527
yllysylaspartic acid (11), prepared as previously described,5 was condensed with N"-benzyloxycarbonyly-benzylglutamate by means of the p-nitrophenyl ester method,6 and the resulting product was subsequently hydrogenated to give glutamylhistidylphenylalanylarginyltryptophylglycylserylprolylprolyl-N'-formyllysylaspartic acid (111, monoacetate octahydrate. A n d . Calcd for C63H86018N18 * CH3COOH. 8HzO : C, 49.2; H, 6.7; N, 15.9. Found: C, 49.3; H, 7.1; N, 15.6), [ c ~ ] ~ O D -73,6'(water); Rrl 0.20, R r 20.28; amino acid ratios in an acid hydrolysate Glu~.o~Hiso.92Pheo.s7A r g ~ . ~ ~ G l y ~ . ~ ~ S e r ~ . ~ ~ P r o ~average . ~ ~ L yrecovs~,~~~sp~.~~ ery 100%. This partially protected undecapeptide (111) was allowed to react with Nu-t-butoxycarbonylmethionine p-nitrophenyl ester,7 and the resulting product was treated with trifluoroacetic acid to give methionylglutamylhistidylphenylalanylarginyltryptophylglycylserylprolylprolyl-N'-formyllysylaspartic acid (IV) (Anal. Calcd for C68H95019N19S. CH3COOH.6HzO: C, 50.0; H, 6.6; N, 15.8. Found: C, 50.1; H, 7.2; N, -66.6' (water); R f l 0.1 1, R f 2 0.25; amino 15.5), [@OD acid ratios in an acid hydrolysate Met~.ojGlul.olHisl.04Pheo.g7Argo. g2Gly1. ooSero. 97Pro1.nLysl .lIAspI.02 ; average (8) See, for example, C. H.DePuy and C. A. Bishop, J . Am. Chem. recovery 100%. It was confirmed in a preliminary exSoc., 82, 2532 (1960); C. H. DePuy and R. E. Leary, ibid., 79, 3705 (1957); S. C. J. Olivier and A. P. Weber, Rec. Traa. Chim., 53, 869 periment that N'-formyllysine survived the action of tri(1934); W. N. White, C. D. Slater, and W. K. Fife, J. Org. Chem., 26, fluoroacetic acid mostly unchanged. 627 (1961). (9) See for comparison Y. Okamoto, T. Inukai, and H. C. Brown, This partially protected dodecapeptide (IV) was J. Am. Chem. Soc., 80, 4972 (1958); J. F. Norris and C. Banta, ibid., Na-t-butoxycarbonyl-NG-nitroarcondensed with 50, 1804 (1928); N. C. Den0 and W. L. Evans, ibid., 79, 5804 (1957). ginine8 by the mixed anhydride p r o ~ e d u r e .lo~ , The William N. White, Edgar E. Moore resulting product was treated with anhydrous hydrogen Department of Chemisfry, University of Vermont fluoride according to Sakakibara and Shimonishill to Burlington, Vermont 05401 remove the t-butoxycarbonyl group and the nitro Receiued Nocember 13, 1967 group from the arginine r e s i d ~ e . ~It~ .is~ ~known that the formyl group on model peptides is not affected Studies on Peptides. XX. Synthesis of the by this treatment. l 3 The desired partially protected Octadecapeptide Corresponding to the Entire tridecapeptide, arginylmethionylglutamylhistidylphenylAmino Acid Sequence of Monkey alanylargin y ltry pt ophylglycylser ylpr o 1y 1pro 1y 1-N' f o r6-Melanocyte-Stimulating Hormone1p2 myllysylaspartic acid (V, [ a I 3 O ~-36.7' (1 N acetic acid), Rr30.17, contaminated with a trace amount of the Sir : side reaction product from the mixed anhydride reacThe structure of 6-melanocyte-stimulating hormone tion, presumably a ethoxycarbonyl derivativel4,l5 of IV, (I) from monkey pituitary glands was elucidated by Rr30.40), was then allowed to react with N"-t-butoxyin 1961. We wish to report the synthesis Lee, et carbonylprolyltyrosine azide derived from the correof the octadecapeptide which embodies the entire Anal. Calcd for sponding hydrazide (mp 174-176'. amino acid sequence of this hormone. C I ~ H ~ ~ OC,~ 58.2; N ~ : H, 7.2; N, 14.3. Found: C, H-Asp-Glu-Gly-Pro-Tyr-Arg-Met-Glu-His-Phe-Arg-Trp-Gly- 57.6; H, 7.3; N, 14.3), and the resulting product was Ser-Pro-Pro-Lys-Asp-OH subsequently treated with trifluoroacetic acid to give the Choice of the protecting groups for the a-amino and partially protected pentadecapeptide, prolyltyrosylguanidino group of arginine which is adjacent to the arginylmethionylglutamylhistidylphen y la1a n y 1a r g i n y 1methionine residue and the e-amino group of the lysine residue at position 17 determined the strategy toward ( 5 ) H. Yajima, Y . Okada, Y.Kinomura, and E. Seto, Chem. Pharm. Bull. (Tokyo), 15, 270 (1967). the total synthesis of this peptide hormone. (6) M. Bodanszky, Narure, 175, 685 (1955); Acta Chim. Hung., 10, The formyl group was selected for the protection of 335 (1957). the a-amino group of lysine. Thus, histidylphenyl(7) K. Hofrnann, W. Haas, M. J. Smithers, and G . Zanetti, J . Am. alanylarginyltryptophylglycylserylprolylprolyl-Nf-form- Chem. SOC.,87, 631 (1965). certed bond breaking and making in the transition state. Thus, the rearrangement of hydrazoaromatics must involve nitrogen-nitrogen bond cleavage to form an intermediate which then rebonds to yield the products. The effect of substituents on the rate of reaction also argues against a one-step concerted process. Most reactions of this type are only weakly affected by substituents.8 The large substituent effect observed is entirely consistent with a process in which an extranuclear bond breaks in the rate-determining step generating a center which can relieve its electron deficiency by resonance with the ring and the substit~ e n t . Therefore, ~ the stepwise mechanism for the benzidine rearrangement is again indicated. In conclusion, product and substituent effect data obtained in a study of the rearrangement of N,N'-dimethylhydrazobenzene strongly favor a mechanism for the benzidine rearrangement in which the nitrogennitrogen bond of the hydrazo compound is broken before the carbon-carbon or carbon-nitrogen bonds in the products are formed.
-
(1) Peptides and their derivatives mentioned in this communication are of the L configuration. Rf1 values refer to the system l-butanolacetic acid-water, 4 : l :5 on paper chromatography. R f * and Rf3 values refer to the system of I-butanol-pyridine-acetic acid-water, 4 : 1 :1 :2 and 15 :10 :3 :12, respectively, on thin layer chromatography (Kieselgel G, Merck). Column chromatography on carboxymethylcellulose was used extensively for purification of these peptides with ammonium acetate or pyridine acetate buffers as eluent. (2) Part XIX of this series: H. Yajima and K. Kawasaki, Chem. Pharm. Bull. (Tokyo), in press. (3) T.H. Lee, A. B. Lerner, and V. B.-Janusch, J. Biol. Chem., 236, 1390 (1961). (4) K. Hofmann, E. Stutz, G. Spiihler, H. Yajima, and E. T. Schwartz, J . Am. Chem. SOC.,82, 3727 (1960).
(E) K. Hofmann, W. Haas, M. J. Smithers, R. W. Wells, Y. Wolman, N. Yanaihara, and G. Zanetti, ibid., 87, 620 (1965). (9) Th. Wieland and H. Bernhard, Ann., 572, 190 (1951). (10) J. R. Vaughan, Jr., and R. L. Osato, J . Am. Chem. Soc., 73, 3547 (1951). (11) S. Sakakibara and S. Shimonishi, Bull. Chem. SOC.Japan, 38, 1412 (1965). The authors wish to express their sincere appreciation to Dr. S. Sakakibara for offering his equipment for this experiment. (12) J. Leonard and A. B. Robinson, J. Am. Chem. Soc., 89, 181 (1967). (13) S. Sakakibara, Y. Shimonishi, Y. Kishida, M. Okada, and H. Sugihara, Bull. Chem. SOC.Japan, 40,2164 (1967). (14) A. R. Emery and V. Gold, J. Chem. Soc., 1443, 1447 (1950). (15) Th. Wieland, B. Heinke, K. Vogeler, and H. Morimoto, Ann., 655, 189 (1962).
Communications to rhe Editor
528
tryptophylglycylserylprolylprolyl-N'-formyllysylaspartic possesses the lysine residue at position 6 instead of the acid (VI. Anal. Calcd for C88H123023N25S. 2CHoCOarginine residue present in the monkey 0-MSH. This O H . 9 H 2 0 : C, 49.9; H, 6.8; N, 15.8. Found: C, difference required a different synthetic approach to 50.6; H, 7.1; N, 15.0), [a!I3OD -60.7"(1 Nacetic acid); monkey P-MSH from that of Schwyzer, e? al., as outR f 3 0.47; amino acid ratios in an acid hydrolysate lined above. During the course of this investigation, we have Pro3.17 Arg1.8~ Metoag4 Glul.01 His0.97Phel.ooGlyOsg7Sero.84LySl.o~ASP1.00; (average recovery 89 %). found that Nu-benzyloxycarbonyl-NG-nitroarginylThe N-terminal protected tripeptide, N"-t-butoxycarmethionine methyl ester could be reduced t o arginylbonyl-0-t-butylaspartyl-y-t-butylglutamylglycine(VII, methionine methyl ester by catalytic hydrogenation dicyclohexylamine salt. Anal. Calcd for C24H41' over a palladium catalyst in the presence of boron OION~.C~~H C,~60.7; ~ N : H, 9.0; N, 7.9. Found: C, trifluoride etherate. 2 2 This procedure offered an 60.7; H, 9.3; N, 8.1), mp 128-130", [ a ' J Z-15.3" 2~ in alternate synthetic approach to this hormone which methanol, was prepared by the p-nitrophenyl ester possesses the particular amino acid sequence of arginylmethod in a stepwise manner from the C-terminal glymethionine. These results will be published in the cine. This peptide (VII) was condensed with VI by future. means of the N-hydroxysuccinimide ester method. l 6 Acknowledgment. The authors wish to express their The resulting product was then treated with trifluoroappreciation to Professor S. Uyeo of this faculty for his acetic acid to form aspartylglutamylglycylprolyltyrosylencouragement during the course of this investigation. arginylmethionylglutamylhistidylphenylalanylarginylR. Schwyzer, B. Iselin, H. Kappeler, B. Riniker, W. Rittel, and trypt ophylglycylserylprolylprolyl-N'-formyllysylaspartic H.(21) Zuber, Hela. Chim. Acta, 46, 1975 (1963). acid (VIII, [aI3O~ -63.6" (1 N acetic acid); Rf3 0.49; (22) M. Okarnoto, S. Kirnoto, T.Oshirna, Y.Kinornura, K. Kawaand ~H. .Yajima, Pharm. Bull. (Tokyo), IS, 1621 (1967). amino acid ratios in an acid hydrolysate A s ~ ~ . ~ ~ saki, G ~ ~ ~ Chem. Glyz.05 Pr03.~~Tyr0.7~ Argl.,, Met0.93Hisl.00 Phel.ooSero.98Haruaki Yajima, Yoshio Okada Lysl.06; average recovery 96 %), which was subsequently Yasuhiko Kinomura, Hideo Minemi treated with 5% aqueous hydrazine acetate at 37" for Faculty of Pharmaceutical Scietices Kyoto Unioersity, Sakyo-ku, Kyoto, Japan 48 hr to remove the "-formyl group from the lysine Received October 23, I967 residue. The use of aqueous hydrazine acetate or hydroxylamine hydrochloride in pyridine for the removal of the formyl group from N'-formyllysine has been demonstrated recently in the synthesis of CY-MSH The Two-step Polar Cycloaddition of Sulfonyl Isocyanates to Carbodiimides from the [ 11-N'-formyllysine]-CY-MSH derivative. l7 Care was taken to prevent possible oxidation of the Sir: methionine residue by performing the reaction in the The polar 1,2-cycloaddition reaction of ketenes t o presence of thioglycolic acid. vinyl ethers shows stereospecificity, thereby indicating The purified octadecapeptide corresponding to the that this reaction proceeds via a concerted one-step entire amino acid sequence of I ([CYI~OD -50.7" (1 N In a recent article by Proskow, et d.,a acetic acid); amino acid ratios in an acid hydrolysate evidence has been presented that in some cases the 1,2Asp1.96 Glu1.97 Gly1.94Pro3.02Tyr0.79Arg2.01Meto.76 His1,08cycloaddition reaction of 1,2-bis(trifluoromethyl)-1,2P h e ~ . ~ ~ ~ e r ~ . average ~ ~ L y recovery s ~ . ~ ~ ;88 %) exhibited dicyanoethylene to vinyl ethers occurs via an intera single spot on thin layer chromatography (Rf30.46) mediate, as indicated by the loss of stereospecificity, and behaved as a single component on paper electroWe wish now to report spectral and chemical eviphoresis in pyridine acetate buffers at two different dence for a two-step polar 1,2-cycloaddition reaction pH values (3.5 and 6.8). of heterocarbon double-bond systems which occurs The MSH potencies (expressed as MSH units/ in the addition of arenesulfonyl isocyanates to dialkylgram) of the synthetic peptides, determined according carbodiimides. For example, on addition of areneto Shizume, et al., l8 using frog skins from Rana pipiens, sulfonyl isocyanates to dialkylcarbodiimides in benzene were as follows: 111, 6.0 X IO6; IV, 1.9 X IO6; V, 1.8 or carbon tetrachloride an immediate reaction occurs, X lo8; VI, 2.2 X 10l2; VIII, 2.0 X IO9; and synthetic as evidenced by the appearance of two double-bond I, 2.5 X 10'0 (lit.19 natural monkey fl-MSH, 3-5 X absorptions at 1869 (medium) and 1724 cm-' (strong), IO9). It is noteworthy that the partially protected respectively, which gradually disappear as the reaction pentadecapeptide VI is nearly as active as the best progresses. The formation of an intermediate prodpreparation of natural a-MSH20 and addition of the uct can also be observed by nmr spectroscopy. acidic tripeptide to this pentadecapeptide (VI) causes On mixing t-butylmethylcarbodiimide and p-toluenesome detrimental effect as far as MSH activity is consulfonyl isocyanate in carbon tetrachloride the Ncerned. methyl signal of the carbodiimide is shifted from 2.9 The structurally related fl-MSH from bovine origin to 3.32 ppm, indicating attachment of the tosyl isowas synthesized by Schwyzer, et al. 2 1 This hormone cyanate to the less hindered nitrogen adjacent to the (16) G . W. Anderson, J. E. Zimmerrnan, and F. M. Callahan, J. Am. methyl group. The N-methyl signal at 3.32 ppm Chem. SOC..86. 1839 (1964). gradually decreases and new N-methyl signals appear (17) H. Yajima, K. Kawasaki, Y.Okada, H. Minami, I
