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
