and the filtrate was acidified to Congo red with concentrated HCl. The acidic aqueous solution was then extracted for 48 hours with ether and from the ether extract 17 mg. of crude acids were obtained. From the above acids it was possible to isolate 4 rng: of adipic acid, m.p. 147-150”, by crystallization from ethyl alcohol. From the mother liquors 1 mg. more of adipic acid was obtained. The adipic acid was converted into its di-fl-naphthylamide as described and 7 mg. of pure di-p-naphthylamide melting at 266-267” were obtained.

HOFMANN,

MELVILLE,

were purified by sublimation material melted at 188-190”. C12H2103N&J. (287.4)

AND

DU

at 165-170”

Calculated. Found.

517

VIGNEAUD

(0.01 mm.).

C 50.16, “ 50.53,

H 7.37, “ 7.64,

The sublimed

N 14.62 “ 14.19

C9Hl;ONaS.HCI. (251.74)

Calculated. Found.

C 42.95, “ 43.15,

H 7.20, “ 7.17,

N 16.68, “ 16.80,

Cl 14.03 “ 14.20

Triamine, C~Hl~N&--The sulfate of the triamine was prepared from the amine hydrochloride. 12 mg. of the latter were hydrolyzed in the usual manner with Ba(OH)z. 11 mg. of the triamine sulfate were obtained which melted at 249-252” with decomposition. (CsH1JT3S)2.3H$04.2H~0. (708.81)

Calculated. Found.

N 11.85, “ 11.57,

S 22.62 ” 22.74

The sulfate of the triamine was also obtained from the urethane. 39 mg. of the urethane were hydrolyzed with Ba(OH)s and the reaction product isolated as described for the preparation of the diaminocarboxylic acid sulfate. The 35 mg. of sulfate obtained could be crystallized from a mixThe pure material melted at 249-252” with ture of water and methanol. decomposition. The picrolonate of the triamine, CBH&I$, was prepared by mixing an aqueous solution of the sulfate with a saturated solution of picrolonic acid. The yellow picrolonate, which decomposed at 250“, was purified by crystallization from aqueous ethanol. CsH19~\‘3S.3CloI~s05T~. (981.8)

Calculated. Found.

C 46.49, “ 46.19,

H 4.41, “ 4.50,

S 3.27 “3.14

The t,ribcnzoyl derivative OCthe triamine was prepared as follows : 10 mg. of the triamine sulfate were dissolved in I cc. of water and were benzoylated The oily triby shaking with an excess of benzoyl chloride and N KOH. benzoyl derivative that separated was extracted with chloroform. The chloroform solution was washed with N KOH, 2 N HCl, and water, was dried over sodium sulfate, and was evaporated to dryness. The tribenzoyl


Preparation of Amine Hydrochloride, C9H170N3S. HCl-66 mg. of crude urethane were dissolved in 3 cc. of concentrated HCl and the solution was heated on the steam bath for 2 hours. The dark brown solution was then evaporated to dryness in vacua and the residue was taken up in water and evaporated again to free it from excess HCl. The material was then taken up in 2 cc. of water, was decolorixed with a small amount of charcoal, and the clear solution was evaporated to dryness in vacua. The crystalline residue was purified by crystallization from dilute ethanol. 32 mg. of needles melting at 265-270” with decomposition were obtained. From the mother liquors another 10 mg. of material were obtained which melted with decompositibn at 260-270”.

518 derivative methanol

ADIPIC

ACID

FROM

BIOTIN

was obtained as clusters of prisms which and ether melted at 194-195”. C2gH3103NaS. (501.6)

Calculated. Found.

when crystallized

from

C 69.44, H 6.23, N 8.37 “ 68.99, “ 6.41, “ 8.01

The authors wish to express their appreciation to Dr. Julian R. Rachele of this laboratory for carrying out the microanalyses. SUMMARY

Oxidation of the diaminocarboxylic acid derived from biotin, either with nitric acid or with permanganate, yields adipic acid as one of the oxidation products. That one of the carboxyl groups of the adipic acid is the original carboxyl group in the biotin molecule is indicated by the non-formation of adipic acid on oxidation of the amine formed by a Curtius rearrangement of biotin hydrazide. BIBLIOGRAPHY

1. Hofmann, K., Melville, D. B., and du Vigneaud, V., J. Am. Chem. Sot., 63, 3237 (1941). 2. du Vigneaud, V., Hofmann, K., and Melville, D. B., J. Am. Chem. Sot., 64, 188 (1942). 3. du Vigneaud, V., Hofmann, K., Melville, D. B., and Rachele, J. R., J. Biol. Chem., 140, 763 (1941). 4. Hofmann, K., Melville, D. B., and du Vigneaud, V., J. Biol. Chem., 141,207 (1941). 5. Melville, D. B., Hofmann, K., and du Vigneaud, V., Science, 94, 308 (1941). 6. Kiigl, F., and Pons, L., 2. physiol. Chem., 269,61 (1941). Chem., 269, 81 (1941). 7. Kiigl, F., and de Man, T. J., Z. physiol.


The tribenzoyl derivative was also prepared from the picrolonate. 22 mg. of the picrolonate were dissolved in 15 cc. of hot water, the solution was acidified to Congo red with dilute HCl, and the picrolonate was decomposed with wool. The wool was separated and was washed repeatedly with hot water and the filtrates and washings were concentrated to a volume of 1 cc. in vacua. The solution was then benzoylated as described above and the benzoyl derivative isolated and crystallized. 5 mg. of benzoyl derivative were obtained, which melted at 194-195’. Oxidation of Triamine-50 mg. of the triamine sulfate were oxidized with potassium permanganate as described for the oxidation of the diaminocarboxylic acid. 10 mg. of crude ether-soluble oxidation product were obtained. Fractionation of this material by the procedures used. for the purification of the crude adipic acid fractions yielded no adipic acid. The same negative results were obtained in our earlier experiments on smaller amounts of material with nitric acid as the oxidizing agent, as stated in the preliminary note.

ADIPIC ACID AS AN OXIDATION PRODUCT OF THE DIAMINOCARBOXYLIC ACID DERIVED FROM BIOTIN Klaus Hofmann, Donald B. Melville and Vincent du Vigneaud J. Biol. Chem. 1942, 144:513-518.

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