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COMMUNICATIONS TO THE EDITOR
3862
eficiency should be decreased by deuteration of the inhibitors. It is noteworthy that isotope effects of a considerable magnitude have been observed in the chain carrying step of air oxidation‘ and in the degradative chain transfer reaction in allyl acetate polymerization.2 It has been found that N-D-N-methylaniline and N-D-diphenylamine give oxidation curves, with cumene and tetralin as substrates in chlorobenzene solution, which are congruent with those observed in the presence of the corresponding undeuterated compounds. An illustrative example is shown in Fig. 1.
N
CHJ””TH~ 11
oxidation in chlorobenzene. During the early part of the reaction the color of the Wurster catwn appeared in the aqueous phase. The ion was positively characterized by measurement of its visible absorp3.20 i tion spectrum. The inhibition continued but with t a very low eficiency and eventually the dye dis2.80 appeared from the solution. These observations indicate that hydrolysis of the complex occurs 2.40 r according to equation 4 and the result represents
t
2.00
t
In-ROS.
+ HtO J _ In.+ f ROzH + OH-
(4)
the detection of gross amounts of an intermediate species which has neither undergone hydrogen abstraction nor attachment of ROY to a specific carbon atom of the inhibitor. It is very evident that these formulations permit wide extension in the field of radical chemistry in solution. Examples too numerous to cite present .OSo: ,040 themselves, A few especially significant related problems or observations are, (1) the postulated o t ,p” complex formation in the chain transfer reaction 0 20 40 60 80 of growing polystyrene radicals in bromobenzene Time in minutes. solution,s (2) the stereospecific free radical addition Fig. 1.-Oxidation of cumene (2.4M) in chlorobenzene a t of hydrogen bromide t o 1-cyclohexenyl bromide16 62.5’ inhibited by CeHsNHCHp (0) and Cd%NDCH* (0), and ( 3 ) the general problem of the mechanism of initiated by 1.01 X 10-2Mazoisobutyronitrile; inhibitor aromatic substitutioo by free radical^.^ concentrations, 3.33 X 10-8 M . Acknowledgment.--We are highly indebted to the B. F. Goodrich Company for their generous As a specific test for complex formation we have studied the action of tetramethyl-p-phenylenedi- support of this study. (5) F.R.Mayo, Tms JOURNS, 75,6133 (1953). amine (I).
a- 1.60 1
.r..-I.J...
(6) H. L. Goeting, P. 1. Ahell and B. F. Aycock, ibid., 74, 3588 (1952). (7) See D. I. Relyea and D. F. DeTar, ibrd., 7 6 , 1202 (1954), and previous papers in the series and G. S . Hammond, J. T Rudesill and F. J. Modic, ibid., 75, 8029 (1951),for examples of discussions pertinent to the iubject
Despite the fact that this compound contains no “labile” hydrogen it is a powerful inhibitor and stops two oxidation chains3 in both nitromethane and chlorobenzene solution with tetralin as a substrate. Furthermore, in nitromethane the purple color of the Wurster cation I1 is developed during the early part of the inhibition period and is subsequently dissipated. In an especially dramatic experiment water was included as a second phase in a repetition of the (1) R. A. Max and F. E. Deatherage, . I . A m . Oil Chcm. Soc., 48, 110 (1951). (2) P. D. Bartlett and F. A. Tate, THISJOURNAL, 76, 91 (1953). (3) The stoichiometric asslgnmcnt Is made on the basis of the length of the induction period. This is made possible by the use of appropriate efficiency factors for the production of radicals from azo-bis-isobutyronitrile by two independent methods which will be reported at an early data.4 (4) 6.9. Hammond, C . E. Boozer, J. N. Sen .nd C. E. Hamilton
uaprtbllrbd obrwvntioor.
DEPARTMENT OF CHEMISFRY CHARLESE. BOOZER IOWA STATECOLLEGE OF AGRICULTURE AND MECHANIC ARTS AMES, IOWA GEORGES. HAMMOND RECEIVED JUNE4, 1954
Sir:
THE CONVERSION OF CHOLESTEROL TO PREGNENOLONE IN BOVINE ADRENAL HO M 0GENATES
The transformation of cholesterol to 17-hydroxycorticosterone and corticosterone has been demonstrated in the isolated perfused bovine adrenal.? Based upon in vitro perfusion studies, the sequence of reactions shown in Fig. 1 was postulated to account for cholesterol conversion to corticoids. (1) Aided in part by the Research and Development Board, Office of the Surgeon General, Department of the Army, under Contract No. DA-49-007-M D-255. (2) 0. Hechter, et al., Recenl Progress i m Hormone Research, 8, 215 (1951). (3) 0.Hechter, Ciba Foundation, “Colloquia on Endocrinology,” 7 , 161 (1053). Edited by 0. E. Wolstenholme, Little. Brown, Boston, Moss.
July 20, 1954
COMMUNICATIONS TO THE EDITOR
3863
All of the reactions illustrated, Pregnenolone 17 OH Progesterone +17 OH DOC ----f 17 OH Corticosterone with the single exception of the conversion of cholesterol to Progesterone Dreznenolone. have now been I ichTeved in adrenal homogenL DOC Corticosterone ates.% The lack of the experiFig. 1. mental evidence for the cholesterol -+ pregnenolone reaction and apparent ab- active zones were detected on the paper with the sence of pregnenolone in adrenal tissue constitute following properties : zone (A) had mobility similar the major weakness of the reaction sequence shown to progesterone in that it traveled 13-20 cm. from the starting line, and exhibited a A4-3 ketone groupin Fig. 1. To obtain information on this point, CI4 labeled ing as revealed by ultraviolet scanning, and an cholesterol was incubated with adrenal homogen- orange dinitrophenylhydrazine reaction. Zone (B) ates and the resulting products fractionated by was similar to pregnenolone in that it travelled 4-7 systematic paper Chromatography. Under our cm. down the paper and showed the antimony conditions, we have regularly observed radioactive trichloride reaction characteristic of hydroxyproducts similar to pregnenolone and progesterone steroids.6 Zone (C) remained a t the origin and in terms of paper chromatographic behavior and gave color reactions characteristic for corticocolor reactions ; corticosteroid production by these steroids. Zone (B) was studied further as follows: after adrenal homogenates is more variable. In this communication we wish to describe results of an elution from paper, the residue was rechromatoexperiment wherein pregnenolone has been identi- graphed using the ligroin-propylene glycol system fied as one of the products of cholesterol metab- for 16 hours, and the radioactive zone of polarity equivalent to pregnenolone containing a total of olism. Cow adrenal glands (60 g.) obtained fresh a t the 14,000 ct. per rnin. was then eluted and chromatoslaughter house were homogenized in a Waring graphed on a 1 g. (ht. 10 cm.) silica gel column. blendor for 90 seconds with 120 ml. of ice-cold Following percolation of hexane and benzene phosphate buffer of the composition: M/150 through the column, about 1-2 mg. of a crystalline phosphate buffer fiH 7.4, diphosphopyridine nucleo- material containing 12,000 ct./min. was eluted tide ( 5 X AT), adenosine triphosphate, nicotin- with benzene-ethyl acetate (9 : 1). The infrared amide, magnesium sulfate, and sodium fumarate spectrum of this solid dissolved in carbon disulfide M ) . To the was identical to that of an authentic sample of (all a t a concentration of 5 X homogenate 200 pg. of cholester01-4C~~ (5 X lo6 pregnenolone. Upon acetylation of 50% of the ct./min./mg.) in 0.2 ml. of propylene glycol was total sample, the radioactive material now had added and the mixture incubated for 3.5 hours a t mobility similar to that of pregnenolone acetate in 37', using 100% oxygen as gas phase. The C14- the ligroin-propylene glycol system. The absorpcholesterol sample was homogeneous in the ligroin- tion spectrum over the range 230 to 600 millipropylene glycol4 and heptane-phenyl cellosolve5 microns, of the sulfuric acid chromogen of the paper chromatography systems. At the end of acetate eluted from the paper was identical to that incubation, the homogenate was extracted exhaus- of an authentic sample of pregnenolone acetate. tively with acetone, ethyl acetate and chloroform; The total radioactivity of the eluted acetate, deterthe total extracts were combined and distilled to mined in duplicate using 2% of the total acetate dryness in vacuo. The crude lipoid residue was per determination, was found to be .5,600 ct. per first fractionated by silica gel chromatography. rnin. using flow-gas counter. By comparison of This separated the radioactive fractions from the the sulfuric acid chromogen peaks (325 and 412 bulk of non-labeled materials which were eluted millimicrons) of two concentrations of authentic first from the column by hexane and benzene. pregnenolone acetate and of the isolated material, Most of the cholesterol was then eluted with a the total amount of pregnenolone acetate in the benzene-ethyl acetate mixture (9 : l), and the sample was estimated to be 0.5 mg. The calcuremaining products of greater polarity stripped lated specific activity of the free pregnenolone is from the column with ethyl acetate. -411 of the 12.7 X lo3 ct./min. mg. This value is based radioactivity in the (9 : 1) benzene-ethyl acetate upon the assumption that the intensity of absorpeluate from the silica gel column was found to be tion a t the peak studied is a direct function of the associated with the cholesterol zone using the concentration. The estimated conversion of cholesheptane-phenyl cellosolve system. Upon sub- terol to pregnenolone on the basis of counts added sequent purification through the digitonide and is about 1Yc. Consideration of the data indicates recrystallization, the specific activity of the choles- that: (1) the bulk of the cholesterol added as CI4 terol isolated from the homogenate a t the end of labeled substrate remained unchanged but is conthe experiment was found to be 4.5 X lo3 ct./min. / siderably diluted by cholesterol already present in mg. using a flow gas counter, counting for 10- the gland; ( 2 ) the pregnenolone isolated as a transformation product appears to have a specific minute intervals. The ethyl acetate eluate from the column was activity higher than cholesterol isolated a t the chromatographed on paper using the ligroin- end of the experiment. This result is consistent propylene glycol system for 4 hours. Three radio- with the view that cholesterol in the adrenal gland cannot be considered as a homogeneous metabolic (4) K.Savard, J . B i d . Chem., 404,457 (1953).
t \ f
(6) R.Nehet and A. Wettotein, Rclr. Chim. Acta, 88, 278 (1952).
( 6 ) H.Rosenkrantz, Arch. Biochcm. Bioahyr., 14, 1 (1953).
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C O M M U N I C A T I O N S TO THE
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EDITOR
pool from which transformation products are iormed. The metabolism of cholesterol and its conversion to pregnenolone by adrenal homogenates have been consistently demonstrated in three additiondl experiments in approximately the same yield. The full details of these studies will be published subsequently. \Ye should like to thank Dr. Harris Iiosenkranz and &It-. Paul Skogstroni for the analysis and interprekdtions (of the infrared spectra.
vin ureidosuccinate and dihydrodrotate. Ureidosuccinate has been pictured as arising from argininosuccinate, based on the finding that the ureide carbon of citrulline3 as well as carbon dioxide4 contribute to position-2 of tissue pyrimidines in the pigeon. In the rate and in rat liver homogenates, no such incorporation from citrulline could be demonstrated, and this failure was attributed to active degradation of citrulline to urea in mammalian k-er. In the present study, pigeon and rat \\.ORCESTER FOUNDATION FOR 9 S A B A ~liver slices have been compared in regard to inEVPERIMENTAL BIOLOGY 0 HECHTER corporation into orotic acid of the ureide carbon of SHREWSBURT, MASS. D STO\E citrulline and the aniidine carbon of arginine. RECEIVED h f A Y 18, 10% L(+)-Citrulline was prepared from uren-CI4 -~ by the method of Kurtz.6 I,( +)-hrginine.HCl Fellow of the Kellogg roundatlon On leave from the ?\llnlqtry (7 was synthesized from cyanogen bromide-CI4 via of Agriculture, England 0-methylisouronium chloride.'j Both materials had specific activities of 2.84 X 106 c.p.111. per MASS SPECTRUM OF OCTABORANE mmole. Incubation was carried out essw tially Sir: according to Reichard7 using 15-18g. of pigeon or From recent mass spectrographic studies' of rat liver slices, 50 ml. of Krebs-Henseleit bicarbonvarious boron hydrides we have observed the mass ate medium supplemented with 90 mg. of glucose, spectrum of an octaborane, the existence of which 50 nig. of sodium ATP and 15 mg. of carrier orotic was first postulated by Burg and Schlesinger2 acid. Each bath also contained either 0.1 mmole of ~ - c i t r u l l i n e - C ~ ~0.5 mmole of L-aspartate or from vapor tension measurements. 0.1 mmole of L-arginine.HC1-C14 0.5 mmole of fumarate. After 4 hours of incubation at R i " , . I orotic acid was recovered from each deproteinized medium as described by R e i ~ h a r d . ~The product was characterized by ultraviolet absorption spectrum and m.p. (341-343') after recrystallization from water. All samples were combusted, and counted as barium carbonate under a thin-window 85 90 95 100 Mass number, m/e. Geiger-Mueller counter. 3t5
+
Fig. 1.-Partial
ma45
spectrum of octaborane.
The mass spectrum of octaborane from mass numbers 85 to 100 is given in Fig. 1. The dominant peak occurs a t mass number 93, and double ionimtion peaks are found in the region of mass numbers 44-48. The sharp cut-off in peak heights a t mass number 100 suggests that the composition of the octaborane is BaHI?, thus indicating that this compound belongs to the group of the (more) st>tijleboron hydrides3 In addition to the above findings we have been ahle to confirm Norton's finding of n ~ n a b o r a n e . ~
RADIOACTIVITY
+
TABLE I OROTIC ACID FROM LIVER SLICESTUDIES
O F ISOLATED
R a t liver, Pigeon liver, c.p.m. per milliatom C
Substrate L( +)-Citrulline-Cl*
+ )-Citrulline-C14 L(+ )-Argini~ie-C~~
I.(
3,390 3,040 0"
0 0 0 0
L(+)-Arginine-C14 Zero means
