2646
CORIMUKICATIONS TO THE
EDITOR
Yol. 79
Anal. Calcd. for ClsHlaCleN~O~:C, 31.33; H, 1.75; nil. of ice-water with vigorous stirring. The resultant peacliFound: C, 31.92; H , 1.83; N, 4.68; colored solid was collected by filtration and recrystallized C1,36.40. from 95% ethanol to yield 1.55 g. (68%) of ivory platelets, The infrared spectrum showed maxima (5% in chloro- m.p. 128-131". Treatment with decolorizing carbon and form) a t 1764 (C=O); 1560, 1351 (NO?); 1274, 1161, 1130, repeated recrystallization from ethanol yielded an analytical sample of white platelets, m.p. 140-141". 1082, 1020, 966; 815 cm.-' (eel3). A n a l . Calcd. for CsH5ClaN206: C, 31.45; H, 1.48; N, Decomposition of Chloretyl on Heating in Vacuum.--'ll;hen either LY- or 0-chloretyl was sublimed a t 135-140' (0.2 mm.), 8.15. Found: C, 31.70; H , 1.58; N, 8.33. a small amount of unstable colorless compound, m.p. 102' The infrared spectrum (57, in chloroform) showed maxima dcc., was obtained as the more volatile fraction. This sub- a t 1755 (C=O); 1634, 1603 (aromatic); 1554, 1351 (NOZ); stance was tentatively assigned the structure \', 2-methyl-51274, 1163 (C-0); 835 crn.-l (non-enhanced cc13). I t will trichloromethyl-l-oxacyclopentan-2-ol-3-one, on the basis of be noted that the position of the C-0 maxinium IS a t lower analysis and infrared absorption spectrum: '?:Y 3355 (OH) ; frequency than that exhibited bv a-chloretyl niono-3,5-di1751 (C=O); 118.5, 1156, 1139, 1099, 995, 936, 911, 869; nitrobenzoate. 799 c m - ' (CCId. ~. (27) T h e formation of XI may be conceived as Anal. Calcd. for C ~ H I C I ~ O C,~30.86; : H , 3.02. Found: -HCl C , 30.89; H, 2.82. TI. This material soon decomposed on standing a t 23' to a yellow oil, with apparent loss of HC1. The infrared spectrum of the oil is consistent with the structure XI, 6,6-dichlorohexane-2,3,5-trione,which probably exists largely as the enol XII.2i The infrared spectrum showed maxima (5Yo in chloroform) a t 3600-3000 (broad absorption with much fine structure, characteristic of bonded O H ) ; 1720 (C=O); 1642 (conj. C=O); 1602 (C=C); 1423-1416, 0 00 0--H---OO 1365,1320,1240-1213,1115,1098,977,840~m.-~. I/ /Ill I ,I I1 of 2,2 ,2-Trichloroethyl3,5-Dinitrobenzoate.~ ClzCCCHzCCCH3 _J CIzCC CCCH, -ToPreparation a cold solution of 1.42 g. (6.7 mmoles) of 3,5-dinitroH H \ / benzoic acid and 2.54 g. (13.6 mmoles) of p-toluenesulfonyl CH chloride in 20 ml. of cold dry pyridine was added 1.0 g. (6.7 mmoles) of 2,P,2-trichloroethanol. The mixture was XI XI I allowed to stand at 0" for 1.5 hr. arid m s poured into 300 URBASA,ILLINOIS ru', 4.87; C1, 37.00.
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T H E ENZYMATIC CONVERSION OF MEVALONIC ACID TO SQUALENE
of U r i + - - , ATP6 and pyridine nucleotide (Table I ) . The reaction proceeds anaerobically as well as in Sir: air. Experiments bearing on the mechanism of The recent isolation and iclentification of me\-a- the condensation process have been carried out with Ionic acid (DL-/?,G-dihyclroxy-P-methylvaleric acid, 2-C'4,.i-di-T-mevalonic acid. Tritium was in troMVA) a new precursor of ~holesterol'-~constitute duced by partial reduction of @-hydroxy-P-methylan important advance ill the understanding of ter- glutaric acid with LiX1T4. The purified product, A pene and steroid bi:)yeiiesis. In this comrnunica- when conibined with authentic D L - ~ - C ' ~ - M Vand tion, we wish to report the transformation o f iiie~:t- recrystallized several times as the dibenzylethyleneloiiic acid to squalene by soluble yeast enzymes aiitl diamine salt or chromatographed on paper, showed to describe experiments bearing on the mechanism 110 change in the T : C I 4 ratio. Squalene syntheof 1IVA utilization. Particle-free extracts of dried sized by yeast extracts from the doubly labeled baker's yeast have been prepared which catalyze MVX contained T and CI4 in the same ratio as the the conversion of DL-2-c"-lIvAA to a radioactive precursor (Table 11). From the work of Tavorlipid, shown to be squalene by alumina chromatog- mina and Gibbs,' it is known that C1 of mevalonic raphy and by preparation of the thiourea adduct4 acid is lost during the conversion to cholesterol and arid of the crystalline hexahydrochl~ride.~No ster- hence the isoprenoid chain must be formed by ols are synthesized from MYA%under these condi- condensations linking C 5 of one MVA residue (or I tions. Dialysis and treatment with charcoal t o derivative thereof) t o Cz of another. The retenremove cofactors render the yeast extract com- tion of T during squalene formation clearly rules pletely inactive. Activity is restored by addition out a preliminary oxidation of mevalonic acid to 8hydroxy-8-rnethylglutaric acid (or a derivative). (1) D. I:. Wolf, C. 11. Hofirnan, P. E. Aldrich, I€. 11 Skeags, I,. 11. 'Though the unchanged T : C L 4ratio suggests that M'right and K . l'olkers, ' T i n s J O U R N A L , 78, 4499 (1956). (2) P. A , Tavormina, VI. I1 Gihlis and J . W. H u f f . ibiii., 78, 449X hoth hydrogen atoms a t C6 (6-carbon) of MVX iLrc 16, 27 I ( l ' l , - > i l *lI l ~ I ciatiiiii, I r i - ~ ~ h u ~ ] >ridine h i > i ~nuclri,ticle, > A T P a c l e n u ~ i n c~riljhusIJh:lt?. f7) 1'. A . '1':LvoriiiinA : t i 1 < 1 \ I I 1
