SYNTHESIS AND STEREOCHEWISTRY OF BIOTIN AND RELATED PRODUCTS' STANTON A. HARRIS Merck & Company, Inc., Rahway, New Jersey
T m s FAPER is intended as a brief review of the work and animal growth, including man. However, there on the synthesis of biotin and related biologically is no known deficiency disease caused by the lack of active analogs. The history of the isolation, structure, biotin in natural diets. and biochemistry of biotin is covered in four excellent In 1937 Kogl published the correct empirical formula reviews by du V i g n e a ~ d , ~H ~ f m a n n , ~Mel~ille,~of biotin methyl ester--namely, CIIHI~O~N~S. Beand Hertz.6 The reader is referred to the above re- tween 1940 and 1942 the Cornell Medical School Group views for early references on this subject. led by du Vigneaud, Hofmann, and Melville, published Biotin was originally known as a yeast growth factor. a series of papers which was culminated by the formulaIt was isolated in 1936 by Kogl and Tonnis in Holland. tion of the strncture of biotin as shown by formula I. I n 1940, Gyorgy, Melville, Burk, and du Vigneaud At that time the synthesis of biotin was undertaken suggested the identity of biotin with vitamin H and in several different laboratories. The Merck Research coenzyme R. Gyorgy's vitamin H is the anti-egg. Laboratories had couaborated with Dr. dn Vigneaud white injury factor in rats. When rats are fed a diet and his group a t the Cornell Medical School in New very high in raw-egg white, they develop a severe York during the later stages of the structural work. dermatitis. They undergo poor growth and have an I n May, 1943, Harris, Wolf, Mozingo, and Folkers6 abnormal kangaroo-like posture. Administration of of the Research Laboratories a t Merck & Company, biotin produces a rapid return to normal gait and pos- Inc., Rahway, New Jersey, announced that they had ture. The dermatitis disappears and a new growth of obtained synthetic biotin which was identical in all respects to natural biotin. The details of this synhair covers the denuded area. Coenzyme R was described by Allison, Hoover, and thesis, the resolution of &biotin, and the correlation of Burk in 1933 as having growth and respiration pro- the biotin isomers appeared in several papers during moting effects for rhizobium trifolii, a legume-nodule organism. In 1940, Gyorgy, Rose, Hofmann, Melville, and du Vigneaud isolated pure crystalline biotin methyl H i 4 \NH ester from vitamin H liver concentrate and it was H L L shown to be identical with Kogl's product isolated from egg yolks. Kogl has not accepted the identity of these H*( d A ~ ~ C H 2 ) 4 ~ h ~ preparations and he uses the terms a-biotin from egg yolk and 8-biotin from liver. (1) The extreme potency of biotin is noted by the fact 1944 and 1945 by Harris, Wolf, Mozingo, Anderson, that less than one part of biotin in 5 X 10" parts of the Arth, Easton, Heyl, Wilson, and Folkers.' Their medium are necessary for the yeast-growth assay. In synthesis is illustrated by the accompanying reactions. rats, one milligram of biotin is equal to 2000 daily Cysteine, 1 1 , a naturally occurring sulfur amino acid curative doses. Biotin is necessary for all bacterial was condensed with chloroacetic acid to give compound This paper was taken from a talk given a t the Eighth Summer 111. After benzoylation and esterification the comConference of the New England Association of Chemistry Teach- pound was treated with sodium methoxide when a ring ers, Middlebury College, Middlebury, Vermont, August 24, 1946. "ARRIS, 9. A,, WOLF,D. E., MOZINGO, R.,ANDFOLRERS, K., DU YIONEAUD, V., Science, 96, 455 (1942).
E"
"OFMANN, K., "Advances in Enzymology," Vol. 111, Interscience Publishers, Inc., New York, 1943. 4 MELVILLE, D. B., "Vitamins and Hormones," Val. 11, Academic Press, Inc., New York, 1944. 6 HERTZ, R., PAg~io2.Rev., 26, 479 (1946).
S k e , 97, 447 (1943). HARRIS,S. A,, WOLF,D. E., MOZINMJ,R., ANDERSON, R.C., h, G. E., EASTON,N. R., HEYL, D., WIWN, A. N., AND FOLKERS, K., J. Am. Chem. Soc., 66, 1756, 1757, 1800, (1944); 67,2096,2100, 2102 (1945).
JOURNAL OF CHEMICAL EDUCATION
twatmmt with ii Hosrnmnnd cutul.vnt and hyrlrogrn IWS conwrtcd to the aldehvdr XI. The condensation reaction between the ketotetrahydrothiophene, VII, and the aldehydo acid, XI, using piperidine and acetic acid as a catalyst, is shown by the reaction VII + XIII. The unsaturated ketone was treated with hydroxylamine in pyridine solution to give the unsaturated oxime, ,XIV. Compound XIV contains the complete carbon skeleton two nitrogen atoms, and the sulfur atom. It was then
closure took place to give compound VI. Treatment with hydrochloric acid in an aqueous acetic acid solution caused decarboxylation to take place to yield compound VII. Compound VII contains the tetrahydrothiophene ring, one amino group, and one potential amino group in the ketone. The synthesis of the side chain is illustrated by the reactions VIII + I X -+ X + XI. Glutaric acid anhydride was treated with methyl alcohol to give glutaric acid methyl ester which was in turn converted to the acid chloride, which on NHn
NH1 NsSCHIAHCOaNa (11)
+ CICHICOINad
NHCOCsHs
Amos
- 1
__f
+acH2c02H
c H s cH,co2H
\s/
I11
(IV) NHCOCsHr
O=
~7
(CH& =O (VIII) NHCOCeH,
CHaOH
---+ HO,C(CHd&O&Ha
ROH HPSO~
C~I~COC AHco,H ~
NHCOCsHs
Pd, H, CIOC(CHl)rCO&Hs -+ OHC(CH3,C01CH8 (x) (XI)
SOCl, d
(1x1 NHCOCaHr
NHCOC6H6
/Go\ NH NH .AR-AH AHz .AH(cHn),cozH
\s/ (XIX)
NhCO,
+ AH1 AH(cH~)~co,H COCL
NHCOCeHr
\S/
(XX)
SEPTEMBER, 1947
461
possible to reduce the oximino group and the double bond to a completely saturated compound. This was partially accomplished by using zinc dust and acetic acid in the presence of acetic anhydride. In this way, two compounds were obtained-namely, XV and XVI. It is to be noted that these compounds differ only by the position of the double bonds. Hydrogenation of these compounds, using a palladium catalyst and hydrogen gas, yielded completely saturated isomeric compounds, XVII. Compound XVII was treated with barium hydroxide a t 140°, as was previously described by du Vigneaud and his coworkers, to yield a diamino carboxylic acid, XIX. Upon treatment of this compound with sodium carbonate and phosgene, a cyclic urea compound, XX, was obtained, having the structure previously designated for biotin. At this point, the synthesis of biotiu was complicated by the fact that it contained three asymmetric carbon atoms. Such a compound should theoretically exist in eight stereoisomericforms. These should exist in four dl or racemic mixtures. Actually, the two unsaturated compounds, XV and XVI, yielded only three series of saturated compounds. The melting points of these series of compounds are represented in Table 1. The series of compounds in the first column yielded dGbiotin. The compounds in columns two and three analyzed the same as the compounds in column one, but they proved to be entirely different stereochemically and the final products were completely inactive biologically.
the synthetic and the natural biotin to be completely equivalent. It was found that strong bases would combine with biotin to yield crystallizable salts. The strongly basic amino acid l(+) arginine proved to be a very satisfactory base to combine with biotin. The d-biotin-1-arginine salt crystallized directly from aqueous isopropyl alcohol, and d-biotin was recovered from this salt in 80 per cent yields. Melville, Dittmer, Brown, and du Vigneaud reported in Science in 1943 the very surprising result that desthiobiotin was as fully active as biotin is by the yeast assay. It was reported that yeast probably resynthesized biotin from desthiobiotin. Desthiobiotin4 had been originally prepared from biotin by the replacement of the sulfur atom with two hydrogen atoms over Raney-nickel catalyst. The three biotins shown in Table 1 were treated with Raney nickel, reaction X X + XXIII. The same compounds were obtained by the alternative series of reactions as illustrated by XVIII + X X I + XXII- XXIII. It was found that dhbiotin yielded dl-desthiobiotin, which had one-half the activity of. that reported for ddesthiobiotiu. The two isomeric biotins yielded another compound which had no activity with yeast and a melting point differing from that of dl-desthiobiotin. Figure 1, taken from the original article' on the stereochemistry of biotin, illustrates the stereochemical interrelationships of the various products obtained during this synthesis of biotin. Products represented by structures (a) and (d) were not obtained experimentally.
Nomenclature end Melting Points of the Isomeric Compounds in the Biotin Synthesis dl-180, X y
dl-Allo, XVI
186-186
163-163'
' !b
Dehydro ester
dl-, "C.
Dismido ester, XVII 155 Diamido acid, XVIII 232 Sulfate of diammo acid, XIX 249-250 232 Biotin, XX
I
dl-Allo,
I
dl-epi-Allo-,
"C.
"C.
172-113 195
185-187 189-190'
228-230 194-196+
Fuses above 195+
(XVIII)
283-285
* The mixed meltingpoint of these two compounds was
182-
184".
+The mixed melting point of these two compounds was depressed to about 180'.
Although dl-biotin contained one-half the biological activity of natural biotin, it could not be compared directly with natural biotin until it had been resolved into its two components. This resolution proved somewhat difficult because it would not combine with the ordinary alkaloids to form crystallizable salts. It was first resolved by combining it with optically active mandelic acid as an ester and fractionally crystallizing to separate the two compounde. M e r hydrolysis of this ester, a biotiu was obtained which was identical in melting point, optical rotation, crystal structure, and all other physical properties with that of natural biotiu. Microbiological and rat assays showed
1
coc1,
COCI*
&-AH
-
1
1
CH-CH
462
JOURNAL OF CHEMICAL EDUCATION
dl-Biotin was synthesized in excellent yields from the diamino acid by treatment with phosgene. The other two isomers, dl-allobiotii and dl-epiallobiotin, were not obtained in such good yields, apparently because they were not stable to recrystallization from water. This observation was supported by the fact that dl-allobiotin was hydrolyzed in an aqueous solution containing one mol of sulfuric acid to the corresponding dl-allodiamino acid sulfate and carbon dioxide by boiling for a few minutes. Under the same conditions, The only other synthesis of biotin which has been dl-biotin liberated no carbon dioxide and was recovered unchanged from the reaction mixture. After removal reported in the literature appeared in 1945 in the of the sulfur atoms the dl-desthiobiotiu and dl-allodes- Heluetica Chimica Acta by Griissner, Bourquin, and thiobiotin were found to he stable under these con- Schnide9 of the Hoffmann-LaRoche Laboratories in ditions, which indicated that the instability of the urea group in dl-allobiotin and dl-epiallobiotin was due to the strained transfusion of the two rings. It was thus concluded that dl-biotin had a cis ring structure, as
Switzerland. Their synthesis is very different from that reported from the Merck Laboratories, as shown in the accompaning reactions XXVI to XXXIX, inclusive. They also obtained three racemic isomers in their synthesis. On an exchange of specimens of samples between the two laboratories it was found that their dl-p-biotin was the same as dl-biotin. Their Figure 1. Th. st-ochernice1 Int....lationahips of th. Vari.3". p-biotin and dl-iso-pbiotin did not compare with Products Obtened during t h e Synthesis of Biotin either allobiotin or epiallobiotin. They have since 'Th. .nantiorn.,rph. are omitted born this diamam for rrirnplicity: reportedqhat these two isomers do not yield either R C6HsCO-. CHxO. H. or --CO--: R1 (CH%)ICO~H. * The confi(yurF.tion of the .ids c h d n has not been ost8bli.h.d. dl-desthiobiotin or dl-allodesthiobiotin and, therefore, they must be structurally different rather than stereoshown by formula XXIV. Likewise the other two chemically diierent from biotin. biotin isomers must have transfused rings as shown by Two syntheses of dl-desthiobiotin appeared in the formula XXV. The formation of the strained ring compounds isomeric to biotin was a very surprising Journal of the American Chemical Society in 1945. The result. The fact that they were obtained a t all is h t , as shown by reactions XL to XLVI, was by Wood explainedtby their very low solubility in water under and du VigneaudIo of the Cornell Medical Laboratories the conditions of the experiment. They were so un- and the second, as represented by XLVII + LIV,was stable that they could not be recrystallized satisfac- by Duchinsky and Dolan" of the Hoffmann-LaRoche torily from water or other solvents. Biotin can be Laboratories in Nutley, New Jersey. Dr. Hofmann recrystallized in excellent yield. The relationship of has reported in several papers from the Ciha Laborathese isomers of biotin can best be demonstrated by GR~SSNER, A,, BOURQUIN, J. P., AND SCHNIDER, O., Helu. the use of models. The above work, plus the assump Chim. A&, 28, 517 (1945). A,, BOURQUIN, J. P., AND SCANIDER, O., &id., 29, tion that hydrogen is added only in the cis manner to 7709 GR~SSNER, (1946). the double bond, leads one to the conclusion that biotin lo WOOD, J. R., AND nu VIONEAUD, V., J. Am. Chem. Soc., 67, has the cis, cis structure. Figure 2 shows a picture of -21n -- 11945i ,- - -- ,. DUSCAINBKY, R., AND DOLAN,L. A,, ibd., 67,2079 (1945), such a model of biotin.
+
I
-
SEPTEMBER. 1947
463
the tohl synthesis -of dhxybiotin. This synthesis is shown by reactions LV to LX, inclusive. In this oxygen series the trans-amino derivatives did not give
tones and later from the University of Pittsburgh on the synthesis of oxybiotin. Paper No. V P reported l2
HOPMANN, K., ibid., 67, 1459 (1945).
ROOC-CH4Hz
I?-
ROOC-CH-CH2
A
AH
I
>,
xI
AH X (XXVIII)
ROOC-CH-CHz
ROOC-
I
AH x (XXX)
I
bx -
- 4 H
A1
AH X (XXIX)
NH,. NH . O M H + X z
ROO
NH2.NH.0C
AH-;4 -
I
(XXXII)
+
CHdCOGHA (Z) H02CCH-(CHJ&O*H
1. Saponification 2. Bromination
i 2H-
Br(CHWOsC2H6
J
3. Amination 4. Decmboxylation
(XLI)
HCl &OzH
CHaCOyH-(CHJECOSH
IH
X (XXXIII)
a (C%GHE),CH(CH~)~CO~C~H~
NHn (XLII)
CHd+H-(CHi
I
ROOC-CH-CH,
(XXXI)
L
HOO
I
(XXVII)
ROOC-CH--CH,
A 2-
HIN.OC - H
N=C-C-CH
(xxvn
ROOC4H-CH,
d 2-
1 ' /3 -
0-CH
ROOCC-CH
ROOC-CH-CH2
484
JOURNAL OF CHEMICAL EDUCATION
the cyclic compounds as did the sulfur series. The into biotin. This does not appear to be the case with most surprising and unexpected part of this work was oxybiotin. Hofmann developed the differential assay that dboxybiotin for certain organisms had activity procedure for oxybiotin and biotin and by its use was equal to that of dGbiotin. Hofmann concluded that able to demonstrate that yeast utilized the oxybiotin oxybiotin and biotin have identical spacial configura- molecule as such and does not transform it into biotin. tions and that .the two compounds differ from one This differential assay was based on the fact that oxianother only in the nature of one of the hetero atoms. dizing agents convert biotin to an inactive sulfone and In the case of desthiobiotin, the yeast transformed it do not attack oxybiotin.
CH,COCH,COaC.H, KOH~NO~ (XLVII) CHGOCH=NOH $Pd, Hz [CHaCOCH,NH,I J.HCNO 0
il\NH .4, C H s1 HN/
(L)
HN02
CH1COC(=NOH)CO2C~H5 (XLVIII)
I
Pd, H*
-
[CH&OCH(NH4CO2C2H,1 JHCNO 0 HN/
alkali
C H s
e
\NH
I
C--COnC2Hs =
(XLIX)
AICL~C~CO(CH~ICOGH~
0
II
HN/?NH
tor ~d
HN/
8
\NH
__f
(LI) CH,d-=&-CO(CHd,CO,CH,
H , ,cH, ,oc, ) H ,& c=== LH ,c
(LID JPt, H, 0
0 HN (LIV) CHs
H,CsOOC
AH--CH(CHd,CO,H '
HN
hydrolysis t-
CH8-L-hH(CHd6COsC,Hs
COOC,H6
HOOC 2 Steps
COOH
di. HA
L(cH,),--CH,oH
(LIII)
