0.33) from Cr(Hz0)61++ (mainly) and Cr(H20)5- servable formation of organo-metallic coiiipounck. c1++(partition coefficient 0.13). Separation was If-ork on the reduction of halogen compounds by also effected by ion-exchange chromatography on chromous salts is continuing.j Dowex 50-X4 resin and elution with 1 M perchloric ( 5 ) This work was supported by a grant from the Sational Rese.irch acid. The ion C ~ ( H Z O ) ~ C I(green) ++ was eluted Couucil of Canada. first, followed by Cr(H20)5CH2Ph++(yellow to DEPARTMETT OF CHEMISTRY orange-yellow) whilst Cr(H20)6+++remained on the UNIVERSITY F. L. :INET OF OTTAWA ONTARIO E. LEBLAYC column. All the above operations were carried out OTTAWA, RECEIVED APRIL 15, 1937 in an atmosphere of oxygen-free nitrogen and as rapidly as possible. Solutions of the benzylchromium compound were yellow to brownish-red depending on the concen- THE BIOSYNTHESIS OF SQUALENE F R O M MEVALONIC ACID tration, and the spectrum of the solution had a low Sir: intensity maximum at 540 mp and a high intensity maximum a t 358 mp. Decomposition of the Tavormina, et al., I have reported the incorporacomplex in the absence of oxygen by heating or by tion of 2-Cl4-labeledmevalonic acid (3,s-dihydroxykeeping for several days gave bibenzyl; in the pres- 3-methylvaleric acid) into cholesterol by honiogeence of oxygen benzaldehyde was the main product. nates of r a t liver. These investigators have also The compound was decomposed very rapidly by provided evidence that the carboxyl carbon of the sulfur dioxide b u t was stable to carbon dioxide. substrate is almost completely converted to carbon Hydrogenation in presence of a palladium catalyst dioxide, and therefore not incorporated into cholesgave toluene. Solutions of the compound reacted terol.2 very quickly with aqueous mercuric chloride with IVe have confirmed the finding t h a t 2-C1:-mevalonic acid3 is converted to cholesterol, and in addi720 change in pH of the solution to give benzylmercuric chloride and c r ( H ~ o ) ~ + +(not + CT(H*O)~- tion have demonstrated t h a t i t is rapidly converted C1++) in equimolecular amounts. Therefore the to squalene by homogenates as well as by the supercompound must contain one benzyl group per natant fluid obtained from such preparations folchromium atom and two positive charges. Hence lowing high speed it is a Cr(1II) complex and its stability is best raEmploying homogenates of r a t liver and X!'4tionalized by the normal octahedral complex struc- mevalonic acid, we have repeatedly isolated CI4ture mentioned before. I t differs, as might be ex- squalene into which has been incorporated 10 to pected, from the chloride complex in having a tend- 20"%, of the substrate. Squalene recovered from ency to dissociate in a homolytic fashion to give several of the experiments was combined and puri~ was subbenzyl radicals and chromous ion. An alternate fied by chromatography on a l ~ m i n a . It structure which can be considered for the compound sequently chromatographed on silicic acid, eluting is a complex of the tropyliunl ion and Cr(1) in the with an increasing gradient of benzene in petroleum manner') of dibenzene-Cr(1) but with only one ring ether. The latter process yields squalene as a single involved per chromium atom, although this is some- radioactive peak. In contrast to cruder preparations of squalene. what unlikely on the above evidence. However. such a structure can be excluded because all the this product is completeIy degraded by ozonolysis carbon atoms would then become equivalent and i t to acetone, levulinic acid and succinic acid. These was found t h a t a-methyl, o-methyl- and p-methyl- substances were separated and further degradetl. benzyl halides gave different compounds on reduc- *ketone was converted t o iodoform and acetic acid, and the acetic acid degraded by the Schmidt tion with chromous perchlorate. The levulinic acid was converted to iodoReduction of benzyl chloride with chromous chloride in hydrochloric acid gave toluene rather form and succinic acid. The succinic acid was dethan an organo-metallic compound. This can be graded by the Schmidt technique to carbon dioxide understood from the work of Taube4 as the ini- and ethylenediamine. 1111 of the degradation prodtially formed complex, Cr (H20)4C1CH2Ph+, will ucts were converted to carbon dioxitle and counted have chloride ion as well as water molecules in the as barium carbonate. The radio-analyses (Table I) demonstratetl t h a t cotjrdination sphere and such a chlorine atom favors reduction by bridging4 with the reducing agent. approximately SOYp of the isotopic carbon of the The resultant Cr(I1) complex is then no longer sub- squalene is present in the methyl carbons of acestitution-inert and dissociates to benzyl anions tone and in carbon 3 of levulinic acid. The sucwhich react with the solvent to give toluene. cinic acid sample, representing the central four carTherefore, the isolation of Cr(H20)4CH2Phf+ in bons of squalene. contained only a sinall amount of the chromous perchlorate reduction is due to the isotope. It is clear that only one of the methylene fact that this compound (like Cr(Hz0)6i+T but groups of the levulinic acid was appreciably labeled. unlike Cr(H20)6Cl++and Cr(H20)&+) is reduced (1) P. A. Tavormina, 11. H. Gibbs and J. W Huff, THISJ O U R N A L . by C r + + extremely slowly, as well as t o the sub- 78, 4498 (195G). ( 2 ) P. A . T a v o r m i n a arid 11.II. Gibbs, zbid., 7 8 , ti210 (195b). stitution-inertness of dipositive complexes of CrL?) T h e authors expreis their apprecjativn t o M e r c k Sharp a n d (111). Ih~li1nef u r a generoils i i i l i p i s l a i i r l c d mevalmic acid u ~ e ( l111 t h e s e .Illy1 chloride and phenacyl chloride were readily r jjrri t u r ~ i i s ( 1 ) I:. 1)ituri. 1'. A . C < > l ) r v 1 V. 1 j L V a n i i . i i i i c l S (;iLriri, .I / j ) t , i reduced by chronious perchlorate. b u t without cihctf
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COMMUNICATIONS TO THE EDITOR
May 20, 1957
SPECIFIC
ACTIVITYO F
TABLE I DEGRADATION
THE
c l4-SQUALENE
PRODUCTS O F
Degradation product
Specific activity, c.p.m. per mg. C
Acetone Carbonyl carbon CHI3 Succinic acid -COOH -CH2Levulinic acid CHI3 (carbon 5) -COOH (carbons 1 and 4 ) -CH2- (carbons 2 and 3 )
306 10 360 70 30 80 150 17 28 310
[
&Ha
I
CoA involves the intermediate formation of AcAcS-CoA by thiolase, which catalyzes reaction 1 . followed by deacylation of AcAc-S-CoA t o A C A C . ~ - ~ However, a direct deacylation of synthetic AcAc-SCoA is not catalyzed by these enzyme fractions. 2Ac-S-Cor\
and i t is probable from the data that only one of the methyl carbons of the acetone fragment contained C14 (Fig. 1). C "H3C=CHCH2C*H2C=CHCH2C*HzC=CHCHs-
1
265 1
XcXc-S-Co;\
+ HS-CoA
(1)
We find that AcAc synthesis by partly purified ox or chicken liver enzyme preparations from ilcS-CoA (generated by phosphotransacetylase from acetyl phosphate and CoA-SH3) requires the addition of a mono- or dithiol compound. Among active thiols, ( j=)-DT06 has the greatest activity, half maximum activation of Acdc synthesis occurring with 1 X M (*)-DTO compared to 3.5 X X BAL and 2 X M GSH. These liver fractions also catalyze the reactions ( 2 ) , (3) (f)-S-.kcAc-DTO
+ H20 +ACAC+ ( f ) - D T O + +
AcAc-S-Pn (or CoA) ( & ) - D T O J_ hc-S-Pn (or Coli) (&)-S-Xc-DTO
(2)
(3)
CHI CHD distribution of isotopic carbon in biosynthetic squalene.
Mono- ( j= ) -S-AcAc-DTO was synthesized by reFig. 1.-Principal acting one equivalent of diketene with (j=)-DTO. Presumably it is the 8-ester, since acetic anhydride The distribution of isotope in the squalene sug- and DTO have been shown to give 8-S-Ac-DTO.' gests that the mevalonic acid was not decarboxyl- While DTO does not give a positive nitroprusside ated to yield a five carbon intermediate prior to assay for sulfhydryl,8~9we find the monothiol, monocondensation. If a five carbon intermediate is thioester form does. Thus, monoacylation of DTO first produced, the compound would have to react results in the appearance of one sulfhydryl equivaasymmetrically to give the observed isotope dis- lent (by nitroprusside assay), as well as one thioestribution. This possibility has not been excluded ter equivalent (measured with hydroxylamine or by the data presented here. Whether mevalonic optically a t 240 mp). The synthesis and breakacid is or is not the biological precursor of squalene down of mono-S-acyl-DTO compounds can thereand cholesterol, there appears to be little break- fore be measured by nitroprusside assay. down and re-condensation prior t o polymerization. The thioesterase catalyzing reaction 2 is assayed I t is likely that decarboxylation occurs during or optically by following the decrease in light absorption a t X 310 mp or X 240 mp, since ( f ) - S after polymerization. Complete details will be presented in a subse- AkAc-DTOhas an absorption spectrum characterquent publication. istic of acetoacetyl thioesters.l@,ll I t is present in Acknowledgment.-This work was supported in liver but apparently not in other tissues. The cnpart by grants from the National Heart Institute zyme has been purified 40-fold from chicken liver of the National Institutes of Health, the American and shown to be different from AcAc-SG thioesterHeart Sssociation, and the American Cancer So- ase.12 It hydrolyzes S-AcAc and S-A4c esters of ciety. DTO, M T O and BAL (Table I). As measured optically a t X 310 m p , liver and heart DEPARTMENT O F BIOCHEMISTRY SCHOOL OF MEDICINE FRANK DITURI enzyme fractions catalyze a disappearance of AcXcUSIVERSITYOF PENNSYLVASIA SAMUEL GURIX S-Pn and AcXc-S-CoA, provided DTO (or certain PHILADELPHIA, PENSSYLVANIA mono- or dithiols) is added. IVith DTO, the deDEPARTXESTO F BIOCHEMISTRY is BAYLORUNIVERSITY JOSEPH L. RABINOIVITZ crease in E310 due to disappearance of XcA%c-SK HOUSTON, TEXAS accompanied by a simultaneous increase in Ezra, RECEIVED MARCH16, 1957 signifying an increase in total thioester concentration. Balance studies (Table 11) show that for each mole of Achc-S-Pn disappearing, two moles of E . R. Stadtman, XI. DoudoroE and I?. Lipmann, J . Bioi. C h e m . , SOME ENZYMATIC REACTIONS O F 6,8-DITHIOLOC- 1 9 1(3), 377 (1951).
TANOIC (DIHYDROLIPOIC) ACID AND ITS ACETOACETIC THIOESTER'
Sir:
Evidence has been presented that AcAc2 synthesis in soluble liver fractions from a source of Ac-S(1) Supported by grants from the U. S. Public Health Service
F u n d of the Research Corporation. ( 2 ) .4hiire\ i:rtir,n>. li,X-dithioloctanoic (dihydrolipoic) acid, DTO; mr~nvthi(,liict:iii(~i~ :ici
