July 20, 1954
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the dichloride. The powder diagram was relatively simple and easily could be recognized in mixtures. The pattern was unchanged when the compound was heated in vacuo a t any temperature up t o 700°, regardless of the time of heating. Above 700°, however, a further disproportionation took place, and the tantalum pattern became evident in the powder diagrams with increasing predominance until only the metal remained. In none of the powder diagrams were there lines other than those of tantalum or those attributed to the dichloride. Moreover, none of these products was pyrophoric, but all appeared stable in dry air. I n all of these experiments considerable care was exercised with respect to purity of the reagents, and in the exclusion of oxygen. Nevertheless we were unable to produce the pyrophoric material described by the previous investigators. We have concluded that tantalum subchloride, if it does exist, cannot be prepared by the thermal decomposition of the higher chlorides.
6
Fig. 1. LABORATORIUM VOOR ANORSANISCHE E N A. E. VAN ARKEL PHYSISCHE CHEMIE Z. J. CERYCH cholesterol derived from 3-C14-DMAshould contain W. J. MIJS OF LEIDEN UNIVERSITY C14, GOand LEIDEN, HOLLAND U.SPITZBERGENsix labeled carbon atoms: Cq, &, GO, G6.Each of these should have R S.A. 27/6 or RECEIVED MAY6, 1954
UTILIZATION OF BRANCHED CHAIN ACIDS IN CHOLESTEROL SYNTHESIS
Sirs: It has been suggested‘ that in steroid biogenesis isoprenoid intermediates are formed from three molecules of acetate and that a likely product of this condensation is @-methyl 6-hydroxyglutarate which could furnish five carbon intermediates on decarboxylation.2 In support of these views it has previomly been found that isovaleric acida and the polyisoprenoid hydrocarbon squalene4 are considerably more effective as carbon sources for cholesterol than acetate. We have now synthesized the following branched chain acids : 3-C14-@methyl-P-hydroxyglutaric acid, 3-C14-cis-@-methylglutaconic acid, 3-C14-@-hydroxyisovaleric acid and 3-C14-@-dimethylacrylicacid (DMA) and tested their utilization in cholesterol synthesis. The sodium salts of the isotopically labeled acids were mixed with a stock diet and fed to rats a t a level of 0.25 mmole. per 100 g. of rat per day for two days. Cholesterol was isolated from the livers and analyzed for C14. All of these acids were found to furnish carbon for cholesterol synthesis, but with the exception of DMA, were less efficient than acetate. On the other hand, after the feeding of DMA the specific activity (S.A.) of liver cholesterol was 4-6 times greater than after the feeding of equimolar quantities of acetate. This result has prompted us to investigate the distribution of DMA carbon in the steroid molecule. If six isoprene units condense to form squalene (cf. Fig. 1) which cyclizes to the steroid structure,6 then (1) J. WUrsch, R. L. Huang and K. Bloch, J . B b l . Clem., lS6, 43Q (1962). (2) K Bloch, Harvey Lectures, Series 48, p. 68 (1952-63). (3) I . Zabin and K . Blach, J . B i d . Chcm., 186, 131 (1950). (4) R. G. Langdon and K. Bloch, THISJOVRNAL, T4,1869 (1964). ( 5 ) R . B,Woodward and K.Bloch, TEXR JQURNAL. ‘IS, 2028 (1963).
4.5 times that of the total molecule. If on the other hand DMA had first been degraded to acetate, then twelve sterol carbons should be labeled and have a S.A. 2.25 times that of cholesteroL6 Degradation of cholesterol formed from DMA showed (see Table) that Clo and (2%had specific TABLEI DISTRIBUTION O F el4 I N CHOLESTEROL FORMEDFROM 3-c”-~-DIMETHYLACRYLIC ACID, C.P.M.* AS INFISITELY THICKSAMPLES OF BARIUMCARBONATE. Found
-CalculatedAS
B)
100 .. .. el0 440 450 225 e 2 5 360 450 225 * Calcd. a Calcd. for six labeled C atoms per molecule. for twelve labeled C atoms per molecule. Cholesterol
activities which are 80 and 95%, respectively, of those which the hypothesis of five carbon intermediates predicts. These results preclude prior transformation of DMA to acetate and also to acetoacetate since Blecher and Gurin’ have shown that in cholesterol formed from l-C1* or 4-C14 acetoacetate the C l 4 distribution is indistinguishable from that given by acetate. The biosynthesis of DMA first detected in guayule leaves,* has now been demonstrated in liver homogenates by Rudneys and a similar observation has been made in this laboratory. The distribution of acetate carbon in DMA as observed by Rudney is identical with that in the terminal five carbon atoms of the cholesterol side chainlo and hence in full accord with the rule assigned here to DMA. Since in the (6) H. N. Little and K . Bloch. J . B i d , Chem., 183, 33 (1950). (7) M.Blecher and S. Gurin, Fed. Proc., I S , 184 (1954). (8) B. Arreguin, J. Bonner and B. J. Woad, Arch. Biochrm., 81, a 4 (1961). (9) H. Rudney, Fed. Proc., 18,286 (1954). (10) J. Wmch, R . L. Huang and E. Bloch,J . Bioi. Chcm., 106,489 (1952).
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Vol. 76
present experiments the 6-carbon dicarboxylic acids and P-hydroxyisovalerate were poorly utilized, their participation in cholesterol synthesis and their metabolic relation to DMA remain unsettled.
three hundred and fifty samples examined to date, over fifty have shown effectiveness a t or below 0,276 in diet. During the course of our studies with CzHS-S-R DEPARTMENT O F BIOCHEMISTRY KONRAD BLOCH compounds we encountered the report by Del UNIVERSITY OF CHICAGO L. C. CLARKE Pianto* on the antituberculosis effect noted after CHICAGO 37, ILLINOIS ISAACH A R A R Y ' ~injecting a combination of sodium ethyl thiosulfate RECEIVED APRIL29, 1954 and mercaptobenzothiazole derivatives in guinea (11) This work was supported by a grant from the Life Insurance pigs. In our hands s3dium ethyl thiosulfate alone Medical Research Fund. was more active orally than by subcutaneous (12) Postdoctoral Fellow, American Cancer Society. injection in mice. Table I is intended to show the relative activities of representative members of some of the classes of THE ANTITUBERCULOSIS ACTIVITY OF SOME compounds covered in this investigation. On the ETHYLMERCAPTO COMPOUNDS basis of data obtained by direct comparisoiis in Sir: it may be said that S-ethylcysteine (I. and In the course of studies dealing with the phenom- mice, DL) is a t least twice as active as pyrazinamide and enon of nitrification in soil' i t was noted that proliferation of nitrifying organisms was inhibited several times more effective than p-aminosalicylic acid.3 Compound I1 was equally effective against by several alkyl mercapto acids. This led to the isonicotinic acid hydrazide resistant and sensitive testing of (3-ethylmercaptopropionic acid (I) for strains of Mycobacteria. antibacterial activity. Only slight activity was found in witro against several gram-positive and TABLE I gram-negative bacteria. Definite protection was, ANTITUBERC~LOSIS ACTIVITY OF SOME CzH&-R COMhowever, afforded to mice infected with the H37Rv POUNDS strain of Mycobacterium tuberculosis (human type) Antituberculosis activity when fed 0-ethylmercaptopropionic acid a t a level of 0.2% in diet ad libitum. Further tests with other I CzH6-S-CHzCH2COOH t compounds of this type indicated that the ability I1 CzHb-S-CH&H(NH2)COOH (I, of a given compound to inhibit proliferation of and DL) 111 C~I-Is-S-CH~CI-T(~H~)CO~T~nitrifying bacteria is not a sufficient qualification for in vitro activity in experimental tuberculosis. .1 0 A systematic investigation of the relationship of I \' structure to in vivo antituberculosis activity in V derivatives of I was next undertaken. Substitution VI of various terminal alkyl groups (methyl through VI1 octadecyl) on the sulfur atom gave activity only VI11 with the CzH6-homolog. Aryl and heterocyclic IX moieties were equally disappointing. Variation of x the distance between sulfur and carboxyl to give XI mercaptoacetic through merwptovaleric acids indicated that the P-relationship between sulfur and carboxyl was essential. Replacement of carboxyl (2) Enrico Del Pianto, Riccra sci., a0, 83 (1950). by -CHtOH, -CHO, -COOR was achieved with (3) M. Solotorovsky, e1 ol., to be published. retention of activity. Elevating the oxidation LABORATORIES OF THE H . D . BROWN state of sulfur to sulfoxide or sulfone destroyed the RESEARCH DIVISION OF MERCK & Co., INC., A . R . MATZVK capacity to inhibit the experimental tuberculosis. CHEMICAL H J . BECKER Replacement of the sulfur atom in I by oxygen or RAHWAY,N. J. J . P. COXBERE nitrogen was also deleterious. I n an effort to J . M. CONSTANTIN find compounds with more acceptable properties, MERCKINSTITUTEFOR M. SOLOTOROVSKY S-ethyl-L-cysteine (11) was early tested and shown THERAPEUTIC RESEARCH, RAHWAY, N. J . S. WINSTEN E. IRONSON to be worthy of practical consideration on the basis RESEARCH INSTITUTE, MONTREAL GENERALJ . H . QUASTEL of efficacy and acute and chronic toxicity studies. CANADA Concurrently with the study of the structure HOSPITAL,MONTREAL, RECEIVED JUNE 14, 1954 requirements for efficacy in I, attention was given to the possible metabolic fate of P-ethylmercaptopropionic*acid in the animal body. This seemed especially pertinent in view of the lack of in vitro OXIDATIVE PHOSPHORYLATION IN THE CYTOCHROME SYSTEM OF MITOCHONDRIA' activity. With the testing of ethyl disulfide a significant increase in efficacy over I was noted. Sir: Results of the above tests opened a broad horizon 'It least two and probably three phosphorylations for the study of CzH6-S-R compounds. In general, are coupled to the passage of a pair of electrons structural modifications which decreased the tend- from reduced diphosphopyridine nucleotide to ency for cleavage of the C2H6S-linkage decreased oxygen via the respiratory chain in isolated liver the antituberculosis activity. Of the more than [ I I Supported by grants from the Nutrition Foundation, Inc., and
+
(1)
W. T.Brown, J . H. Quastel, P. G. Scholefield, J. Appl Micro-
bial.. in press.
Ndtiondl Institutes of Health.
S.O.N.is fellow of E H. Petersen
I'ounddion and recipient of Fulbright travel grant.
