Nov. 5, 1961
SYNTHESIS OF BIOLOGICALLY ACTIVENONADECAPEPTIDES
[CONTRIBUTION FROM THE
HORMONE RESEARCH LABORATORY, UNIVERSITY OF
44.49
CALIFORNIA, BERKELEY, CALIF.]
Synthesis of a Biologically Active Nonadecapeptide Corresponding to the First Nineteen Amino Acid Residues of Adrenocorticotropins'" BY
CHOH
HAOLI, JOHANNES MEIENHOFER, EUGEN SCHNABEL, DAVIDCHUNG, TUNG-BIN LO AND JANAKXRAMAN RAMACHANDRAN RECEIVED APRIL6, 1961
The synthesis of a nonadecapeptide, ~-seryl-~-tyrosyl-~-seryl-~-methionyl-~-glutmyl-~-histidyl-~-phenylalan~-~arginyl-~-tryptophyl-glycyl-~-lysyl-~-prolyl-~-valyl-glycyl-~-lysyl-~-lysyl-~-arginyl-~-arginyl-~-proline, which has an amino acid sequence identical with the first 19 residues from the NH2-terminus of adrenocorticotropin ( A C T H ) , is described. The synthetic nonadecapeptide has been shown by bioassay t o possess high ACTH as well as melanotropic (MSH) activities.
Adrenocorticotropic hormone (ACTH) in highly purified form has been isolated from sheep,lb pig2 and beef pituitaries. The complete structures of the p ~ r c i n e ovine6 ,~ and bovine6 hormones, which are straight-chain polypeptides composed of 39 amino acids, have been established. In Table I , which summarizes the known structural data on the hormones isolated from these three species, differences in composition and in the arrangement of amino acids at positions 25-33 are evident among the three. From these data, i t can be inferred that the removal of that portion of the COOHterminal sequence comprising positions 25-39 would not impair the adrenal-stimulating activity. Indeed, in the late 1940's i t already was suspected that the active portion of ACTH was a relatively small polypeptide, after it had been demonstrated that digestion of the ACTH protein with pepsin did not destroy its activity,l and now there is some experimental evidence4 to indicate that the biological potency of ACTH probably resides in the core consisting of the first 24 amino acid residues in the whole peptide molecule. It is obvious, however, that the minimal structural requirement for activity in the ACTH polypeptide molecule can be established with certainty only by the chemical synthesis of a peptide possessing adrenocorticotropic activity. I n this paper is presented an account of the synthesis of a biologically active nonadecapeptide, L-seryl-L-tyrosyl-Lseryl - L methionyl - L glutamyl - L - histidylL - phenylalanyl - L - arginyl L - tryptophylglycyl - L - lysyl - L - prolyl - L - valyl glycyl - Llysyl-L-arginyl-L-arginyl-L-proline (V), which has an amino acid sequence identical with the first 19
-
-
-
-
(1) (a) This Communication is Paper X X I V of the adrenocorticotropins (ACTH) series; (b) C. H. Li, I. I. Geschwind, A. L. Levy, J. I. Harris, J. S. Dixon, N. G. Pon and J. 0. Porath, Nature, 178, 251 (1964); C . H. Li, I. I. Geschwind, J. S. Dixon, A. L. Levy and J. I. Harris, J . Biol. Chem.,213, 171 (1955). (2) P. H. Bell, J . A m . Chcm. Soc., 76, 5565 (1954); R. G. Shepherd, K . S. Howard P. H. Bell, A. R . Cacciola, R . G. Child, M. C. Davies, J. P. English. B. M. Finn, J. M. Meisenhelder, A. W. Moyer and J. van der Scheer, ibid., 78, 5051 (1956). (3) C. H. Li and J. S. Dixon, Science, 124, 934 (1956). (4) K.9.Howard, R. G. Shepherd, E. A. Eigner, D. S. Davies and P. H. Bell, J . A m . Chem. Soc., 77,3419 (1955); R. G. Shepherd, S. D. Wilson, R. S. Howard, P. H. Bell, D . S. Davies, 9. B. Davis, E. A. Eigner and N. E. Shakespeare, i b i d . , 78, 5067 (1956). (5) C. H. Li, I. I. Geschwind, R. D . Cole, I. D . Raacke, J. I. Harris and J. S. Dixon, Nature, 176, 687 (1955); J. Leonis, C. H. Li and D . Chung. J . A m . Chem. Soc., 8 1 , 419 (1959). (0) C. H . Li. J. S. Dixon and D. Chung, ibid., 80, 2587 (1958); Biochem. Biophyr. A d a , 46, 324 (1961). (7) C . H. Li, Conference on Metabolic Aspects of Convalescence. Josiah Macy, Jr. Foundation, N. Y . , 17th Meeting, 1948, p. 114.
residues from the "2-terminus of ACTH. A short account of the synthesis has already appeared.' The nonapeptide Na-carbobenzoxy-Ne-tosyl-Llysyl -L -prolyl-L- valyl -glycyl - Ne- tosyl-L-lysyl-Netosyl- L-lysyl - NG-tosyl-L-arginyl-NG-tosyl-L-arginyl-L-proline methyl ester(1d) was prepared by couplingthep-nitrophenyl ester Ibof the protected tetrapeptide Na-carbobenzoxy-Ne-tosyl-L-lysyl-L-prolylL-valyl-glycine with the product which was obtained by catalytic hydrogenation of the protected pentapeptide Na-carbobenzoxy-Ne-tosyl-L-lysyl-N'tosyl - L lysyl - NG-tosyl-L-arginyl-NG-tosyl-L-arginyl-L-proline methyl ester (IC). Peptide IC was synthesized by the stepwise lengthening of the chain one amino acid a t a time starting from the COOH-terminal proline methyl ester. A similar stepwise procedure performed by the nitrophenyl ester methodg has been employed recently for the syntheses of oxytocinlO and lysinevasopressinll and their 2-phenylalanine analogs. l2 In our synthesis we employed dicyclohexylcarbodiimide coupling13during the first two steps, since we did not succeed in preparing a stable nitrophenyl ester of Na-carbobenzoxy-NG-tosyl-L-arginine.It was thought that this nitrophenyl ester might possibly be formed when Na-carbobenzoxy-NG-tosylL-arginine was treated with p-nitrophenol in the presence of dicyclohexylcarbodiimide13~ 14; however, only the corresponding lactam could be isolated.16 This lactam formation was also reported when peptide synthesis was performed with dicarbobenzoxy-L-arginine'B by the dicyclohexylcarbodiimide method. For the last two steps in the preparation of the protected pentapeptide IC, Na-carbobenzoxy-Netosyl-L-lysine p-nitrophenyl ester" was employed. The dipeptide intermediate (Na-carbobenzoxyNO-tosyl-L-arginyl-L-proline methyl ester) was obtained in crystalline form. Attempts a t crystal-
-
(8) C. H. Li,J. Meienhofer, E. Schnabel, D. Chung, T. B. Lo and J . Kamachandran, J . A m . Chcm. Soc., 82, 5760 (1960). (9) M. Bodanszky, Nature, 176, 685 (1955); Acta Chim. Hung.. 10, 335 (1957); A n n . N . Y . Acad. Sci., 88, 655 (1960). (10) M. Bodanszkp and V. du Vigneaud, 1.A m . Chcm. Soc., 81,6688 (1959). (11) M. Bodanszky, J. Meienhofer and V. du Vigneaud, ibid., 82, 3195 (1960). (12) M. Bondanszky and V. du Vigneaud. ibid., 81, 1258 (1959). (13) J . C. Sheehan and G. P. He-, ibid., 77, 1067 (1955). (14) M. Rothe and F. W. Kunitz, Ann. Chcm. Liebizs, 609, 88 (1957). (115) E. Schnabel and C. H. Li, J . A m . Chem. Soc., 82, 4576 (1960). ( 1 6 ) L. Zervas, T. T . Otani, M. Winitz and J. P. Greenstein, ibid., 81, 2878 (1959).
4450
C. H. LI, J. MEIENHOFER, E.SCHNAREL, D. CHUNG,T. Lo
AND
J. RAMACHANDRAN Vol. 83
TABLE I STRUCTURAL DIFFEREBCES AMONGXDRENOCORTICOTROPISS ISOLATED FROM PIG,SHEEP AND BEEF PITUITARY GLANDS Structure of bovine ACTH
Ser-Tq-r-Ser-Met-Glu-His-Phe-Arg-Tr~-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val 1
2
3
4
5 6
7
8
9
10
11 12 13 14
15 16 17 18 19 20
*"
I L~s-Val-Tyr-Pro-Asp-Gly-Glu-Ala-Glu-Asp-Ser-~la-Glu-Ala-Ph~-Pro-Leu-Glu-Phe 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Species
Laboratory
Amino acid residue in position
25 26
27 28 29
30 31 32 33 a"
Pig
American Cyanamid C O . ~
Asp-GIy - Ala-Glu-Asp-&u-Leu- Ala-Glu 2"
I
Sheep
University of Calif.6
Ala-Gly-Glu-Asp-Asp-Glu-Ala-Ser-Glu NHz
Beef
University of Calif.6
Asp-Gly-Glu-Ala-Glu-4sp-Ser- Ala-Glu
I
lization of the other intermediates have so far fied protected pentapeptide ICafter hydrogenation proved unsuccessful; consequently, countercur- with the tetrapeptide nitrophenyl ester Ib. rent distribution has been used a t various stages The nonapeptide I was hydrogenated cataof the synthesis for the purification of the inter- lytically and then allowed to react with the hexamediates. Na-carbobenzoxy-~-benzyl-L-glutamylpeptide The protected tetrapeptide IYa-carbobenzoxy- Im - benzyl - L - histidyl - L - phenylalanyl - NGNe - tosyl - L - lysyl - L - prolyl - L - valyl - glycine tosyl-L-arginyl-L-tryptophyl-glycine(11) via the (Ia) was synthesized by coupling crystalline L- mixed anhydride procedure with isobutyl chloroprolyl-L-valyl-glycine methyl ester with carbo- formate. l8 The resulting pentadecapeptide I11 benzoxy-N'-tosyl-L-lysine by means of the p- was then purified by countercurrent distribution nitrophenyl esterg method. Carbobenzoxy-L-pro- in the system consisting of toluene-chloroformlyl-L-valine was allowed to react with glycine methanol-water (5 :5 :8 :2 by volume) for 242 transmethyl ester, with dicyclohexylcarbodiimide as fers. The protected pentadecapeptide I11 was isothe condensing agent ; the resulting protected lated from those tubes falling within the theoretical tripeptide, carbobenzoxy-L-prolyl-L-valyl-glycine distribution curve corresponding to the main peak methyl ester, I7 was obtained in crystalline form which had a distribution coefficient ( K )of 0.34. and in high yield. The protected tripeptide ester Two slightly different routes of synthesis were was hydrogenated to give a free crystalline tri- used in the preparation of peptide 11. For the peptide ester. This material was allowed to react first, Na-carbobenzoxy-NG-tosyl-L-arginyl-Lwith Na-carbobenzoxy-N'-tosyl-L-lysinep-nitro- tryptophylglycine methyl ester15 was subjected phenyl ester in ethyl acetate to give the crystalline to catalytic hydrogenation, and the product was protected tetrapeptide ester. Saponification of then allowed to react with Nu-carbobenzoxy-Imthis tetrapeptide derivative yielded crystalline benzyl-L-histidyl-L-phenylalanine 9-nitrophenyl Ne - carbobenzoxy - N' - tosyl - L - lysyl - L- ester. The resulting protected pentapeptide ester prolyl-L-valyl-glycine (la) in 93% yield. The was saponified and the resulting acid then was subp-nitrophenyl ester of I a was prepared with the use jected to catalytic hydrogenation; the hydroof dicyclohexylcarbodiimide13~14; the product was genated product next was coupled with the p-nitroobtained in crystalline form in good yield. phenyl ester of carbobenzoxy--y-benzyl-~-glutamic Preparation of Nm-carbobenzoxy-Nf-tosyl-L-ly- acid. The second route of the synthesis of peptide syl - L - prolyl - L - valyl - glycyl - Nf - tosyl - L- I1 differs from the first only in the saponification lysyl - Ne - tosyl - L - lysyl - NG - tosyl - L - arginyl- step. For this step, the Nu-carbobenzoxy-NGNG-tosyl-L-arginyl-L-proline (I) by saponification tosyl-L-arginyl-L-tryptophyl-glycinemethyl ester of the protected nonapeptide ester Id presented was saponified, hydrogenated and then condensed some difficulties. If the alkali was not allowed to with carbobenzoxy-Im-benzyl-L-histidyl-L-phenylreact long enough, the saponification was incom- alanine p-nitrophenyl ester. This method was used plete. On the other hand, prolonged reaction with in order to avoid degradation of the protected the alkali caused considerable damage to the pentapeptide ester during saponification; morematerial, so that increasing amounts of decomposi- over, the tripeptide acid is easier to purify than tion products were obtained. We found that the the pentapeptide acid. The remainder of the synmost satisfactory procedure to follow was to allow thesis was carried out in the same manner as in the the alkali to react for one or two hours and sub- first procedure. sequently to separate the ester and the acid either The protected pentadecapeptide I11 was subby extraction or countercurrent distribution; the mitted to catalytic hydrogenation and the product unreacted ester was then re-saponified in the same was allowed to react with peptide IV, Nu-carbomanner. The protected nonapeptide acid I also benzoxy - L - seryl - L - tyrosyl - L - seryl - L - methiowas prepared in good yield by coupling the saponi- ninhydrazide, by the azide procedure. Peptide I V was obtained by the condensation of carbobenzoxy(17) K Hofmann E Sturz, G Spuhler, H Yajima and E T S c h n d r t z J A m Chrvi For
82, 3727 (19GO).
(18) J R Vaughan Jr
and K. L Osatn, ibid , 7 4 , 676 (1952).
Nov. 5, 1961
SYNTHESIS OF BIOLOGICALLY ACTIVENONADECAPEPTIDES
4451
Tos Tos Tos Tos
Tos
I
I
l
l
1
I, 2-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-ProOH OBzBz Tos 2, Z-Glu-I!Iis-Phe-Arg-Try-GlyOH, I ( CH&CHCHzOCOCl
I1 OBzBz Tos Tos Tos Tos Tos Tos 111, Z - G I l u -1 H i s - P hIe - A r g - T r y - G l y - ~ ~ ~ s - P r o - ~I a l -I~ l y -I ~ y sI- ~ y s - A ~ g - ~ r g - P r o O H
2, 2-Ser-Tyr-Ser-MetNHhTH2, HKOz IV Tos
Tos
I
I
Tos Tos Tos Tos
I
I
I
I
Z-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Try-Gly-Lys-Pro-~'al-Crl~-Lys-Lys-Arg-Arg-ProOH
4 Na, I
liq. NH,
V, H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Try-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-ProOH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Fig. l.--Outline of the synthesis of ~-serpl-~-tyrosyl-~-seryl-~-methionyl-~-glutamyl-~-histidyl-~-phenylalanyl-~-argin~l~-tryptophyl-glycyl-~-lysyl-~-prolyl-~-valyl-glycyl-~-l~syl-~-lysyl-~-argin~~l-~-arginyl-~-proline; Z, carbobenzoxy; Bz, benzyl; Tos, p-toluenesulfonyl.
L-seryl-L-tyrosine azide with L-seryl-L-methionine methyl ester similar to the procedure employed by previous workers. l9 The protected nonadecapeptide was treated with sodium-liquid ammoniaN; the crude nonadecapeptide was desalted and then purified by countercurrent distribution in two solvent systems. Figure 1 presents an outline of the synthetic steps involved in the preparation of the nonadecapeptide. Even though countercurrent distribution was used extensively for purification of the intermediates in the course of our synthesis, the final products were still difficult to obtain in crystalline form. In only one instance, peptide 11, was crystallization achieved after purification by countercurrent distribution. In Table I1 are given the distribution coefficients of the large peptide fragments obtained during the synthesis.
acid is quite likely to be racemized in the course of the reactions that are carried out by means of the mixed anhydride procedurela and the p-nitrophenyl ester method.g I n the preparation of the nonapeptide I, the p-nitrophenyl ester of the Nucarbobenzoxy - Ne - tosyl - L - lysyl - L - prolylL-valyl-glycine was allowed to react with the pentapeptide NE-tosyl-L-lysyl-NE-tosyl-L-lysyl-NGtosyl - L - arginyl - NG - tosyl - L - arginyl - L -proline. For the synthesis of the pentadecapeptide 111, the mixed anhydride of the hexapeptide 11, which has glycine as carboxyl terminal residue, was used for reaction with the hydrogenated peptide I. Finally, since there is no evidence that azide coupling involves any racemization,22 this procedure was used to link the final tetrapeptide IV to the hydrogenated pentadecapeptide I11 to form the protected nonadecapeptide V. During the preparation of the various peptide TABLE I1 fragments, much effort was directed toward obDISTRIBUTION COEFFICIENTS ( K ) OF VARIOUSSYNTHETIC taining crystalline intermediates in the hope that PEPTIDE s it will reduce the amount of racemized product. Peptidea Solvent systemb K In those peptides where arginine was present, howI Toluene 0.26 ever, attempts a t crystallization were unsuccessful I1 Carbon tetrachloride .I6 except for the dipeptide Na-carbobenzoxy-NGI11 Toluene .34 tosyl-L-arginyl-L-proline methyl ester, and the IV Pyridine .08 hexapeptide 11. V 2-Butanol .58 In the solvent system consisting of 2-butanola See Fig. 1. * Toluene, CHC13-CBHSCH3-CHaOH0.5% trichloroacetic acid, peptide V distributed in HzO ( 5 : 5 : 8 : 2 ,by volume); carbon tetrachloride; CHClaaccordance with the theoretical curve for a partiCClr-CHaOH-Hz0 (3: 1:3:1, by volume); pyridine, 0.1% acetic acid-1-butanol-pyridine (11:5 :3, by volume); 2tion coefficient of 0.58. Peptide V also was subbutanol, 2-butanol-0.5Yo trichloroacetic acid. jected to amino acid analyses by both the paperfluorodinitrobenzene and the chromatoIn order to avoid the danger of racemization graphic procedure2j as well as by spectrophotoduring the synthesis, we planned the coupling metric methodzEfor the quantitativea determination reactions to involve optically inactive carboxyl terminal glycine (as has been done in the synthesis of tyrosine and tryptophan ; these analyses gave of melanocyte-stimulating hormone derivatives21), (22) M. Goodman and 0. W. Kenner, Advances i n Prolein C h e m . , since i t is known that any carboxyl terminal amino 12, 465 (1957). (19) K. Hofmann, A. Jlihl, A. E. Furlenmeier and H. Kappeler. J . A m . Chem. Soc., 79, 1636 (1957). (20) V. du Vigneaud and 0. K. Behrens, J . Biol. Chem., 117, 27 (1937) (21) R . Hofmann, A n n . N. Y. Acod. Scz., 88, G89 (1960)
(23) F. Sauger, Biochcm. J . , 39, 507 (1945). (24) A. L. Levy, Nature, 174, 126 (1954). (25) D. H. SpacZman, W. H. Stein and S. Moore, Anal. Chcm., SO, 1190 (1958). (26) T. W. Goodwin and R. A. Morton, Biockem. J., 40, 628 [ 1946).
4452
c. H.
LI, J.
~ ~ E I E I \ " O F E R ,E. S C H N A B E L ,
D. CHUNG,T .
L O AND
J. RAMACHANDRAN VOl. 83
the composition in molar ratios which are consistent with the theoretically calculated values (mol. wt. 2346.7)
a 95% confidence limit for the assay in the range of 3 3 4 9 . When the synthetic nonadecapeptide was tested for adrenocorticotropic activity by the Ser~Tyr~Met~Glu~His~PhelArg~Try,Gly,Lys,Pro2Valladrenal ascorbic acid depletion method, 39 it was found to have a potency of 74 U.S.P. units per nig. Successive hydrolysis of peptide V by trypsin, if injections were given subcutaneously, and a chymotrypsin and leucine aminopeptidase caused potency of 35 units per mg. by the intravenous complete degradation of the peptide V to its con- route. It may be r e ~ a l l e d ' - ~ ~ that * ~ . ~ native ~ stituent amino acids with the exception of the adrenocorticotropins from various species possess arginyl-proline bond a t the C-terminus. Ap- an ACTH activity of 80-150 U.S.P. units per mg. parently, the arginylproline linkage is not only as estimated by both in vivo and in vitro methods. resistant to tryptic digestion as is the case with Thus, peptide V, which has an amino-acid sequence the adrenocorticotropins4-6 but also is unaffected corresponding to the first 19 amino acid residues of by leucine aminopeptidase. the adrenocorticotropins, has approximately 50% It is well that adrenocortico- the ACTH potency of the native hormone. It will tropins possess an intrinsic melanocyte-stimulating be of interest to ascertain the minimal structural activity. Indeed, one of the melanocyte-stimulat- requirement for full adrenocorticotropic potency ing hormones, a-MSH, from pig28 and horsez9 and to obtain synthetic peptides which possess pituitaries, consists of 13 amino acids and contains either minimal ACTH activity, or none a t all, but a sequence identical with ACTH except that the which exercise other physiological functions ina-amino group is blocked with an acetyl residue cluding lipolysis, adipokinesis, erythropoiesis, etc. and the C-terminus with an amide. The synthesis Experimental4' of a-MSH in its natural form has been achieved N4hrbobenzoxy-NG-tosyl-L-arginyl-L-proline Methyl recently by Guttman and BoissonnasIaO and its methyl ester (7.0 g.), prepared from a glutaminyl and formyl-lysyl analog has been Ester.-I,-Proline solution of L-proline methyl ester hydrochloride," was synthesized by Hofmann, et aLal Moreover, a dissolved in acetonitrile (50 ml.) along with Na-carboheptapeptide sequence, L-methionyl-L-glutamyl- benzoxy-No-tosyl-Gargininelb (23.3 g.). The solution was cooled to 0' before the addition of dicyclohexylcarbodiimidz L - histidyl - L - phenylalanyl - L - arginyl - L(12.5 g.). After being allowed to stand for 6 hours at 0 tryptophyl-glycine, which occurs in preparations and 40 hours a t room temperature, the heavy precipitate of ACTH as well as in the synthetic nonadeca- (dicyclohexylurea) was filtered off and washed once with hot peptide, forms a part of the structure of P-MSH acetonitrile and twice with boiling acetone. When the solufrom pig, a 2 # 3 3 beefa4and humana6pituitary glands. tion was concentrated in wcuo, the product crystallized in the form of prisms. From the mother liquor, more of It was therefore to be expected that peptide V the protected dipeptide was isolated by evaporating to should exhibit melanocyte-stimulating activity. dryness, dissolving the material in ethyl acetate, and washing By the in vitro frog skin assay procedure36peptide in the usual manner with acid and base; yield 20.6 g. (72%), V was shown to possess an MSH activity of 1.4 m.p. 151-152'. A sample was recrystallized from much X lo7 units per gram. Moreover, a single dose of ethylacetate; m.p. 152-153', [ c I ] ~ ~ D-39.0' (c 1, methanol). d . Calcd. for CnHsaO,NaS (573.7): C, 56.5; H , 6.15; 0.1 microgram of the peptide caused a change in N,A12.2; S, 5.59. Found: C, 56.8; H, 6.01; N, 12.0; S, melanophore index37 in hypophysectomized R a m 5.42. Pipiens of from 1+ to 3+ within one hour. It is N-Carbobenzoxy-N G-tosyl-L-arginyl-No-tosyl-L-arginylthus clearly evident that peptide V has an MSH L-proline Methyl Ester.-Na-Carbobenzoxy-NQ-tosyl-Larginyl-i-proline methyl ester (19.2 9 . ) was suspended in potency comparable to that of the native adreno(150 ml.) and concentrated hydrochloric acid corticotropins. As far as we are aware, this is the methanol (3 ml.) containing palladium black (freshly prepared from first synthetic peptide which possesses both adreno- 4 g. of PdC12). Hydrogen was passed through the solution corticotropic and melanotropic activities in high which was stirred vigorously with a Vibro-Mixer." After potency; hence, i t represents the most convincing 4 hours the catalyst was filtered from the clear solution and the solvent was removed by evaporation in vacuo. The evidence yet obtained in favor of intrinsic melano- residual colorless oil was dissolved in acetonitrile (100 ml.), cyte-stimulating activity in adrenocorticotropins. and hTa-carbobenzoxy-NG-tosyl-L-argininelS(15.5 g.) was According to the results of bioassay by the in added along with triethylamine ( 5 nd.). The solution was vitro adrenal method, peptide V was found to have cooled and dicyclohexylcarbodiimide (8.25 g.) was added. being allowed to stand for 6 hours a t 0' and for 34 an ACTH activity of 40 U.S.P. units per mg. with After hours at room temperature, the precipitated dicyclohexyl(27) C. H. Li, P. Fgnss-Rech, I. I. Geschwind, T. Hayashida, 0 . Hungerlord, A. J. Lostroh, W. R. Lyons, H. D. Moon, W.0. Reinhurdt and M. Sideman, J. E r p . Mcd., 106, 335 (1957). (28) J. I. Harris and A. B. Lerner, Nature, 179, 1346 (1957). (29) J . S. Dixon and C. H. Li, J. Am. Chcm. Soc., 82,4568 (1960). (30) St. Guttmann and R. A. Boissonnas, Efelv. Chim. A d a , 42, 1257 (1959). (31) K. Hofmann, H. Yajima and E. T. Schwartz, J. Am. Chcm. Soc., 82, 3732 (1960). (82) J. I. Harris and P. Roos, Nafurc. 178, 90 (1956); Biochcm. J . . 71, 434 (1959). (33) I. I. Gcschwind, C. H. Li and L. Barnafi, J. Am. Chcm. Soc., 1 8 , 4494 (1956); 79, 620 (1957). (34) I. I. Geschwind, C. H. Li and L Bsrnafi. ibid., 79, 6394 (1857). (35) J. I. Hurris, Nafurc, 184, 167 (1959). (36) K. Shizume, A. B. Lerner and T. B. Fitzpatrick, Endacrinol., 64, 553 (1954). (37) L. T. Hogben and D. Slome, Proc. Roy. SOC.(London), BlOB, 10 (1931). (38) M. Saffran and A. V. Schally, E n d o c r i n d . , 66, 523 (1955).
urea was removed by filtration and the solvent was removed by evaporation in wacuo, leaving a yellow oil, which was dis(39) M. A. Sayers, G. Sayers and L. A. Woodbury, i b i d , 42, 379 (1949). (40) R. Guillemin, ibid., 66, 819 (1960). (41) Melting points were performed on a Fisher-Johns melting point apparatus and are uncorrected. Microanalyses were performed by the Microanalytical Laboratory, Department of Chemistry, University of California, Berkeley. Ail samples for microanalyses were dried in an Abderhalden drying pistol with PIO, a t 77' fur 16 hours a t 0.3 mm. pressure. Paper chromatography was carried out on Whatman No. 1 filter paper a t room temperature; the solvents used were 1-butanol-acetic acid-water (BAW) in a ratio of 4 : l : l and sccbutyl alcohol-1070 N H I (SBA) in a ratio of 85: 15; the location of the peptide .pot was either revealed by the ninhydrin reagent or the chlorine method.'# (42) H. Zahn and E. Resroth, Z. anal. Chem., 148, 181 (1956). (43) Vihro-Mixer, A. G. Fuer chemic-Apparatehau, Zurich, Model El.
Nov. 5 , 1961
SYNTHESIS OF BIOLOGICALLY ACTIVENONADECAPEPTIDES
4453
solved in ethyl acetate. The solution was washed in the in ice-cold saturated potassium carbonate solution (50 ml.) usual manner with acid and base and dried over MgSO4. and extracted with three 50-ml. portions of dichloromethane. The product, which was obtained by evaporation of the The dichloromethane extract was dried over anhydrous sodium sulfate and the dried material was added to a solusolvent, was isolated by precipitation from acetone-ether. in dichloromethane The product was obtained in the form of a white amorphous tion of carbobenzoxy-~-prolyl-~-valine~~ powder, yield 21.5 g. (73.5%), m.p. 112-120", [ ( Y ] ~ ~ D(17.4 g. in 150 ml.). The solution was cooled to 0" and dicyclohexylcarbodiimide (10.4 g.) was added. This solu-31.0' (c 1, methanol). The product was further purified by countercurrent dis- tion was allowed to stand for 15 hours a t 5' and the dicyclotribution in the solvent system methanol-water-chlorohexylurea that formed then was filtered off and washed with form-carbon tetrachloride (4: 1:5:5). After 240 transfers dichloromethane. The combined filtrate and washings two peaks were well separated. The first peak with K = were washed successively with 0.1 N HC1, water, 1 M 0.17 represented less than 5% of an impurity, whereas the ",OH and water and dried over anhydrous sodium sulfate. second peak with K = 0.41 followed closely the theoretical The solution was concentrated to a volume of 150 ml. and distribution curve and represented the tripeptide derivative an equal volume of petroleum ether (b.p. 30-60") was which was recovered in SO% yield, m.p. 115-120", [ c Y ] * ~ D added to yield a product in crystalline form; yield 19 g. (goyo), m.p. 109-110". A sample was recrystallized for -32.Oo(c 1, methanol). Anal. Calcd. for C,oHuOioN&z (884.0): C, 54.3; H , analysis from dichloromethane-petroleum ethef; m.p. 111-112', [cY]~~D -90.4' ( c 1.6, methanol); in paper 6.04; N, 14.3; S, 7.25. Found: C, 54.3; H, 6.21; N, chromatography, Rr BAW = 0.81 and Rr SBA = 0.79. 14.4; S, 7.10. NeCarbobenzoxy-Nt-tosyl-L-lysyl-NQ-tosyl-L-arginyl-NQ- Anal. Calcd. for C21HZpOeNa (419.5): C, 60.1; H, 6.97; tosyl-L-arginyl-L-proline Methyl Ester.-The tripeptide N, 10.0. Found: C, 60.0; H , 6.80; N, 10.1. Na-Carbobenzoxy-N~-tosyl-L-lysyl-L-prolyl-L-v~ylbase was prepared from N-carbobenzoxy-NQ-tosyl-Larginyl-NQ-tosyl-arginyl-L-proline methyl ester (19.0 g.) glycine Methyl Ester.-Carbobenzoxy-L-prolyl-L-valylby hydrogenation as described above and dissolved in ace- glycine methyl ester (14.0 g.) was dissolved in 150 ml. of tonitrile (110 d . ) ; to this solution was added Xa-carbmethanol, and to this solution, palladium-black (freshly prepared from 3 g. PdC12) was added. Hydrogen wzs benzoxy-Ne-tosyl-L-lysine p-nitrophenyl ester" (12.0 g.). After the mixture had been allowed to stand for 40 hours at passed through the solution while it was being rapidly room temperature, the solvent was evaporated in vacuo. stirred by a Vibro Mixer.43 After 8 hours the catalyst was The residual oil was dissolved in acetone (100 ml.) and the removed by filtration and the solvent removed in vacuo. solution was slowly poured into ether (900 ml.) with vigor- The methyl ester of the tripeptide base crystallized in the ous stirring. This precipitation procedure was repeated form of needles, m.p. 140-142'. The needles were immediately dissolved in ethyl acetate (100 ml.) along with twice, yielding 21.0 g. (84?Jo) of a white amorphous powder, Na-carbobenzoxy-Ne-tosyl-L-lysine p-nitrophenyl ester" m.p. 110-115", [ c Y ] * ~ D-30.5'(c 1,methanol). Anal. Calcd. for C H H ~ ~ O ~ S (1166.4): N ~ ~ S ~C, 54.6; H , (18.9 g.). After this mixture had been allowed to stand 6.14; N, 13.2; S, 8.26. Found: C, 54.8; H, 6.33; N, for 60 hours a t room temperature, a gelatinous precipitate formed. This precipitate was dissolved by the addition 13.1; S, 8.05. of more ethyl acetate (400 ml.) with slight warming. The N~-Carbobenzoxy-N~-tosyl-L-lysyl-N~-tosyl-L-lysyl-NQsolution was washed successively with 0.75 N ammonia tosyl-L-arginyl-No-tosyl-L-arginyl-L-proline Methyl Ester.The free tetrapeptide base was prepared from Na-carbo- (5 times), water, 1 N hydrochloric acid, and water, and dried benzoxy-Ne-tosyl -L - lysyl NQ tosyl L - arginyl- No tosyl- over magnesium sulfate. Concentration to a small volume (30 ml.) yielded a product which crystallized in the form arginyl-L-proline methyl ester (1.99 g.) by hydrogenation of needles. Recrystallization from ethyl acetate gave a as described above and dissolved in acetonitrile (5 ml.); yield of 17.6 g. (76y0) Na-carbobenzoxy-Ne-tosyl-L-lysylto this solution was added Na-carbobenzoxy-Ne-tosyl-LL-prolyl-L-valyl-glycine methyl ester in the form of white lysine $-nitrophenyl ester" (1.14 g.). After the mixture needles, m.p. 139-140', [ C Y ] % -79.0' (c 1, methanol); had stood for 45 hours a t 35', the solvent was evaporated in vacuo and the residual material was purified by three in paper chromatography, Rr BAW = 0.91 and Rr SBA = 0.73. successive precipitations from acetone-ether. The product Anal. Calcd. for Ca4Hl,0gNaS (701.8): C, 58.2; H, was obtained in the form of a white amorphous powder, yield 2.24 g. (91%), m.p. 108-113", [ ( Y ) ~ ~-29.0' D (c 1, 6.75; N, 9.98; S, 4.56. Found: C, 58.3; H, 6.69; N, 9.91; s, 4.79. methanol). Anal. Calcd. for GH89016N& (1448.7): C, 54.7; H , N~-Carbobenzoxy-N~-tosyl-L-lysyl-L-prolyl-L-valyl6.19; N, 12.6; S, 8.85. Found: C, 54.5; H, 6.19; N, glycine.-The protected tetrapeptide ester (4.9 9.) was dis12.7; S,8.84. solved in acetone (15 ml.). The solution was cooled in ice The compound withstood all attempts a t crystallization. and 2 N sodium hydroxide ( 7 ml.) was added dropwise over In order to check its purity, a sample was subjected to a period of 20 minutes. After being stirred for 1 hour a t countercurrent distribution in the solvent system chloro- room temperature the solution was acidified with dilute form-toluene-methanol-water (5: 5 :8:2). After 405 trans- hydrochloric acid. The crystalline precipitate was recrystalfers, the distribution followed closely the theoretical curve lized from ethyl acetate which had been saturated with water; yield 4.46 g. (93%) of white needles, m.p. 109-llOo, of K = 0.45. [a]"D -73.0' ( c 1, methanol); in paper chromatography, N~Carbobenzoxy-N~-tosyl-L-lysyl-N~-tosyl-L-lysyl-NQtosyl-L-arginyl-NQ-tosyl-L-arginyl-L-proline.-The protected Rr BAW = 0.78 and Rr SBA = 0.38. pentapeptide ester (3.15 9.) was dissolved in acetone (10 Anal. Calcd. for C J ~ H ~ ~ O(687.8): ~ N ~ S C, 57.6; H , ml.) and the solution was cooled in ice; 2 N sodium hy- 6.60; N, 10.2; S, 4.66. Found: C, 57.5; H, 6.59; N, droxide (2.1 ml.) was added dropwise over a period of 20 10.4; S, 4.60. minutes. The solution was stirred for 50 minutes longer a t N~-Carbobenzoxy-Nc-tosyl-L-lysyl-~prolyl-L-valylroom temperature. It then was acidified with 1 N hydro- glycine p-Nitrophenyl Ester.-NaCarbobenzoxy-Ne-tosylchloric acid (5 ml.) and the solid precipitate was filtered, L-lysyl-L-prolyl-L-valyl-glycine (4.82 9.) was dissolved in washed thoroughly with water, and dried; yield 3.1 g. ethyl acetate (30 ml.) along with p-nitrophenol (1.07 g.). The material was subjected to countercurrent distribution The solution was cooled to 0' and dicyclohexylcarbodiin the system consisting of chloroform-toluene-methanolimide (1.59 9.) was added. After the mixture had been water (5:5: 8 : 2). After 348 transfers, two well-separated stirred for 1 hour a t 0' and for 5 hours a t room temperature, major peaks were obtained with values for K of 0.47 (un- acetic acid (0.1 ml.) was added and the almost solidified saponified ester) and 0.93 (acid). Decomposition impuri- mass was diluted with ethyl acetate (100 ml.). After the ties traveled faster; recoveries: 50% acid, 25% ester, 10% diluted mixture had been heated to the boiling point, the other impurities. The acid (1.57 9 . ) was obtained in the insoluble dicyclohexylurea was removed by filtration and form of an amorphous white powder, m.p. 124-126', was then washed repeatedly with boiling ethyl acetate. [nl%. -28.4' (c 1,methanol). _ From the combined filtrate and washings, the solvent was Anal. Calcd. for C&HmO;sNlpS4 (1434.7): C, 54.5; H , removed by evaporation in vacuo and the residue was crystal6.11; N, 12.7; S, 8.94. Found: C, 54.3; H, 6.12; N, lized in the form of white prisms from absolute ethanol (70 12.6; S, 8.76. ml.) to which a few drops of acetic acid had been added; Carbobenzoxy-L-prolyl-L-valyl-glycine Methyl Ester.Glycine methyl ester hydrochloride (9.42 9.) was dissolved (44) R . L.M. Synge, Biochem. J . , 48, 89 (1948).
-
-
-
-
yield 5.16 g. (91%), m.p. 152-153', CY]^'.^^) -40.5' ( C 2, tliinethylformamide). Anal. Calcd. for CS9H4&N& (808.9): C, 57.9; 13, 5.98; N, 10.4. Found: C,58.0; H,6.08; N, 10.4.
dissolved in acet.onitrile (5 ml.) with 0.05 inl. of triethylamine; to this solution was added Ne-carbobenzoxy-Netosyl-L-lysyl-L-jprolyl-L-valyl-glycine nitrophenyl ester (178 mg.). After the mixture had been allowed to stand for 45 hours a t room temperature, the solvent was removed i i z N~-Carbobenzoxy-N~-tosyl-L-lysyl-L-prolyl-L-valylvacuo and the product purified by two successive precipitaglycyl-N~-tosyl-L-lysyl-N~-tosyl-~-lysyl-N~ - tosyl - L - arginyl- tions from acetone-ether; yield 258 mg. (91c/o) of n white NG-tosyl-L-arginyl-L-proline Methyl Ester.--The free base of the pentapeptide ester was prepared from Na-carbobenz- amorphous powder, m.p. 185-137', [ojz5.%-37.4' (c' 1, oxy-Ne-tosyl-L-lysyl-N~-tosyl-L-1ysyl- No - tosyl - L - arginpl- methanol). Carbobenzoxy-Im-benzyl-L-histidyl-L-phenylalanine NG-tosyl-L-arginyl-L-proline methyl ester (4.35 9.) by hydrogenation as described above and dissolved in acetonitrile p-Nitrophenyl Ester.-Carbobenzoxy-Im-benzyl-L-histidylL-phenylalanine'j (3.71 g.), was suspended in 150 nil. of (30 rnl.); t o this solution was added Na-carbobenzoxy-Neacetonitrile; p-nitrophenol (1.17 g.) was added and the tosyl-L-lysyl-t-prolyl-L-valyl-glycine p-nitrophenyl ester (2.67 g.). After the mixture had been allowed to stand for mixture was cooled to 0". Dicyclohexylcarbodiimide (1.44 g.) was added and the mixture was stirred for 2 hours a t 60 hours at room temperature, the solvent was evaporated in vacuo and the residual product was precipitated twice OD, and then for 48 hours at room temperature. The prefrom acetone-ether, yielding 5.76 g. (96%) of a white cipitate, which contained both the p-nitrophenyl ester and amorphous powder, m.p. 119-121", [o]"D -42.0' (c 1, dicyclohexylurea, vias filtered and stirred with 250 ml. of hot acetone. The acetone suspension mas cooled and the methanol). dicyclohexylurea was removed by filtration. After the Anal. Calcd. for C9iH126022N18S5 (1984.4): C, 55.1; H, acetone had been allowed to evaporate a t rooin temperature. 6.40; X, 12.7; S, 8.08. Found: C, 54.9; H, 6.21; N , the product crystallized. The p-nitrophenyl ester w3s 12.8; S, 8.05. then filtered, washed with cold acetone, and dried (111.p. The product resisted all attempts at crystallization. .4 152-15.5", yield 3.08 6.); from the mother liquor, :inother sample (1.25 g.) was subjected t o countercurrent distribu- 0.48 g. was recovered; total yield 3.60 g. (i9%,). After tion in the solvent system consisting of chloroform-toluenerepeated crystallization from acetone, the nieltltlg point methanol-water (5:5:8:2). After 435 trausfers, one rose t o 164-166", [ c ~ ] ~ $3.6" ~ D ( c 1, methanol), [a125n main peak (K = O B ) , which followed the theoretical distri- 19.0" (c 1, dimcthylformaxnide). bution curve, and two very small peaks on either side were Anal. Calcd. for C36H330iN5 (647.7). C, 66.8; H, 6.13; obtained. From the main peak 850 mg. (65%) of maS,10.8. Found: C,(i6.9; H,5.34; N, 10.7. terial was recovered with m.p. 119-121", [a]"D -43.2" Carbobenzoxy-Im-benzyl-~-histidyl-~-phenylalanyl-NC(c 1, methanol). The protected nonapeptide ester also was prepared by tosyl-L-arginyl-L-tryptophyl-glycine Methyl Ester.-N"methyl ester15 (3.25 the mixed anhydride procedure, with isobutyl chlorofor- Tosyl-L-arginyl-L-tryptophyl-glycine g.) was dissolved in 25 ml.of dimethylformamide, and carbomate.45 The free base of the pentapeptide ester was prebenzoxy-Im-benzyl-L-histidyl-L-phenylalanine p-nitrophenyl pared from its carbobenzoxy derivative (1.45 g.) by hydroester (3.02 g . ) mas added, as well as a few drops of triethylgenation as described above, and then dissolved in peroxidefree tetrahydrofuran (20 ml.). Xa-Carbobenzoxy-Ne-tosyl- amine to ensure basicity. .-liter the mixture had been allowed to stand a t room temperature for 24 hours, the diL-lysyl-L-prolyl-L-valyl-glycine (0.69 g.) was dissolved in methylformamide was evaporated in vacuo and the residue peroxide-free tetrahydrofuran (25 ml.) and the solution was cooled t o -15". Triethylamine (0.14 m l . ) and iso- was dissolved in acetone and precipitated with anhydrous butyl chloroformate (136 mg.) were added and the mixture ether, giving a product, in a yield of 4.1 g., which was homugeneous in paper chromatography. By inems of countc;was stirred for 15 minutes a t -10". The cooled pentacurrent distribution in the toluene system, some minor peptide solution then was added. After this mixture had impurities were removed and the product with n partition been allowed t o stand at room temperature for I5 hours, of 0.55 was obtaimd in the amount of 3.1 g. the triethylammonium chloride was filtered off and the coeficient ( K ) solvent removed by evaporation iiz ' i a c z ~ o . The residual oil (637,). The material was dissolved in acetone, ~vhich was allowed to evaporate slowly a t rooin temperature, \vas isolated by two successive precipitations from acetoneaud then in methanol, which was evaporated in the same ether; yield 1.75 g. (88y0)of a white amorphous powder, manner; this yielded a material mhich melted at 144-146' 111.p. 120-124', [ o I z 5-42.3O(c ~ 1, methanol). with sintering from 14%'; [ C Y ] ~ ~-22.5' D ((. 2 , ineth:itiol), N~-Carbobenzoxy-N~-tosyl-L-lysyl-L-prolyl-L-valyl[ a j ? ;-~2 j o ( c 1, acetone). glycyl-Ne-tosyl-L-lysyl-Ne-tosyl-L- lysyl - NG tosyl- L - arginylAnal. Calcd. for CjiHeaNliOioS(1094.23): C, W . 6 ; 13, NG-tosyl-L-arginyl-L-proline (I). a . By Saponification of the Corresponding Methyl Ester.-The protected nonapep- 5.81; N, 14.1. Found: C, 63.4; H, 5.61; N, 14.2. Carbobenzoxy-Im-benzyl-L-histidyl-L-phenylalany1-N~tide ester (2.0 g.) was dissolved in acetone (20 ml.) and the solution was cooled in ice; 2 Ai sodium hydroxide (1.25 ml.) tosyl- L-arginyl- L - tryptophyl -glycine .-Carbobenzoxy - I m benzyl-L-histidyl-L-phenylalanyl-SG-tosyl-L - arginyl-L-trypwas added dropwise over a period of 20 minutes. After the solution had been stirred for 2 hours more at room tophyl-glycine methyl ester (2 g.) was dissolved in 20 ml. temperature, water (25 ml.) was added until the solution of 95% aqueous acetone a t 0" and N NaOH (4.0 ml.) was became slightly turbid. It was washed with ethyl acetate added slowly at 0". The reaction mixture was kept a t 0" for 25 minutes with stirring and for an additional 30 minutes with very gentle shaking t o avoid formation of The water layer was acidified with dilute hydrochloric acid. a t room temperature, and then 4.0 mI. of 1 LVHCIwas added. Paper chromatography revealed that the saponification was The fine precipitate was collected, washed thoroughly with water, and dried over P205-KOH; yield 1.51 g. (7670), m.p. complete and that some degradation had occurred. The acetone was evaporated in vacuo and the residue was washed 134-136', [ c Y ] ~ ~-37.4" D (c 1, methanol). Anal. Calcd. for ClgOH12~02~N&j(1970.3): C, 54.9; H, with water and dried (yield 2 g.). The dried material was then submitted to countercurrent distribution in the carbon 6.34; N, 12.8. Found: C, 55.1; H,6.11; N , 12.8. tetrachloride system for 100 transfers. The main peak with To check the purity of this product, a sample was sub- K = 0.26 was isolated by precipitation with anhydrous jected to countercurrent distribution in the system consisting ether from a dirnethylformamide solution; yield 1.5 g. of chloroform-toluene-methanol-water ( 5 :5 : 8 : 2 ) for 482 (7770), m.p. 144-148", [cY]'~D -19.5' ( c 1, methanol); transfers. One single peak, which had a theoretical distri- in paper chromatography, R fJ R A = 0.42. bution curve with K = 0.75, was obtained. Anal. Calcd. for C j ~ H ~ l O 1 ~ N l l S 1 1080.2): ~ H ~ O ( C, G1.3; b. By Nitrophenyl Ester Method.-Na-CarbobenzoxyH;5.74; N, 14.0. Found: C, 61.1; H , 5.62; N,1.3.8. Pie-tosyl-L-lysyl-Xr-tosgl- L-lysyl- NG-tosyl-~ - a r g i n y lNGCarbobenzoxy-~-benzyl-L-glutamyl-Im-benzyl-L-histidyltosyl-L-arginyl-proline (287 mg. j was submitted t o catalytic r~-phenylalanyl-NG-tosyl-~~-arginyl-I,-tryptophyl-glycine (11). hydrogenation ,a:; described above and the base formed was -Carbobenzoxy-Im-benzyl-L- histidyl- L - phenylalanyl - NGtosyl-L-arginq.l-L-tryptophyl-glycine ( 1.08 g. ) mas dissolved (4.5) J. I
