March 5, 1959
COMMUNICATIONS TO THE EDITOR
1259
6- (2-amino-3 4,5-trimethylbenzoyl) -valeric acid VI, m.p. 146-149’. (Found: C, 68.56; H, 7.84; N, 5.50), and by alkali to 2,3-trimethylene6,7,S-trimethyl-4-quinoloneVII, darkens above 300’. (Found: C, 80.90; H, 7.64; N, 5.86). These transformations of I1 through IV and V to V I or VI1 parallel a precisely analogous series of reactions starting with tetrahydro~arbazole.~ The ultraviolet and infrared spectra of V-VI1 are notably similar to those of the lower homologs derived from tetrahydrocarbazole. The location of the methyl groups in the structure I1 and those of its derivatives was proven by synthesis of I1 from hemimellitene. The latter (14) E. V. Baldes, Biodynamico No. 46, 1 (1939). afforded an adduct with ethyl azodicarboxylate, (15) As the experiments reported here were completed, we learned m.p. 151-152’ (Found: C, 60.10; H, 7.62; a t a lecture delivered by Dr. R. A. Boissonnas that the same analog of oxytocin was prepared and studied in his laboratory simultaneously N, 9.60), which was converted to 2,3,4-trimethylhut independently from our work. phenylhydrazone, m.p. 105-106’ (Nz), too un(16) After this communication was submitted for publication, stable to analyze. The structure of this compound Professor H. B. van Dyke found that 2-phenylalanine oxytocin shows was demonstrated by its hydrogenation over Raney milk ejecting activity of about 60 units per mg. DEPARTMENT OF BIOCHEMISTRY MIKLOSBODANSZKYnickel to 2,3,4-trimethylaniline, characterized as its N-acetyl derivative. Cyclohexanone 2,3,4CORNELL UNIVERSITY MEDICALCOLLEGE VISCENTDU VIGNEAUD trimethylphenylhydrazone, oily solid, too unstable XEW YORK,N. Y. RECEIVED JANUARY 12, 1959 to analyze, when boiled in acetic acid under nitrogen, afforded 11, isolated as its picrate, m.p. and mixed m.p. 171-172’(d.). From this picrate I1 EVIDENCE OF A 1,C-METHYL MIGRATION itself and from the latter 1,2,3-trimethylcarbazole DURING A FISCHER WACTION and I V , V, VI and VI1 were prepared. Their Sir: m.pts. mixed m.pts. and spectroscopic properties An investigation of the structure of a compound identified them with samples obtained from I as isolated some years ago’ as its picrate from the starting material. product of the action of boiling acetic acid on cycloAlthough i t is possible to rationalize the formahexanone mesitylhydrazone (I) has now disclosed tion of I1 from I by means of a series of three that the compound is not “1,2,3,4-tetrahydro- consecutive 1,2 methyl shifts, along with the re6,S,12-trimethylisocarbazole” (111)) but is in- quired accompanying reactions, a single 1,4 shift stead 6,7,8-trimethyl-1,2,3,4-tetrahydrocarbazole of methyl, although to our knowledge unprece(11). dented, seems to US to offer a superior explanation. Mesitylhydrazine, m.p. 60-61’ (Nz), was pre- In either instance the shift would start with an pared by a new synthetic method based on the intermediate VIII, a homolog of one for which addition of mesitylene to ethyl azodicarboxylate.2 evidence has been offered previously.6 A single The adduct, m.p. 159-160’ (Found: C, 61.67; 1,4 shift of methyl, through transition state I X , H, 7.61; N, 9.579 was converted to mesitylhy- would yield a second intermediate from which I1 drazine by boiling ethanolic potassium hydroxide. could be formed by previously suggested routes6 I, m.p. 45-47’ (N2), from mesitylhydrazine and cyclohexanone in the absence of solvent, was too unstable to analyze. Under nitrogen, boiling acetic acid converted I to 11, isolated as its picrate, m.p. 171-172’ (d.), as reported.’ (Found: C, 57.61; H, 5.00; N, 12.87.) The tetrahydrocarbazole, from the picrate and sodium hydroxide, was extremely air-sensitive, m.p. 92-98’ (Nz), insuf(4) B. Witkop and J. B. Patrick, THISJ O U R N A L , 73, 2188, 2198 ficiently stable to analyze. Chloranil in xylene (1951). under nitrogen converted I1 t o 1,2,3-trimethyl( 5 ) R. B. Carlin and D. P. Carlson, ibid., 79, 3605 (1957). (6) See R. B. Carlin, W. 0. Henley, Jr. and D. P. Carlson, ibid., carbazole, m.p. 127.5-128.5’. (Found: C, 85.32; H, 7.37; N, 6.85) ; infrared and ultraviolet curves 79, 5713 (1957). OF CHEMISTRY ROBERTB. CARLIN very similar to those of carbazole. Exposure in DEPARTMENT ether solution of I1 to air produced ll-hydroperoxy- CARNEGIEIXSTITUTEOF TECHNOLOGY 13, PENNA. MEADS. MOORES 6,7,S-trimethyl-1,2,3,4-tetrahydrocarbazolenine IV, PITTSBURGH RECEIVEDDECEMBER 24, 1958 m.p. 134’ (d.). (Found: C, 72.46; H, 7.81; N, 5.77)) which isomerized in ethanol solution to 9,10,1l-trimethyl-l-benzazonidine-2,7-dione V, m.p. 171-172’. (Found: C, 73.08; H, 7.90; N, 5.55). AMINO ACID INCORPORATION INTO LIPOIDAL MATERIAL BY CELL-FREE LIVER PREPARATIONS The latter was converted by acid hydrolysis to
dose of 607. (Anal. Calcd. for C43Ho6N12011S2: C, 52.1; H, 6.71; N, 17.0; mol.wt., 991. Found: C, 52.1; H, 6.83; N, 16.9 ;mol. wt.14940). From these data we can conclude that the phenolic hydroxyl group of oxytocin contributes strongly to the activity of the hormone but is not essential for biological activity.lB,l6 Acknowledgements.-The authors wish to thank Mr. Joseph Albert for the microanalyses, Mr. David De Peter for the determination of the molecular weight, Miss Dade Tu11 for the biological assays, and Mr. David N. Reifsnyder for technical assistance.
(1) C. S. Barnes, K. H. Pausacker and W. E. Badcock, J. Cham. SOC.,730 (1951). (2) R. Huisgen. F. Jacob, W. Siege1 and A. Cadus, Ann., 690, 1 (1954). ( 8 ) Sample first prepared by Dr. Robert J. Laufer.
~
Sir : The incorporation of amino acids into cellular constituents via adenosine triphosphate-amino acid activation has been studied by many workers,
COMMUNICATIONS TO THE EDITOR
1260
Vol. 81
particularly Zamecnik and his collaborators.1 TABLE I1 Relative specific T h a t additional reactions may be involved in activity in amino acid incorporation has been suggested by microsomal Protein Lipid Treatment Hendler2 upon noting a substantial incorporation lOOb 1006 None (control) of labeled amino acid by minced hen oviduct into Ribonuclease (0.1 mg./ml.) 42 107 lipoidal material similar to “pr~teolipids.”~ ATP and 3-phosphoglycerate omitted 21 115 We have examined the incorporation of labeled p-Chloromercuribenzoate ( M) 15 SG amino acids into lipid soluble material by cell-free 13 393d CrotoxinC(20 pg./nil.) preparations of rat liver. Phenylalanine was found Conditions as in Table I. Microsomes were analyzed to be incorporated readily into such material in both microsomes and microsome-free soluble cyto- after incubation of whole supernate. * Measured specific activity of the two fractions separately set equal to 100. plasmic supernate alone or together (Table I). Kindly furnished by Dr. 11. Fraenkel-Conrat. This TABLE I R a t liver homogenates prepared according to Zamecnik and Keller‘ with medium Ah were centrifuged a t 10,000 X g for 10 min. t o yield the whole supernate, 3.5 ml. of which was incubated a t 37” for 2 hr. in SZ:COZ(95:5) with 5 pmoles of ATP, 50 pmoles of 3-phosplioglycerate and 1.15 prnoles ( 5 pc.) of ~~-plienj.lalanine-3-C‘~; total volume, 5 nil. Microsomes and soluble supernate were prepared before or after incubation by centrifuging the whole supernate a t 105,000 X g. Both the microsomes obtained after 30 min. centrifugation. suspended in cold medium A , and the first supernate were centrifuged an additional 60 min. Protein and lipid were prepared from the trichloroacetate-precipitated material essentially as described by Siekevitz.6 Fraction incubated
Whole supernate
C.p.m./rng.e hZicroson~al Protein Lipid
390 21s (14,200)b (3860) 136 714
Soluble supernate Protein Lipid
36 (5130)
stimulation has been verified repeatedly.
Tryptophan and leucine were also incorporated into lipid while glycine, lysine and valine were incorporated to a much smaller degree. Support of this work by a grant from the National Science Foundation (G-5685) is gratefully acknowledged. PAPERNo. 1371 FROM THE JOSEPHL. HAINISG AGRICULTURAL EXPERIMENT STATION DEPARTMENT OF BIOCHEMISTRY, PURDUE USIVERSITY TOSHIO FUKVI WEST LAFAYETTE, INDIASA BERNARD L4XELROD RECEIVEDDECEMBER15, 195s JOURNAL
334 (1002) Microsomes .. .. .. .. 39 567 Soluble supernate e Net incorporation. Blanks containing trichloroacetic acid initially and incubated for zero and 2 hours were less than 10% as radioactive. * Figures in parentlieses indicate total c.p.m. in each fraction.
Sir:
P. C. Znmecnik, M. L. Stephenson and L. I. Hecht, Proc. Nall. Sci., U .S., 44, 73 (195s). R. W. Hendler, Science, 128, 143 (1958). J. Folch and M. Lees, J. Bid. Chrm.. 191, 807 (1951). (4) P. C. Zamecnik and E. R. Keller, ibid., 209, 337 (1954). ( 5 ) E.B.Keller and P. C. Zamecnik, i b i d . , 221, 45 (1956). (0) P. Siekevitz, i b i d . , 196, 549 (3952). (7) J. Hirsch and E. H. Ahrens, Jr., ibid., 233, 311 (1958). ( 8 ) W. Stepka, in Corcoran, “Methods in Medical Research,” The Year B u d l’iillishers, Chicdgu, 1052, Vul. 5, p . 25.
Upton, ibid., 224, 47 (1957). (3) I. E. Bush, J . Endocrinol., 9,95 (1953). (4) P. K. Bondy, G. V. Upton and G. E. Pickford, Nofurc, 1 7 9 , 1354 (1957). (5) G. Pincus and E . B. Romanoff,“Ciba Colloquia on Endocrinol..” J. & A. Churchill, Ltd., London, 8,97 (1955). ( 6 ) J. Tamm, 1. Beckmanu and K. D. Voigt, A c f a Endocrinol., 97, 403 (1958). (7) C. 5. 0 . R . Morris a n d D. C. \Tilliams, “Ciba Colloquia on Eililucrinul..” 8, 157 (1955j.
ISOLATION O F CORTISONE AND CORTISOL FROM THE PLASMA OF PACIFIC SALMON
(0NCO RH YNCH US NE RK A )
Cortisol (I) has been identified as the principal adrenal-cortical hormone in the peripheral plasma of the normal human’ where it occurs at a concentration of approximately 10 pg. per 100 m1.2 It A major portion of the radioactive lipid-soluble is also the principal steroid in the blood of several material was separated from other lipids by silicic animalsa and has be.en tentatively identified in acid c h r ~ m a t o g r a p h y . ~ This fraction, which was carp p l a ~ m a . ~ httempts to find cortisone (11) free of phosphorus, yielded on hydrolysis (6 hr in adrenal vein5 and peripheral human blood’ HC1, l l O o , 18 hr.) a number of amino acids includ- have been unsuccessful. Occasional blood samples ing radioactive phenylalanine, which was identi- have been reported to contain small amounts of a fied by co-chromatography on paper (butanol- “cortisone-like” substance,lJ but the evidence is acetic acid-water8) and radioautography with inadequate to establish identity. authentic phenylalanine. Milder conditions ( 2 We wish to report the isolation of I and I1 from V : HCI, looo, 2 hr.) released negligible amounts of Fraser River sockeye salmon (0. ?zerka) just prior phenylalanine. A mixture of the labeled lipoidal to their arrival on the Adams River spawning fraction with C14-phenylalanine was resolved by grounds. Blood was obtained from (ca). 100 fish paper chromatography. The radioactivity of the by severing the caudal artery. Ethyl acetate lipoidal substance was not diminished by extensive extractable steroids were obtained from 1760 ml. washing with a solution of non-radioactive phenyl- of plasma by the usual procedures. Chromatogalanine. raphy on purified Whatman no. 1 paper was perT h a t the incorporation of the amino acid into formed in three solvent systems known to resolve the lipid did not occur via the “conventional” I and I1 from closelyre lated steroids; (CHCla: pathway of incorporation into protein was proven C6Hal:1-forniamide (111), toluene-propylene glycol by the following: Microsomes were not required, (IV) and butyl acetate:ethylene glyco1:HzO 20:l :l. as noted above. Conditions which interfered with The corresponding U.S.P. reference standards and the “conventional” pathway did not curtail in- I and 11 were located on chromatograms both by corporation into the lipid-soluble material (Table (1) I. E. Bush and A. A. Sandberg, J. Biol. Chcm., ZO6, 783 (1953). 11). (2) P. K. Bondy, D. Abelson. J. Scheucr, T. K. L. Tseu and V. (1) Acad. (2) (3)
