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COMMUNICATIONS TO THE EDITOR
VOl. 81
CONTROLLED FORMATION OF cis- AND transDECALIN-9-CARBOXYLIC ACIDS BY CARBONYLATION
carboxyl appears more crowded than a methyl group, which in analogous cases is known to reverse Sir: the usual order of stability and to favor the cisKoch and Haaf' have described the preparationof decalin system over the trans.2,3s485 In strong enough acid to make all the steps decalin-9-carboxylic acid (Soy0cis) from @-decalol, sulfuric and formic acids. In a recent use of this reversible, equilibrium is approached; K = (V),' preparation, we have found that products all the (117) = 4-1 1 at 5", depending upon the composition way from 84% cis (V) to 90% trans (IV) can be of the unisolated lOy0 of the material. This is obtained, the only variable being the amount of consistent with the value 1.5 at 250" obtained for fuming sulfuric acid included in the reaction 9-methyl-1-decalone. This work was performed under a grant from the medium (see Table I). National Institutes of Health. TABLE I COMPOSITIOS OF DECALIN-9-CARBOXYLIC ACID IS PREPARATIONS BY UNIFORM PROCEDURE 41.5 g. 98% H2SO4;4.6 g. 88% formic acid; 0-5", 1-13 hr. 30y0 oleum, g.
Sone
5 10 15 20 40 (Isomerization product)
% tvans (10.29 a)
70 cis (11.26 p )
90 74 56 19 5 7 5 9
10 22 5 41 62 5 84 79 87 5
loo-(% (%
cis)
lvans)
-
0 3 5 3 18 5
(2) W. E. Bachmann, A . Ross, 4 . S. Dreiding and P. A. S. S m i t h , J . Oig. Chem., 19, 222 (1951). ( 3 ) A. Ross, P. A. S. Smith and A. S . Dreiding, i b i d , 20, 905
(1955). (4) S . 1,. Allinger, ibid., 21, 915 (195G). ( 5 ) W. G. Dauhen and K. S. Pitzer in N e w m a n , "Steric E f f e c t s in Organic Chemistry," John Wiley a n d Sons, N e w York, S . Y . , 1956, pp. 30, 31.
DEPARTMEKT OF CHEMISTRY RICHARD E. PIXCOCK UNIVERSITY ERNSTGRIGAT HARVARD PAULD . BARTLETT CAMBRIDGE 38, MASS. RECEIVED XOVEMBER 10, 1959
11 13 5
3 5 ON THE MECHANISM OF THE ENZYMATIC
DECARBOXYLATION OF ACETOACETATE' For the rapid assay of the total acid fraction calibration curves were prepared by the infrared Sir. examination of six synthetic mixtures of the puriIn order to investigate the mechanism of the enfied isomers (cis, m.p. 122"; trans, m.p. 135") a t zymatic decarboxylation of @-ketoacids,we have extotal concentrations of 25 mg. of acid per cc. of amined the oxygen exchange which accompanies carbon disulfide. Estimates made separately from the decarboxylation of acetoacetic acid, labeled in the "trans" peak a t 10.29 p and the "cis" peak a t the carbonyl group with 0'8, in the presence of the 11.26 agreed within 3% on isomer composition crystalline2 decarboxylase from Clostridium acetoof the products made with 0-10 g. of fuming sul- butylicum. furic acid present, but indicated the presence of Acetone was labeled with OI8 by allowing it to 1 1 1 8 z of a further isomer from the more strongly stand with enriched water (Stuart Oxygen Co., acid media. l.4yoO X 8 and ) a trace of sodium hydroxide. Ethyl X sample of the acid assaying 90Yo trans was acetoacetate was saponified in 2 ill potassium hyintroduced into a mixture of 41.5 g. of 9Sso sulfuric droxide in enriched water, and the potassium acetoacid and 20 g. of fuming sulfuric acid (3Oyo), acetate, labeled in the carbonyl and carboxyl groups, along with 5 g. of formic acid, over a period of 10 was obtained by evaporation i n nacuo and purified by minutes. After 1.5hours at 5" decalin-9-carboxylic precipitation of an alcoholic solution with ether. acid was recovered in 90% yield, having the com- The Ola content of the acetone was determined position 87.5 czs, 9yo trans. Thus kinetic control mass spectrometrically by measuring the ratio of the of carbonylation leads to trans-decalin-9-carboxylic 5s and GO peaks, using a Consolidated model 21acid, eventual equilibrium favoring the cis isomer. 103C mass spectrometer; the cracking pattern of Models show that the trans-acyliuni ion IT, the acetone established its purity. Xcetone froin the enzymatic decarboxylation and from control co LOOII experiments was swept froin the reaction mixture with a stream of air through ;in ice-water COII,-,-\,,-.--z I/\ J +-. denser, and trapped a t - 78". ;1s;unple of acetone vapor then was prepared for illass spectrometry by II I\ equilibrating liquid and vapor a t 0". Some water undoubtedly was present in the liquid sample, but I \ < 0011 earlier work3has shown that, in the absence of buffers, exchange between acetone and water at low temperatures is very slow. The internal consist111 v ency of the results further supports this conclusion. Control experiments with potassium acetoacetate with the C-C=O+ atoms in a straight line, has were conducted by freezing the solution after 2.5 little axial-axial interaction and should be rapidly (1) This research w a s supported in p a r t by a grunt from t h e Xstionxl formed without strain. On the other hand, in the trans acid IV, or its protonation product, the axial Institutes of H e a l t h .
c2J
17
+ k"L' >
eo
a7
(1) H Koch and W Haaf, Aizgeu. Chem , 70, 311 (1958), 261 (1'358).
.4>171 ,
618,
( 2 ) G. A . Hamilton and F. H. Westheimer, THIS J O U R S A L , 81,2277 (1959). (3) hI. Cohn a n d H. C. Urey, i b i d . , 60, 679 (1938).
Dec. 5 , 1959
6333
COMMUNICATIONS TO THE EDITOR
minutes and lyophilizing to dryness. The resulting salt was decarboxylated by heating the residue with phenol, and the acetone analyzed for Ols. Some of the pertinent data are shown in the table. -----YoExchangeb a t 20’Experimental conditionsa
+ + +
Control Control Decarbox(2.5 min.) (15 min.) ylationC
Acetone buffer 15 65 Acetone 0.45 mg. 40 92 enzyme Acetone 0.90 mg. 57 enzyme Acetoacetate bufferd 25 Acetoacetate 0.45 mg. 45 98.5 enzyme Acetoacetate 0.90 mg. 61 100 enzyme a All reactions were carried out in 2 ml. of 1 hl phosphate buffer, pH 6.5, with 0.05 ml. of acetone or 0 06 to 0.15 g. of potassium acetoacetate. Some of these values are averages The acetone was blown from the solution as soon as i t was formed and was all collected within 2 min. after adding the acetoacetate. An isolated experiment of this type gave a higher value (65%).
+ + +
Inspection of these data shows that the exchange of 01* from the carbonyl group of acetoacetate is an obligatory part of the enzymatic decarboxylation process ; control experiments establish that the direct exchange of olsfrom acetone and acetoacetate, in the presence or the absence of enzyme, is incomplete. The results are consistent with the hypothesis that the reaction proceeds by way of Schiff-base formation? between the ketoacid and the enzyme, but do not of themselves demand this conclusion. Further tests of this hypothesis are in progress. (4) K J . Pedersen, J . Phys. Chem., 33, 559 (1934), proposed asimilar mechanism for t h e non-enzymatic amine-catalyzed decarboxylation of acetoacetate. (5) Holder, National Research Council of C a n a d a Special Scholarship, 1937-1959.
MALLINCKRODT CHEMICAL LABORATORY HARVARD UNIVERSITY GORDON A . HAMILTON‘ CAMBRIDGE 38, MASS. F. H. Q‘ESTHEIMER RECEIVED OCTOBER15, 1959
STEREOCHEMISTRY OF THE FUMARASE AND ASPARTASE CATALYZED REACTIONS AND OF THE KREBS CYCLE FROM FUMARIC ACID TO d-ISOCITRIC ACID’,’
Sir:
be referred5 to as a-OHL,-/3-H2D,-malic acid and a-OHD,-P-H2L,-malic acid. Nuclear magnetic resonance spectroscopic examination6 of this 3-monodeuterio-DL-malicacid gave a coupling conD H.., /...COOH
H, ,COOH
F
I
Hooc’c*o
c
/
I
CH,.COOH
74
HOOc’qOH
H, ,COOH C
OH H,:,COOH
?
4
C HOOC.H,C‘”~“COOH H FIG.1.-Stereochemistry of biochemical route from fumaric acid to &isocitric acid. !L
HOOC.H,C’ ‘COOH
.fE
HO tCOOH H
D
H t D COOH
H COOH
DL- 1
stant of 4.4 =t 0.2 C.P.S. I t is thus identical in stereochemical configuration with the 3-monodeuterio-D-malic acid prepared7 by inversion a t the acarbon atom of the 3-monodeuterio-~-malic acid ~ b t a i n e dby ~.~ fumarase hydration of fumaric acid in deuterium oxide or by nitrous acid treatment,’ with retention of configuration, of the 3-monodeuterio-L-aspartic acid obtained7 by aspartase-catalyzed addition of ammonia to fumaric acid. Thus the stereochemical configuration of the 3monodeuterio-D-malic acid is ~ - O H D , - / ~ - H ~the L,, structure of the product of the fumarase reaction in deuterium oxide is ~ - O H L , - @ - H and ~ L , the product of the aspartase reaction is ~ - N H ~ I , ~ - @ - H * L , . With these configurations in mind, it is now seen that both the fumarase-catalyzed hydration of fumaric acid and the aspartase-catalyzed addition of ammonia to fumaric acid proceed via a trans add i t i ~ n . The ~ t r a m nature of these additions is in
The stereospecific synthetic approach used3 to elucidate the stereochemistry of the cis-aconitase ( 5 ) Using t h e nomenclature of R e f . 3 . (6) I n 2 hf Dd0 solution after replacement of exchangeable protons system has been applied to the fumarase and asparwith deuterium. W e are indebted t o Dr. Paul Lauderbur for this tase systems. determination. The racemate (I), m. 127-128.5”, neut. equiv. (7) A. I. Krasna, J . Bioi. C h e m . , 233, 1010 (1958). A coupling 67 .O, of 3-monodeuterio-~~-mak acid, having the constant of 4 C.P.S. is reported f o r this 3-monodeuterio~n-malicacid. hydroxyl and deuterium in trans configuration, A coupling constant of 6 C.P.S. was found for t h e 3-monodeuterioacid. has been stereospecifically synthesized by the trans L-malic (8) R . A. Alberty a n d P. Bender, THISJOURNAL, 81, 542 (1959). lithium aluminum deuteride opening? of the oxide A coupling constant of 7.1 C.P.S.was found for t h e 3 - m o n o d e u t e r i o - ~ ring of 3,4-epoxy-2,5-dimethoxy-tetrahydrofuran?malic acid. A sample of enzymatically prepared 3-monodeuterioand then acid hydrolysis4 to the dialdehyde and L-malic acid, kindly supplied us b y Dr. Alberty, was found b y us t o a coupling constant of 7.3 C.P.S. nitric acid oxidation of the dialdehyde to %mono- have (9) Previously, Ref. 10, 8 a n d 7 , these additions have been condeuterio-DL-malic acid. The enantiomorphs may sidered t o proceed via cis additions. (1) Support of t h i s work b y g r a n t RG-6245, from t h e National Institutes of H e a l t h i s gratefully acknowledged. ( 2 ) With t h e technical assistance of David Belitskus. (3) 0. Gawron, A. J. Glaid, 111, A. L o M o n t e a n d S. G a r y , THIS JOURNAL, 80,5856 (1958). (4) J. C. Sheehan and B. M. Bloom, ibid., 7 4 , 3826 (1952).
(10) T. C. F a r r a r , H.S. Gutowsky, R . A. Alberty a n d W. G. Miller, THIS JOURNAL, 79, 3978 (1957). Stereochemical assignments were based o n nuclear magnetic resonance conclusions a n d t h e assumption t h a t t h e carboxyl groups of malic acid in t h e solid s t a t e are in a trans relationship. D o u b t s of t h e correctness of this assumption have been raised, ref. 8 , a n d personal communication, R . Alberty.
