THE STRUCTURE OF BIOTIN: A STUDY OF DESTHIOBIOTIN

BY VINCENT DU VIGNEAUD AND DONALD B. MELVILLE*

(From the Deparlwlent oj Biochemistry, Cornell University Medical College, New York City)

AND KARL FOLKERS, DONALD E. WOLF, RALPH MOZINGO, JOHN C. KERESZTESY, AND STANTON A. HARRIS

(From the Research Laboratory of filer& and Company, Inc., Rahway, New Jersey)

(Received for publication, September 25, 1942)

Structural studies on biotin in the previous paper (1) showed that the molecule probably possessed either structure (I) or (II). Structure (I) appeared to be more acceptable than (II) for the interpretation of experi- mental data, particularly the formation of adipic acid from the oxidation

I I CII&- 4’CH I I

CI-1; 2dl~-C~2-CH2-CH2-CH2-C00H

\;/

(1)

0

A NH NH

I I CH---CH

/ \ CH, CH-CHz-CH,-CH,-COOH

\ / S- CH2

(II)

(2) of the diaminocarboxylic acid derived from biotin (3). However, since the oxidative mechanism of the formation of adipic acid might have in- volved the decarboxylation of an intermediary malonic or P-keto acid

* Acknowledgment is made to the 8. M. A. Corporation in appreciation of a re- search grant which has made part of this work possible.

475

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476 DESTHIOBIOTIN

derivative from (II), this structure was not eliminated. Evidence from additional degradation reactions described herein eliminates structure (II) from consideration and establishes the preferred formulation (I) as the structure of biotin.

It was believed that organic sulfides could be cleaved by the Raney nickel catalyst in the absence of a hydrogen atmosphere according to the equation

R-S-R’ Ni(H)

--+ RH + R’H

If this reaction were successful on biotin, the desthiobiotin produced would have either structure (III) or (IV) as based upon structures (I) and (II) respectively for biotin. Just as the ureido ring of biotin is hydrolyzed (3) to the diaminocarboxylic acid, the ureide (III) should yield r,s-diamino-

ii

A NH NH I I

CH- CH I I

CH8 CHZCH~CH~CH~CH~CO~H (III)

0 II

C /\

NH NH I I

CH- CH / \

CH, ‘CHCH2CH2CH2C02H /

CH(

(IV)

pelargonic acid (V) on hydrolysis, and the ureide (IV) should yield b-methyl-e,{-diaminocaprylic acid (VI). The diamino acids (V) and (VI) contain one and two carbon-methyl groups respectively. Thus, a Kuhn-

NH* NH2 NH* NH2 I I I I

CH-CH CH-CH I I / \

CR, CH,CHsCHzCHzCHzCOzH CHs CHCHsCHzCHzCOzH

(V) / CHs

(VI)

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DU VIQNEAUD, MELVILLE, FOLKEXS, WOLF, MOZINGO, 477 KERESZTESY, AND RARRIS

Roth carbon-methyl group determination on the desthiodiaminocarboxylic acid so obtained from biotin would quickly differentiate between struc- tures (V) and (VI). More positive characterization of the desthiodiamino- carboxylic acid could be established by an oxidative cleavage reaction. Pimelic acid would be formed, if its structure were (V), and a-methyladipic acid would be formed if its structure were (VI).

Because of the limited amounts of available crystalline biotin, the sulfide cleavage reaction over Raney’s nickel was tried on “model” compounds. The sulfides (VII), (VIII), and (IX), which possess certain structural fea- tures of biotin, were cleaved to their corresponding sulfur-free products in yields of 65 to 95 per cent on both a macro and semimicro scale. These reactions and the results of subsequent studies on sulfur compounds not related to biotin are described in detail elsewhere.’ When biotin methyl

S-(CH,CH,CH,CH,CO,H), __f 2CH,(CH,),C02H

(VII) CH3SCHzCH2CHC02H CH,CHzCHCOzH

I - I NHCOCBHI, NHCOCJI6

(VII I)

CH,SCH2CH,CH-C=O CHSCHSCH-C=O

II - NH NC&H, AH I!CaHs

‘C’ ‘C’

II II 0 0

(IX) ester was treated similarly, a product containing the same number of carbon atoms and 2 added hydrogen atoms and no sulfur atom was obtained. Whereas earlier studies had strongly indicated the cyclic nature of the sulfide group in biotin, the formation of desthiobiotin provided definite proof of it.

Desthiobiotin methyl ester was hydrolyzed under several conditions by aqueous HCI (4), and the corresponding desthiodiaminocarboxylic acid was isolated as the dihydrochloride. The Ba(OHJ method (3) of hydrolysis yielded the desthiodiaminocarboxylic acid sulfate, and this salt was ob- tained more satisfactorily than the dihydrochloride. A carbon-methyl group determination on the desthiodiaminocarboxylic acid sulfate showed the presence of only one such group, which corresponds to structure (V) and not (VI).

1 Moeingo, R., Wolf, D. E., Harris, S. A., and Folkers, K., unpublished dat,a.

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478 DESTHIOBIOTIN

Oxidative cleavage reactions on the desthiodiaminocarboxylic acid by nitric acid and alkaline permanganate gave low yields of mixtures of adipic and pimelic acids from which the pimelic acid was not separated satisfac- torily on a micro scale. Oxidations with lead tetraacetate and alkaline hypochlorite solution were no better, but oxidation with alkaline periodate solution gave good yields of crude acid from which pure pimelic acid was obtained and identified as such and as its di-p-bromophenacyl ester by com- parison with authentic samples of each.

When the desthiodiaminocarboxylic acid was treated with phenan- threnequinone, the quinoxaline derivative (X) was obtained. Again (l), the oxidized form (X) rather than the dihydro form (XI) was obtained, as

N \ HN

c-c I I

CHs CH2CH2CH2C~2C~~zCOzH

co

N\ /N CH-CH

I I CHs CH~CH~CHZCH,CH~CO~H

(Xl)

shown by analyses and the formation of a characteristic red color with sul- furic acid. The compound showed no optical activity in NaOH solution, in agreement with structure (I) for biotin. The corresponding product from structure (II) would be expected to show optical activity. That the quinoxaline derivative had structure (X) was established by comparing it with an authentic specimen synthesized according to the accompanying reactions.

The quinoxaline derivatives of the synthetic and isolated diaminocar- boxylic acids were synthesized to facilitate the comparison of the two com- pounds, since the quinoxaline derivatives contain no asymmetric carbon atoms. In this way the resolution of the synthetic diaminocarboxylic acid was obviated. The ultraviolet absorption spectrum of the synthetic com- pound was compared with the spectra of the quinoxaline and dihydroquin-

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DU VIGNEAUD, MELVILLE, FOLKERS, WOLF, MOZINGO, 479 KERESZTESY, AND HARRIS

ii CHsCCHzC02CzHb + Br(CH,),C02C2H,

NaOEt ------+ CHaC-CH-C02CzHs

I (iX&CO&zHs

%H; CH !(CH) CO H CnH6OH CzHsONO

3 26 2 Hf+ CH,C(CH,),COzCJI, -

OH OH OH

CH i-&H ) CO C II NHtOH

3 25 225 p----f CH,C-C(CH&CO&zH5 ++

OH- + ca

AH (;H)COCH 3 2 5 2 2 5

oxaline derivatives of 3,4-diaminotetrahydrothiophene (1). The curve of the synthetic compound was similar to that of the quinoxaline derivative of 3,4-diaminotetrahydrothiophene, in agreement with the other data that the synthetic compound was the oxidized form (X).

Therefore, the isolation of pimelic acid as the oxidation product of the desthiodiaminocarboxylic acid and the synthesis of the quinoxaline deriva- tive established structure (V) for the desthiodiaminocarboxylic acid and established biotin as 2’-keto-3,4-imidazolido-2-tetrahydrothiophenevaleric acid, as represented by structure (I). In an accompanying paper (5) structure (I) has been established for biotin by direct demonstration of the presence of a 5-membered sulfur ring with an n-valeric acid side chain attached in the a: position.

EXPERIMENTAL

The biotin methyl ester (m.p. 162-163” corrected, [cy]i5 = +55.5’) used in these studies corresponded exactly in properties to that isolated (6) by other methods.

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480 DESTHIOBIOTIN

Desthiobiotin Methyl Ester (Methyl (4-Methyl-5-imiclaxolidone-W)-capro- ate)-100 mg. of biotin methyl ester were dissolved in 100 cc. of 90 per cent ethanol and refluxed with approximately 5 gm. of Raney’s nickel for 5 hours. The reaction mixture was centrifuged to remove the nickel, which was washed with 125 cc. of hot 95 per cent ethanol in five portions, then with 35 cc. of hot methanol. The combined eluates were concentrated to dryness in vacua at not over 40”. The residue was taken into about 5 cc. of methanol, centrifuged, and the precipitate was washed with an additional 5 cc. of methanol. The methanol solution was concentrated in vacua and the residue was thoroughly dried. Extraction of the dry material with chloroform gave a solution of the crude ester. Insoluble material was col- lected on a filter and the filtrate was concentrated in vacua. The yield was 85.5 mg. Purification was accomplished by sublimation of the crude product at 10” mm. and 100”. The highest melting point observed was 69-70”. [(Y]:* = f2.6” for a 2 per cent solution in chloroform.

CIIHZ~O~N~. Calculated. C 57.87, H 8.83, N 12.27 228.3 Found. “ 57.84, “ 8.83, “ 12.27

It was observed that desthiobiotin, desthiodiaminocarboxylic acid sul- fate, and dihydrochloride had very low positive specific rotations. Even though the removal of the sulfur atom destroys the asymmetry at carbon atom 2 (I), there is no evidence that partial racemization took place at carbon atoms 3 and 4, although the possibility of such a reaction has been considered.

Desthiodiaminocarboxylic Acid Dihydrochloride ({, q-Diaminopelargonic Acid Dihydrochloride)-85 mg. of crude desthiobiotin ester were dissolved in 15 cc. of concentrated HCl and the solution was heated in a sealed tube at 200” for 1 hour. Some darkening occurred with the formation of an insol- uble film on the surface of the solution. The insoluble material was col- lected on a filter and the filtrate was concentrated in vacua to dryness and then twice concentrated with 1 cc. of water to remove traces of HCl. The crude product was dissolved in absolute ethanol, traces of insoluble material were collected on a filter, and the clear solution was concentrated in vacua to dryness. Crystals were obtained by dissolving the residue in a minimum of methanol, and diluting with about 3 volumes of absolute ethanol and then with ether until cloudy. Small clumps of crystals of the diamine hydrochloride were obtained. The yield was 61 mg.; the melting point 180-182”. [cx]“,” = +4.04’ for a 0.75 per cent solution in methanol.

CsH&XzNz02 (261.2). Calculated, C 41.38, H 8.49; found, C 41.36, H 8.66

Desthiodiaminocarboxylic Acid sulfate (< , q-Diaminopelargonic Acid Sul- fate)-20 mg. of desthiobiotin methyl ester were placed in each of eight

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DU VIQNEAUD, MELVILLE, FOLKERS, WOLF, MOZINGO, 481

KERESZTESY, AND HARRIS

small test-tubes. To each were added 400 mg. of Ba(OH)2 and 2 cc. of water. The tubes were sealed and placed in a bath at 140’ for 16 to 17 hours. The contents of the tubes were combined, saturated with Cot, and the barium carbonate was centrifuged and washed. The total aqueous portion was acidified with sulfuric acid until faintly acid to Congo red. The barium sulfate was separated and the filtrate was concentrated nearly to dryness in vczcuo. Addition of methanol gave a crystalline product. The yield was 151.5 mg. The crude product was recrystallized by dissolving it in a minimum of water and diluting with about 3 volumes of methanol. The melting point was 242-243”. [cy]i6 = t-7.75” for a 1.4 per cent solution in water.

CsHnzNzO&. Calculated. C 37.75, H 7.75, N 9.79, C-methyl 5.25 286.4 Found. “ 37.71, “ 7.82, “ 9.78, “ 1.88

Oxidation of Desthiodiaminocarboxylic Acid Sulfate ([ , q-Diaminopelar- gonic Acid Sulfate) with Periodate-During the preliminary studies of oxi- dizing agents and conditions for this oxidation, use was made of the bio- logical method for the determination of pimelic acid (7). In the first experiments, this bioassay indicated the presence of pimelic acid in the crude oxidation products, and in the later ones it aided in the selection of the oxidation conditions.

50 mg. of desthiodiaminocarboxylic acid sulfate were dissolved in 5.5 cc. of water. To the solution were added 1.15 cc. of N NaOH and 2.55 cc. of 0.206 N periodic acid and the mixture was allowed to stand overnight at room temperature. Tests with potassium iodide-starch paper after this period were positive. The mixture was warmed to 40” for 3 hours and finally to 75” for 23 hours to complete the oxidation, or until tests for the oxidizing agent were no longer positive. The solution was acidified with HCl to Congo red and extracted continuously with ether. The extract yielded 22.7 mg. of white solid material. Preliminary purification was accomplished by sublimation in a high vacuum, the last sublimed portion melting at 95-100”.

The crude sublimates from the oxidation of three 50 mg. samples were recrystallized repeatedly from a mixture of ether and petroleum ether. In this way 17 mg. of nearly pure pimelic acid were isolated from the three sub- limates. Recrystallization gave pure material melting at 103-104”. A mixture of this material with a known sample of pure pimelic acid (m.p. 103-104”) melted at 103-104”.

C&H1204 (160.2). Calculated, C 52.49, H 7.56; found, C 52.71, H 7.72

For further identification the p-bromophenacyl ester was prepared. The pure ester melted at 137.5-138.5”. A mixture of the compound with a pure

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482 DESTHIOBIOTIN

known sample of p-bromophenacyl pimelate (m.p. 137-137.5”) showed no depression of the melting point.

C&Hz2Br20c (554.3). Calculated, C 49.84, H 4.00; found, C 50.14, H 4.24

By fractional crystallization from ether-petroleum ether a trace of crude adipic acid was obtained melting at 144.5-150”. The melting point of a mixture of this substance with adipic acid (m.p. 150.5-151”) was 148-150”.

Dibenzoquinoxaline Derivative of Desthiodiaminocarboxylic Acid (W-Meth- yl-3-dibenxoquinoxalinecaproic Acid)-50 mg. of desthiodiaminocarboxylic acid sulfate were converted to the free diaminocarboxylic acid by treatment with the calculated amount of Ba(OH)2. The desthiodiaminocarboxylic acid was dissolved in 15 cc. of ethanol and 43 mg. of phenanthrenequinone were added to the solution. The solution was refluxed for 10 hours on the water bath. A small amount of insoluble material was removed by filtra- tion and the filtrate was concentrated to 2 cc. in vacua. On addition of 3 cc. of water a crystalline material separated. This was washed twice with an alcohol-water mixture and dried. The material, ,47 mg., m.p. 182-186”, was recrystallized from alcohol-water. The recrystallized compound, 44 mg. of pale yellow micro plates, melted at 1866187”. With sulfuric acid the compound produced a deep red color. A 0.9 per cent solution of the compound in 0.04 :r NaOH showed no optical activity in a 2 dm. tube.

Cz,HANz. Calculated. C 77.06, II 6.19, N 7.82 358.4 Found. ‘( 77.39, [‘ 5.95, ‘( 8.14

v-Ketopelargonic Acid-The e-bromocaproic acid was prepared according to the general method of Marvel and coworkers (8). 133.9 gm. of ethyl acetoacetate were dissolved in ethanol to which 23.7 gm. of sodium had been added. A little sodium iodide was added and, after the solution had been heated to boiling, 250 gm. of ethyl e-bromocaproate were added. The solution was refluxed for several hours, the alcohol was distilled, and the product was dissolved in ether and was washed with water to free it from sodium bromide. The product was distilled under reduced pressure; b.p. 144-148” at 0.9 mm.; yield 188 gm. (64.5 per cent).

2.3 gm. of the ester were dissolved in diethylene glycol to which had been added 8 gm. of NaOH dissolved in the minimum quantity of water. This solution was warmed on the steam bath for 30 minutes and the precipitate of sodium carbonate was separated. The mixture was poured into acidified water and extracted with chloroform and benzene. The extract was dried, concentrated, and distilled; b.p. 135” at 0.9 mm.; m.p. 39-40”.

C&H1803 (172.2). Calculated, C 62.76, H 9.37; found, C 62.33, H 9.33

Ethyl c,q-Dioximinopelargonate-Since it was found that the above keto acid was partially esterified by the action of ethyl nitrite and I-ICI, it was

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DU VIGNEAUD, MELVILLE, FOLKERS, WOLF, MOZINGO, 483 KERESZTESY, AND HARRIS

first esterified and then nitrosated. The acid was dissolved in an excess of ethanol and after the addition of 1 cc. of concentrated H2S04 the solution was refluxed for 4 hours. The solution was concentrated, washed with water, and distilled; b.p. 91-96” at 0.4 mm.

CllHJ003 (200.3). Calculated, C 65.97, H 10.07; found, C 65.99, H 9.90

The keto ester was nitrosated by a procedure similar to a method de- scribed (9). 4 gm. of the keto ester were dissolved in a little ethanol and 1 drop of concentrated HCl was added. The solution was heated to 45-50” and 1.54 gm. of ethyl nitrite were added; the temperature was kept near 50”. The solution was allowed to stand until there was no longer a test for nitrite. It was then treated with 2 equivalents of hydroxylamine hydro- chloride and 3 gm. of sodium acetate. After the solution had been heated for 30 minutes on the steam bath and diluted with water, the dioximine crystallized. It was recrystallized from benzene and then from methanol and water; m.p. 107-108”.

CL~HS~O&~. Calculated. C 54.08, H 8.25, N 11.47 244.3 Found. “ 53.68, “ 7.89, ” 11.52

” 53.46, “ 8.03

Ethyl {,T-Diaminopelargonate-6.1 gm. of the dioximino ester were dis- solved in 50 cc. of methanol and 100 cc. of liquid ammonia and hydrogen- ated over 3 gm. of Raney’s nickel at 50-55” and 140 atmospheres for about 2 hours. After removal of the catalyst by filtration, the ammonia was re- moved by concentration under reduced pressure. The residue (3 gm.) was dissolved in 50 cc. of ethanol and treated with sulfuric acid until just acid to Congo red, when the sulfate crystallized; m.p. 274” with decomposition.

C,H2,0,N,S. Calculat,ed. C 42.02, H 8.34, N 8.91 285.4 Found. I‘ 41.48, “ 8.12, “ 9.13

Ethyl 2-Methyl-3-dibenzoquinosalinecaproate-0.005 mole of ethyl j-, q- diaminopelargonate was refluxed overnight with 0.52 gm. of phenanthrene- quinone in 16 cc. of ethanol. The solution was filtered and concentrated to dryness. The gummy residue was crystallized from alcohol and water; m.p. 78-79”. The crystals gave a red color with sulfuric acid.

GH&xO,. Calculated. C 77.69, H 6.77, N 7.25 386.5 Found “ 77.42, “ 6.89, “ 7.55

W-Methyl-S-dibenzoqu&oxalinecaproic Acid-This acid was prepared by the hydrolysis of the ethyl ester (60 mg.) with 1 equivalent of NaOH in water, and by the direct condensation of the {,v-diaminopelargonic acid with phenanthrenequinone. After crystallization from ethanol the melt-

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484 DESTHIOBIOTIN

ing point of the compound was 186-187”. A mixture of this compound with the quinoxaline derivative of the desthiobiotin prepared from biotin melted at 186-187”.

CUHXN~O~. Calculated. C 77.06, H 6.19, N 7.82 358.4 Found. ” 77.05, “ 6.28, “ 8.18

The coworkers at Cornell University Medical College wish to express their appreciation to Dr. J. R. Rachele for microanalyses and to Miss Eleanor Hague for the pimelic acid assays.

The coworkers in the Merck Research Laboratory wish to acknowledge the valuable assistance of Mr. Rickes and Mr. Chaiet on the isolation of biotin, Dr. J. L. Stokes and assistants for microbiological assays, Mr. Anderson and Mr. Easton on synthetic work, Mr. Hayman, Mr. Clark, and Mr. Boos for microchemical analyses, and Mr. Bastedo, Jr., for the ultraviolet absorption determination on the 2-methyl-3-dibenzoquinoxa- linecaproic acid.

SUMMARY

Treatment of biotin with Raney’s nickel catalyst cleaves the sulfur atom and 2 atoms of hydrogen are added. Hydrolysis of this desthiobiotin in acid or alkaline solution gave the corresponding desthiodiaminocarboxylic acid which on oxidation with periodate yielded a dibasic acid identified as pimelic acid. By treatment with phenanthrenequinone the desthiodiamin- ocarboxylic acid was converted to the corresponding quinoxaline derivative which agreed in all its properties with the synthetically prepared compound.

The formation of pimelic acid by oxidation of desthiobiotin and the iden- tity of the synthetic quinoxaline derivative with that obtained from desthiobiotin, in conjunction with other published data, establishes the structure of biotin as given in the accompanying formula.

NH NH I I

CH---CC I I

'Ilig/ CH-CHr--CHr-CHa-CHP-COOH

BIBLIOGRAPHY

1. Hofmann, K., Kilmer, G. W., Melville, D. B., du Vigneaud, V., and Darby, H. H., J. Biol. Chem., 146, 603 (1942).

2. Hofmann, K., Melville, D. B., and du Vigneaud, V., J. Am. Chem. Sot., 83,3237 (1941).

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3. Hofmann, K., Melville, D. B., and du Vigneaud, V., J. Biol. Chem., 141,207 (1941). 4. K6g1, F., and Pons, L., 2. physiol. Chem., 269, 61 (1941). 5. Melville, D. B., Moyer, A. W., Hofmann, K., and du Vigneaud, V., J. Biol. Chem.,

146, 487 (1942). 6. du Vigneaud, V., Hofmann, K., Melville, D. B., and Gyergy, P., J. Biol. Chem.,

140, 643 (1941). 7. du Vigneaud, V., Dittmer, K., Hague, E., and Long, B., Science, 96, 186 (1942). 8. Marvel, C. S., MacCorquodale, D. W., Kendall, F. E., and Lazier, W. A., J. Am.

Chem. Sot., 46, 2838 (1924). 9. Clarke, H. T., et al., Organic syntheses, New York, 10, 22-27 (1930).

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John C. Keresztesy and Stanton A. HarrisMozingo,Karl Folkers, Donald E. Wolf, Ralph

Vincent du Vigneaud, Donald B. Melville,OF DESTHIOBIOTIN

THE STRUCTURE OF BIOTIN: A STUDY

1942, 146:475-485.J. Biol. Chem. 

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