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.
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Vol. s1
~ O M l r l U N I C A T I O h ' STO THE E D I T O R
From the slope of the plot of 1/@ us. 1/'[BH*],the value of k d / k r is found to be 0.050. Consideration of this number compels the conclusion that the triplet state is responsible f o r the chemical reaction. The largest value that kr can be imagined to have is lo9 liter mole-' set.-', the diffusion controlled rate.4 This gives an upper limit of 5 X lo7 set.-' for k d . Fluorescence rate constants for singlet statese are believed to be of the order of l o 8 set.-' (11) (a) S. Englard, F e d . PYOC., 18, 222 (1059) (b) S. Englard, Since benzophenone solutions have no visible fluoperaunal communication. W e wish t o t h a n k Dr. Englard for sending us his manuscript prior t o publication. rescence, the actual rate of non-radiative quenching (12) T h e other results, Ref. 11, are also consistent with t h e scheme of the lowest singlet state must be a t least 10IOsec.-'. presented in Fig. 1. I t is clear that some longer lived state must be in(13) S a t i o n a l Science Foundation Cooperative Graduate Fellow. volved in the measured competition. DEPARTMEKT O F CHEMISTRY DUQCESNE UNIVERSITY OSCARGAWRON Reaction (5) actually must be much slower than PITTSBURGH, PA. THOMAS P.FOSDYI3 diffusion-controlled since a substantial isotope effect is observed when a-deuteriobenzhydrol is used RECEIVED SEPTEMBER 25, 1959 as the hydrogen donor. The value of k d / k r ( D ) is 0.133, which indicates that k r ( H ) / k r ( D ) is 2.7. THE ROLE OF A TRIPLET STATE IN THE In summary, the results indicate that intersystem PHOTOREDUCTION OF BENZOPHENONE crossing to a triplet state must be complete, and Sir: that the triplet is responsible for chemical reaction. Irradiation of benzene solutions of benzopheAcknowledgments.-This work was supported by none and benzhydrol with near ultraviolet light pro- grants from the Film Department of the du Pont duces benzpinacol stoichiometrically according to Company and from the National Science Foundaequation (1). tion.
keeping with the truns nature3 of the cis-aconitase system. The stereochemistry of the Krebs cycle from fumaric acid to isocitric acid may now be that the 3traced (Fig. 1) utilizing the finding11~12 monodeuterio-L-malic acid (from the fumarase rereaction) gives, biochemically, isocitric acid lacking deuterium and utilizing our previously suggested3 scheme for the &-aconitase system.
(C6&)2CO
+ (C6Hs)gCHOH
-
(C&)X-C(
I
1
C6HS)Z
OH OH
(1)
Experiments were carried out using a collimated beam from a Westinghouse SAH800-C Mercury arc Gltered to give a band having a maximum a t 3660 A. and a band pass of 500 A. Photolysis rates were measured by spectrophotometric determination of residual benzophenone and by titration of the pinacol with lead tetraacetate.' Quantum yields were based upon uranyl oxalate actinometry although the reaction of benzophenone with 2-propanol2was used as a secondary standard. Solutions were demm. A series of experiments was gassed to run using 0.1 M benzophenone and varying concentrations of benzhydrol. Graphical analysis showed the l/@ was a linear function of 1/[BH2]. This relationship indicates a simple competition between deactivation of the chemically active state and its reaction with benzhydrol. Since the intercept of the plot is one, physical quenching by benzhydro1 must be negligible. The mechanism shown accounts for these facts. B
-+n + Izu -n+ B 1(singlet) B'
-
--+
B3 (triplet)
(2) (3)
kd
B
+
B3 ----f B (all deactivation steps) (4) kr BHs 2( CBHJ)ZCOH ----f Benzpinacol ( 3 )
Application of steady state kinetics to the concentrations of excited states gives the rate law.
(1) R. Criegee, Ber., 64B,264 (1931). (2) J. N. P i t t s , R . L. Letsinger, R. P. T a y l o r , J. M. Patterson, G. Rechtenwald a n d R. B. M a r t i n , THISJOURNAL,81, 1068 (1959). (3) Light absorption was essentially complete. O t h e r experiments show t h a t t h e q u a n t u m yields are independent of both light intensity a n d ketone concentration.
(4) Calculated by t h e method of Schultzs using a diffusion coef ficient of 10-6 cm.2 see.-' for benzophenone a n d benzh)drul in ben zene. ( 5 ) G . V . Schultr, Z . p h y s i k . Chem., 8, 284 (195ti). (GI M. K a s h a , Disc. F a r a d a y SOL, 9, 11 (19:O). However, see J. W. Sidman. Chem. Ret's., 68, 689 (1958), for a possible lower estimate.
COSTRIBL-TION Fo. 2505
GEORGE S. HAMMOND
G A T E S A S D C R E L L I S LABORATORIES CALIFORNIA ISSTITUTE O F TECHNOLOGY PASADENA, CALIFORXIA WILLIAMAI. MOORE
RECEIVED SEPTEMBER 8, 1059
T H E ADDITION OF NITRONES TO OLEFINS. A NEW ROUTE TO ISOXAZOLIDINES
Sir: The isolation of cis-N-methyl-3-oxa-"-azabicyclo [3.3.0]octane (111) from the pyrolysis o f FI mixture of the isomeric N-methyl-a-pipecoIiI'e oxides' suggests that the unsaturated nitrone I may be an intermediate. We have therefore itivestigated the cyclization of I and a homolog. Monofunctional aliphatic nitrones have iiot been reported, since their preparations generall!r lead to aldol-type dimers.* Nevertheless, tlic reactive nitrone linkage of I, generated in s i t u might well undergo intramolecular addition to the terminal olefin group. Oxidation of an ether solution of N-methyl-N-3hexenylhydroxylamine (V) with excess mercuric oxide afforded 111, characterized as its hydroger! oxalate (m.p. and m.m.p. 82-82.5') and by comparison of the infrared spectra,' in 24% yield. (1) A. C. Cope a n d N. A. LeBel, Abstracts of Papers, 133rd h l e e t ing, American Chemical Society, S a n Francisco, California, April 1318, 1958, p . 6 2 - N : THISJOURNAL, in press. (2) R . B o n n e t t , R . F. C. Brown, V. M. Clark, I . 0. Sutherland and A. T o d d . J . C h e m Sac.. 2094 (1959), present a s u m m a r y of t h e various dimeric structures f o r nitrones including aldolization structures, and report t h e syntheses of several monomeric alicyclic nitrunes. C/. also R. F. C. Brown, V. 31. Clark, I. 0 . Sutherland a n d A . T o d d , ibid , 2109 (l
