Jan. 20, 1959
505
COMMUNICATIONS TO THE EDITOR
cm.-l, accompanying the strong maximum a t 1703 cm.-l (0.01 M s o h . in CC14)7corresponding to “non-interacted” conformations of I. By contrast, under the same conditions, tetrahydro-4H-lthiapyran-4-one (11)8and 1-thiacycloheptan-4-one (III)g exhibited single maxima a t 1716 and 1711 cm.-’, respectively. The dipole moment of the eight-membered ring compound (I),3.81 D in benzene, was higher than that of the seven-membered ring compound (111) (3.04 D; 1.73 D for II).2b I t is important to note that S-Cco interaction occurs to a lesser extent than N-Cco transannular interaction in the electronic ground state by comparison (infrared especially) of 1-thiacyclooctan-5-one with l-methyl-l-azacy~looctan-5-one.~ Finally, the ultraviolet absorption maxima of I in cyclohexane, a t 226 mp ( E 2445) and -232 mp ( E 2150), are associated with excitation of the interacting S-CCO system A,(: 223 mp ( E 695), XES, 233 mp ( E 507). lo (7) N. J. Leonard, M. d k i , J. Brader and H. Boaz, T ~ r JOURNAL, s 7 7 , 623 (1955). (8) E. A. Fehnel and M. Carmack, ibid., 70, 1813 (1948). (9) C. G. Overberger and A. Katchman, i b i d . , 78, 19G5 (1956). (10) E. Fehnel and M. Carmack (zbid.,71’84 (1949)) have suggested earlier that the difference between the ultraviolet spectrum of tetrahydro-4H-l-thiapyran-4-one(11) and those of its acyclic analogs is attributable t o direct interaction between t h e 1,4-atoms in the excited state. (See also V. Georgian, Chemisfvy and I n d u s f u y , 1480 (1957).) If this is correct, the four- to five-fold increase in intensity for the eight membered ring over the six-membered ring may be regarded as manifestation of the greater contribution of transannular interaction in the medium-ring compound, (11) Sinclair Refining Co. Fellow in Organic Chemistry, 1957-1968. Work done under the sponsorship of the Sinclair Research Laboratoiies, Inc.
dehydrogenase, DPN reduction (reaction l), pyruvate dismutation, and reduction of free lipoic acid (reaction 2).a,6 These activities were restored by
CH8COOP03‘
+ Lip(SH)t + COZ (2)
addition of FAD, but not of F M N (Table I). The dihydrolipoic transacetylase activity of the preparation was not affected by removal of flavin. TABLE I REACTIVATION OF SPLIT PYRUVATE DEHYDROGENATION SYSTEM WITH FAD Assay system
Before splitting
Specific activities’ -After splittingWithout With With FAD FAD0 FMNV
870 214 544 228 Lipoic DeHb 486 144 870 214 DismutationC Reaction Id 156 20 62 20 32 64 32 Reaction 2e 90 112 100 110 100 Lip. transac.’ Expressed as pmoles/hr./mg. protein based on assays described previously. * Ref. 7 , P H 7. Ref. 3. Ref. 8. Ref. 6, pH 7, 5 prnoles DL-lipoamide employed. f Ref. 8. 0 Aliquots of split complex incubated 10 min. a t 30’ with FAD or FMN before assay. Final concentration of added flavin in assays was 10-6 to 10-6 M .
These data indicate that FAD is an essential component of the enzyme complex, presumably associated with dihydrolipoic dehydrogenase. The data are consistent with the reaction sequence21a shown for oxidative decarboxylation of pyruvate by the enzyme complex.
+ T P P 2[CHaCHO-TPP] + COz S + SI>. 2HS)R + T P P CHaCOS
THENOYESCHEMICAL LABORATORYKELSOXJ. LEONARD CHzCOCOzH UKIVERSITY OF ILLINOIS THEODORE L. BROWS URBASA,ILLINOIS TERRY W. M I L L I G A N ~[CH3CHO-TPP] ~ RECEIVED DECEMBER 4, 1958
COA-SH
Sir:
ON THE MECHANISM OF OXIDATIVE DECARBOXYLATION OF PYRUVATE
+ COZ + DPNH + H +
(1)
(4)
I_ CHaCOS CHaCOSCoA
+
Extracts of Escherichia coli contain an enzyme system which catalyzes an oxidative decarboxylation of pyruvate represented by reaction 1.1,2-3
acetyl CoA
+
(3)
-3
+
+ HS “>R
HYR FAD + I R FADHl HS R = -CHZCHZCH(CHp)&O-enzyme
(5)
(6)
I
The reduced flavoprotein produced in reaction 6 apparently can interact with DPN (cf. reaction 1) and free lipoic acid (cf. reaction 2).
( 6 ) I. C. Gunsalus, Federalion Proc., 13, 715 (1954). We have obtained highly purified preparations (7) L. P . Hager and I. C. Gunsalus, THIS JOURNAL, 76, 5767 (1953). (250-fold purification) of this system from extracts (8) L. P. Hager, Thesis, University of Illinois, 1953. of the Crookes strain. It is apparently an enzyme (9) During this investigation Dr. V. hlassey. Biochim. el bioghys. ~ o m p l e x and , ~ sediments in the ultracentrifuge (1 acfa, 30, 205 (1958) communicated t o us his significant finding (ref. 9) to 2 hr. a t 144,000 X g) as a dark yellow, fluores- t h a t highly purified diaphorase exhibited strong dihydrolipoic dehydrocent pellet. The complex contains a flavin which genase activity. BIOCHEMICAL has been tentatively identified as FAD. Release CLaYTON FOUNDATION INSTITUTE AND DEPARTMENT OF CHEMISTRY of the flavin by precipitation of the enzyme com- UNIVERSITY OF TEXAS MASAIIIKO KOIKE plex with ammonium sulfate a t pH 3.6 resulted in a ACSTIN,TEXAS LESTERJ. REED decrease in the enzymatic activities : dihydrolipoic RECEIVED NOVEMBER 25, 1958
(1) S. Korkes, e l al., J . Biol. Chem., 193, 721 (1951). (2) I. C. Gunsalus, in “The Mechanism of Enzyme Action,” The Johns Hopkins Press, Baltimore, Md., 1954, p. 545. (3) L. J. Reed, el al., J . Bioi. Chem., 232, 123, 143 (1958). (4) Abbreviations: D P N , diphosphopyridine nucleotide; CoA. coenzyme A; TPP, thiamine pyrophosphate; FAD, flavin adenine dinucleotide; Lip&, free lipoic acid; Lip(SH)o, free dihydrolipoic acid; RSa, protein-bound lipoic acid. (5) R. S. Schweet. PI ol., J . R i d . C h o n . . 196, 563 (1952); 11. R. Sauadi. u l ui , t h i d . , 197, 851 (1!152).
CHELATION AS A DRIVING FORCE IN SYNTHESIS. A NEW ROUTE TO a-NITRO AClDS AND a-AMINO ACIDS
Sir: Dibasic a-iiitro acids (I) are converted in weakly basic i i i c d i a to acid salts which rapidly decarbos-
506
CO?VlMUNICATIONS T O THE
ylate.' I n stronger alkali the acids form divalent salts such as dipotassium nitroacetate,2which does not decarboxylate readily, and which forms typically ionic crystals3 The chelate salts (11) have not been characterized, although their existence R-CHCOOH I
XOp
I
R-CCOO I1 I1
n'
O/',J
II
Metal
I1
was suggested by the effect which many polyvalent metal ions have upon the rate of decarboxylation of nitroacetic acid.'v4 We have now succeeded in preparing the aluminum and magnesium salts of nitroacetic acid, and have utilized the stability of magnesium salts to provide a remarkably simple synthesis of a-nitro acids from primary nitroalkanes and carbon dioxide. Aluminum nitroacetate precipitates when aluminum isopropoxide and nitroacetic acid are mixed in ether solution. The white powder has the composition A12(C2HN04)3. ( s o l ~ e n t ) , ,is ~ insoluble in the common solvents, and is decomposed by aqueous hydrochloric acid to free nitroacetic acid. Magnesium nitroacetate has not been obtained crystalline, but may be prepared in methanol (Amax 272 my, log E 4.03) and shown spectroscopically to be a 1:l complex by the method of continuous variation.6 Magnesium methyl carbonate' may be prepared by saturating a solution of magnesium methoxide in dimethylformaniide with dry carbon dioxide. Treatment of nitromethane with 4 molar equivalents of this solution (2 molar) a t 50" for 4-5 hours resulted in quantitative conversion to magnesium nitroacetate as determined spectrophotometrically. Hydrolysis of the solution with ice and hydrochloric acid led to the isolation of nitroacetic acid, imp. S9-92" (reported 87-S9°,2 91.5-9204) in 63% yield. This result demonstrates that the wellknown decarboxylation of nitroacetic acid is a reversible reaction, the position of equilibrium being completely shifted by chelation. 0ther primary nitroparaffins react similarly. Nitroethane is converted to 2-nitropropionic acid in 49y0 yield under conditions found best for nitroacetic acid, although these conditions may not be optimum in this and other cases. The following amino acids were prepared by hydrogenating the crude nitro acids, prepared as described above, in acetic acid a t room temperature with 10% Pd/C, in the specified over-all yield based on nitroparaffin : ( I ) 1;. J. I'e~lcrseti,7 ' y a n i . I ' u r i i d < i y b u i . , 23, L l l G (1027). ( I ) W. Steinkol)f, D P K , 42, 3035 (1'30D). (:;) 13. J. Suter. lp. J. Llcwellyn and H. S. AIaslen, A d a Cryil , 7 , 146 (I!)> 1). (1) I
EDITOR
VOl. 81
DL-alanine,Sdec. 292-294' (4G%) from nitroethane; DL-a-aminobutyric acid, dec. 283-285" (34%) from 1-nitropropane ; and DL-norvaline, dec. 290-202" (42%) from 1-nitrobutane. ?-Nitropropane, which could not give rise to a species such as 11, fails to undergo the reaction. The use of magnesium methyl carbonate as a carboxylating agent for other active hydrogen compounds is being investigated. (8) T h e amino acids were identified by comparison of their decomposition temperatures and infrared spectra (Nujol) with those of commercial samples. DEPARTMEST O F CHENISTRY UXIVERSITV OF MICHIGAN
MARTINSTILE: HERMASL. FINKBEISEH ANS ARBOR,MICH. RECEIVED DECEMBER 4, 1958
A TRANSANNULAR REACTION IN AN EIGHT-
MEMBERED RING SULFIDE
Sir: IlTe wish to report a novel transannular reaction in a cyclic 8-membered ring sulfide. The carbinol (I), 5-hydroxythiacyclooctane, on treatment with phosphoric anhydride and then treatment of the purified solution (ion exchange) with picric acid, gave the bicyclic sulfonium salt 11, 57%, bicyclo[3,3,0]octane-l-thianium picrate, m.p. 262-264" (microblock, corr.) C, 43.55; H, 4.23; N, 11.73.
r--
L
s
4
i- ~--:
Ls'>
I
I1 0
l\M L / S 4 I11
This sulfonium picrate was identical (infrared spectrum) with the same compound prepared by R. H. Eastman' and G. Kritschevsky, m.p. 2G12G3", mixed m.p. 261-264 (micro-block, corr.) The eight-membered ring ketone (111),250x0thiacyclooctane, b.p. 120-123 (19 mrn.), imp. 50-52" ; C, 35.26; H, 8.39; (2,4dinitrophenylhydrazone, n1.p. 194-195°, N, 17.34) was prepared by the Dieckmann cyclization of ethyl thio-di-nbutyrate with subsequent hydrolysis and decarboxylation (31yo). The infrared spectrum (0.01 AT in CC14) showed a strong normal carbonyl absorption for this compound a t 1707 cm.-', with a shoulder a t 1G90 ctn.-l. We had reported previously a single absorption for the 7-membered (1) We are indebted t o Dr. I
