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ratios identical with those of guanylic acid under similar conditions. Further analyses are listed in the table.
C, 66.2; H , 6.08; N, 5.63), was prepared by COUpling S-benzyl-N-carbobenzoxy-L-cysteine with methyl L-phenylalaninate by the dicyclohexylcarbodiimide procedure4 and also by the reaction oi pCOMPOSITION OF FRACTIOS 111 nitrophenyl S-benzyl-N-carbobenzoxy-L-cysteinAnalyses are on a total eluate concentrated to 50 nil. ate3,5,6with methyl L-phenylalaninate. I was mmol./ hydrolyzed to give S-benzyl-N-carbobenzoxy-LComponent liter cysteinyl-L-phenylalanine (II), m.p. 1.57-158", Guanine 0.41 [CX]~OD -22" (c 2, pyridine), (Anal. Calcd. for C27Total phosphate 0.82 HZ~OJV&~: C, 65.8; H, 5.73; N . 5.69; neut. Labile phosphate 0.39 equiv., 492.6. Found: C, 65.9; H, 5.92; N, Pentose 0.39 5.76; neut. equiv., 491). I1 was converted to a Total amino acids 0 35 mixed anhydride1 by the action of isobutyl chloro0.38 Acid labile amino acids formate and brought into reaction with L-isoleucylIt is apparent that guanine, pentose and amino ~-glutaminyl-~-asparagine(111). 3 The protected acids (ninhydrin on a hydrolysate with 8 N HCI pentapeptide S-benzyl-N-carbobenzoxy-L-cysteinylfor 24 hr. a t 105') were present in a 1:1:1 ratio. L-phenylalanyl-L - isoleucyl- L - glutaminyl-L - aspara-27" (c 0.5, Total phosphate was in a ratio of 2 : l with the other gine (IV), m.p. 233-238", [ c x ] ~ ~ D components but half was released by hydrolysis dimethylformamide), ( Anul. Calcd. for C42H53with 1 N HC1 for 30 min. Guanosine diphosphate 010N7S: C, 59.5; H, 6.30; N, 11.6; neut. equiv., was the major nucleotide found in an alkaline hy- 848. Found: C, 59.7; H , 63.0; N, 11.6; neut. drolysate with a formate c01umn.~ The arrange- equiv., 848), thus obtained was linked? to S-benzyl(V) to ment of amino acids cannot be as a protein or ~-cysteinyl-~-prolyl-~-leucylglycinamide~~~~~ give the protected nonapeptide S-benzyl-N-carboas a single peptide; hydrolysis with 1 N HC1 for 60 min. a t 100" led to the release of the amino benzoxy-L-cysteinyl-L- phenylalanyl - L - isoleucyl- Lacids (acid labile amino acids). Thus the amino glutaminyl- L - asparaginyl - S- benzyl - L - cysteinyl-Lacids must be combined in some type of labile link- prolyl-L-leucylglycinamide (VI), m.p. 247-248 ', [CX]"D -52" (c 1, dimethylformamide), ( A n d . age with guanine nucleotides. ~O 59.7; ~ ~ SH,Z 6.63; : N, These results support the concept that poly- Calcd. for C S ~ N ~ ~ S ~C, nucleotide, non-protein materials have catalytic 12.9. Found: C, 59.6; H, 6.77; N, 12.7). The activity in the hydrolysis of peptides. Cridine protecting groups were removed from VI with nucleotides and hexosamine, previously reported sodium in liquid ammonia and the resulting nonaas minor components of an active fraction from peptide oxidized by aeration to form the cyclic diDowex columns, have not been found in this sulfide (VII), an octapeptide, the 2-phenylalanine material; otherwise, the composition is much as analog of oxytocin. One milligram of VI1 gave in this procedure 18 units of avian depressor acwas reported.6 tivityl0 and 0.08 unit of pressor activity in the (5) R . B. Hurlbert, H. Schmitz, A. F. Brumm and V. R. Potter, rat." VI1 was purified by extraction with alcohol J. Bid. Chern., 209, 23 (1954). and precipitation by ethyl acetate followed by ex(6) F. Binkley, C . K. Olson and C. Torres, Abstracts, 128th Meettraction with pyridine and reprecipitation by ethyl ing, American Chemical Society, Minneapolis, 1955. acetate. The solid thus obtained assayed about 25 DEPARTMENT OF BIOCHEMISTRY EMORY UNIVERSITY FRANCIS BINKLEY units/mg. of avian depressor activity and was furATLAXTA 22, GEORGIA ther purified by countercurrent distribution in a RECEIVED DECEMBER 8. 1958 solvent system of butanol-ethanol-0.050jo acetic acid (4: 1: 5 ) , The distribution curve obtained by Folin color assay12 was in excellent agreement with SYNTHESIS OF A BIOLOGICALLY ACTIVE ANALOG the curve showing biological activity and also OF OXYTOCIN, WITH PHENYLALANINE REPLACING with a curve calculated for the K value 0.68. This TYRO SINE' agreement indicates the presence of a single comSir: pound. After this purification VI1 showed an We have studied the significance of one of the avian depressor activitylO of about GO-70 units per few free functional groups of oxytocin, the phenolic ing. and about 30 units per mg. of oxytocic activity hydroxyl group of the tyrosyl residue, by synthesis when assayed on the isolated rat uterus.13 N o of an analog of oxytocin with tyrosine replaced by phenylalanine. This analog, 2-phenylalanine pressor activity1' was found in the rat a t a total oxytocin,2 was prepared by a method recently used (4) J. C. Sheehan a n d G. P. Hess, THISJOURNAL, 77, 10G7 (1956). ( 5 ) h1. Bodanszky, S u t u r e , 176, 685 (1956); Acta Chirn. Hung., 10, for the synthesis of oxytocin.3 (1957); 31. Bodanszky, hl. Szelke. E. Tomorkeny a n d E. Weisz, Methyl S-benzyl-N-carbobenzoxy-L-cyteiny1-L-336 Chcin. a n d I n d . , 1517 (1955); A c t a Chi?% H u n g . , 11, 179 (1957). phenylalaninate (I), m.p. 106-107", [CX]'~D -37' (G) B. Iselin, W. Rittel, P. Sieber and R . Schwyzer, Hela. Chim. (c 2, dimethylformamide), (Anal. Calcd, for Cze- Acta, 40, 373 (1957). (7) J. P. Vaughan, Jr., THISJOURNAL, 74, 6137 (1952). H300SN2S:C, 66.4; H, 5.97; N, 5.53. Found: (1) T h i s work was supported in p a r t by a grant from t h e hTational Heart Institute, U. S. Public Health Service, Grant h-a. H-1675. (2) In order t o designate various analogs of oxytocin, this numbering is proposed (using t h e reduced form): -CySH-Pro-Leu-Gly(h"z). CySH-Tyr-Ileu-Glu ("2) -Asp "2() 1 2 3 4 5 0 7 8 9 (3) M. Bodanszky and V. d u Vigneaud: in press,
(8) C. Ressler a n d V. d u Vigneaud, ibid., 78, 3107 (1954). (9) 51.Zaoral and J. Rudinger, Chem. L i s t y , 49, 745 (1955). (10) J. M. Coon, Arch. Intern. Pharmacol., 62, 79 (1939). (11) J. Dekanski, B y i f , J . Pharmacol., 7 , 567 (1952). (12) 0. H. Lowry, S . V. Rosebrough, A. L. Farr and R . J. Randall, J . B i d . Chem., 183, 265 (1951). (13) J. H. Burn, D. J . Finney and L. G . Goodwin, "Biological Standardization," Oxford University Press, New York, N. Y., 1950.
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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 t h e 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 t h a t 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 t h a t 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 MEDICAL COLLEGE XEWYORK,N. Y. VISCENTDU VIGNEAUD trimethylphenylhydrazone, oily solid, too unstable 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 OF TECHNOLOGY ether solution of I1 to air produced ll-hydroperoxy- CARNEGIEIXSTITUTE 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,
