A New Synthesis of a Biologically Active Nonadecapeptide

BIOLOGICALLY ACTIVE NONADECAPEPTIDE RELATED TO ACTH,. 2715 distribution in the .... to the first nineteen residues of ACTH has been described...
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July 5, 1964

BIOLOGICALLY ACTIVENONADECAPEPTIDE RELATED TO ACTH,

distribution in the toluene system for 100 transfers. A single peak with K = 0.94 was observed. T h e material isolated from this peak behaved as a single component in paper chromatography: Risam 0.77, R r s m 0.79, yield 1.6 g. (87.9701, m.p. 104-110°, [ a l Z s D -30.0' ( c 1, methanol). Anal. Calcd. for CalHsgNl&014 (1357): C, 54.0; H , 6.61; N , 13.4. Found: C, 54.2; H , 6.69; N, 13.7 Carbo benzoxy-seryl-tyrosyl-seryl-methionyl-~-benzyl-glutamyl - histidyl- phenylalanyl- NG-tosyl- arginyl- tryptophyl- glycylNe- t butyloxycarbonyl lysyl- Nf - t butyloxycarbonyl lysyl-NGtosyl-arginyl-NG-tosyl-arginyl-proline &Butyl Ester (XIII).-The decapeptideg X I 1 (0.35 g., 200 mmoles) was dissolved in 2 cc. of dimethylformamide, and the solution was cooled in ice and stirred with 0.1 cc. of triethylamine. Compound 11 (0.076 g., 200 mmoles) was added and stirring was continued for 1 hr. a t 0". Compound VI11 (0.25 g., 200 mmoles) was then added and the mixture stirred at room temperature for 24 hr. T h e reaction mixture was then kept a t 37" for another 24 h r . T h e solvent was removed in vacuo at 40", and the residue was dissolved in the lower phase of the carbon tetrachloride system and submitted t o countercurrent distribution for 70 transfers. The material in tubes 0-20 was pooled, evaporated t o dryness, washed with water, and triturated with methanol t o yield 0.305 g. of X I I I , which, however, was contaminated with traces of ninhydrin-positive material (unreacted V I I I ) . A sample of XI11 was prepared for analysis by exhaustive washing with methanol until all traces of VI11 were removed; [ a ] " ~-51.5' ( c 2.4, dimethylformamide). Anal. Calcd. for C138H187N2&0S3 (2908): C, 57.0; H , 6.481 K, 14.0. Found: C , 56.6; H , 6.17; N , 13.8. Seryl-tyrosyl-seryl-me thionyl-glutamyl-his tidyl-phenylalanyl-

-

-

-

-

arginyl-tryptophyl-glycyl-lysyl-lysyl-arginyl-arginyl-proline (XV). -Compound X I I I (0.305 8.) was dissolved in 15 cc. of trifluoroacetic acid in a nitrogen atmosphere and kept at room temperature for 1.5 hr. The Jrifluoroacetic acid was removed in vacuo at room temperature and the residue was dried overnight

[CONTRIBUTION FROM

THE

2715

over P2Os-XaOH. The residue was then submitted t o countercurrent distribution in the system composed of l-butanolacetic acid-water ( 4 : 1 : 5 , b y volume) for 80 transfers. A fast moving peak, K = 6.3 (ultraviolet, Pauly and ninhydrin positive), and a slower moving peak, K = 0.7 (ninhydrin positive, ultraviolet and Pauly negative), were seen. The peak with K = 6.3 (XIV) was isolated and found homogeneous in paper chromatography; Ri B . ~ W0.66, wt. 0.175 g. (yield based on XI1 is 337,). Compound XIV (0.16 9 . ) was dissolved in 200 cc. of freshly distilled liquid ammonia and treated with small pieces of sodium until the blue color persisted for 30-40 min. The ammonia was allowed t o evaporate and the residue dried in vacuo over P20aconcentrated HsSOa. The peptide was desalteds on IRC-50 cation exchange resin and eluted with pyridine-acetic acid-water (30:4:66, by volume). The solvent was removed in vacuo a t room temperature and the residue was dissolved in water and lyophilized t o yield 0.11 g. of crude XV. This material was purified by chromatography on a carboxymethylcellulose column (1 X 55 cm.) using continuous gradient elution with ammonium acetate (Fig. 3 ) . The major peak was isolated and rechromatographed on C M C (Fig. 4 ) t o yield, after three lyophilizations, 71 mg. of XV (peptide content determined by- ultraviolet absorption a t 280 mp, 877,). Compound Xl' was found to be homogeneous by electrophoresis on paper a t p H 3.7 (mobility relative t o lysine, 0.81) and on polyacrylamide gel a t p H 4.5 (see Fig. 5). Amino acid analysis is given in Table 11; [ a . I z 5-56.8 ~ ( c 0.5, 0.1 M acetic acid).

Acknowledgment.-This work was supported in part by grants from the National Institutes of Health of the United States Public Health Service (GM-2907). The authors wish to acknowledge the able technical assistance of C. W. Jordan, Jr., Ruth Johnson, Eugene Racz, and W. F. Hain.

HORMONE RESEARCHLABORATORY, UNIVERSITY

OF

CALIFORNIA, BERKELEY, CALIF.]

A New Synthesis of a Biologically Active Nonadecapeptide Corresponding to the First Nineteen Amino Acid Residues of Adrenocorticotropins BY CHOHHAOLI, DAVIDCHUNG, AND

JANAKIRAMAN

RAMACHANDRAN

RECEIVEDJANUARY 24, 1964 A completely new method for the synthesis of a nonadecapeptide with an amino acid sequence corresponding t o the first nineteen residues of A C T H has been described. The biological and chemical properties of the product are in agreement with those of the synthetic peptide reported by us in 1960.

The synthesis of the nonadecapeptide seryl-tyrosylseryl-methionyl-glutamyl-histidyl-phenylalanyl-arginyltryptophyl-glycyl- lysy 1-prolyl-valyl -glycyl- lysyl-lysylarginyl-arginyl-proline, in 1960, not only served to establish the structural features of the ACTH molecule essential for eliciting adrenal response, but also proved conclusively that the melanocyte-stimulating activity exhibited by the adrenocorticotropins is an intrinsic property. Subsequent work from our l a b ~ r a t o r yas ~,~ well as the efforts of other investigators5-' has shown that the nonadecapeptide corresponding to the first nineteen residues of ACTH probably represents the active core of this hormone. Hence i t appeared im'12

(1) C. H. Li, J. Meienhofer, E. Schnabel, D . Chung, T-B. Lo, and J , Ramachandran, J . A m . Chem. Soc., 82, 5760 (1960); 8S, 4449 (1961). (2) All amino acids occurring in the peptides mentioned in this paper are of the L-configuration with the exception of glycine. (3) C. H. Li, D . Chung, J Ramachandran, and B. Gorup, J . A m . Chem. Soc., 84, 2460 (1962). (4) C . H . Li, J. Ramachandran, and D. Chung, i b i d . , 8 6 , 1895 (1963). (5) K. Hofmann. H. Yajima, T-Y. Liu, and N . Yanaihara, i b i d . , 84, 4475 (1962). (6) K . Hofmann, H. Yajima, T-Y. Liu, N. Yanaihara, C. Yanaihara, and J. L. Humes, i b i d . , 84, 4481 ( 1 9 6 2 ) . (7) R. Schwyzer and H . Kappeler, Helv. Chim. Acta, 46, 1550 (1963).

portant to synthesize this peptide by a completely different route and obtain confirmation of the biological properties previously observed. In our original synthesis, the final steps involved the reaction of the NH2-terminal tetrapeptide with the C-terminal pentadecapeptide b y the azide procedure. With the avairability of the crystalline NH2-terminal decapeptide carbobenzoxy-seryl-tyrosyl-seryl-methion yl y -benzyl - glutamyl- histidyl -phenylalanyl -NG-tosyl-arginyl-tryptophyl-glycine8 (V), the protected nonadecapeptide was prepared by the reaction with a suitably protected nonapeptide derivative. T w o different derivatives of the nonapeptide were synthesized. In one, the €-amino groups of lysine were blocked with the t-butyloxycarbonyl group and the guanidino functions of the arginine residues were masked with the p-toluenesulfonyl group. The protected nonapeptide Nu-carbobenzoxy-N:'-t-butyloxycarbonyl - lysyl - prolyl- valyl- glycyl - N' - t - butyloxycarbony1 -1ysyl- N'- t - butyloxycarbonyl -1ysyl -KG-tosyl -ar-

c.

(8) H. Li, J. Ramachandran, D. Chung, a n d B. Gorup, J Soc., 86, 2703 (1964).

A m Chem.

2716

CHOHHAOLI, DAVIDCHUNG, AND JANAKIFUMAN

RAMACHANDRAN

BOC

a

BOC BOC Tos

2 . Lys . P r o . V a l . Gly . OH

t

I

BOC

TQS

?BZ

Z -Ser - Tyr .Ser - Met

~

Glu

.His

~

-

Phe Arg - T r y Gly ~

OB2

70s

Z. Ser-Tyr - Ser - M e t - Glu .His.

H - Ser. T y r . S e r . M e t . Glu I

Fig. 1.-Outline

1

3

4

5

- OH

. His. 6

Phe .Arg

.Try.

t H

i

~

BOC BOC Tos

8

9

Pro

- O!Bu

(111

Tor

BOC BOC Tor :os BOC , , , Lys . Pro .Val .Gly . Lys - Lyr . Arg . Arg

.Pro. 0th

(Ill)

- Pro

([VI

~

OfBu

Dicyclohexylcorbodiirnide

BOC

BOCBOC Tos

V S

- Arg - Pro - OlBu

(vr)

1 . CF,COOH

2. No, Liquid NH3

Phe. Arg . T r y . Gly . Lyr . Pro. V a l . Gly . Lyr . Lys . Arg . Arg - Pro 7

-

Dicyclohexylcorbodiirnide

Gly . Lyr . Pro . Val . Gly . L y s . L y s . Arg

I

Tor

H . L y s . Lys . Arg . Arg

Z - Lyr - P r o . Val . Gly . L y s . i y r . Arg . Arg

(y

Vol. 86

IO

I1

11

13

IP

15

I6

17

18

- OH

(Vll)

I9

of the synthesis of C ~ - ' ~ - A C T: H2 , carbobenzoxy; TOS, p-toluenesulfonyl; BOC, t-butyloxycarbonyl; Bz benzyl.

ginyl-NG-tosyl-arginyl-proline t-butyl ester (111) was synthesized by the reaction of Ne -carbobenzoxy-Ne-tbutyloxycarbonyl-lysyl-prolyl-valyl-glycine (I) with N't-butyloxycarbonyl- lysyl -Nf- t -butyloxycarbonyl -1ysylNG-tosyl-arginyl-NG-tosyl-arginyl-prolinet-butyl esterga

(VIII) was prepared by the reaction of the crystalline tetrapeptide ?P-carbobenzoxy-N\"-tosyl-lysylprolyl-valyl-glycinel (IX) with Nf-tosyl-lysyl-Ne-

tosyl-lysyl-NG-tosyl-arginyl-NG-tosyl-arginyl-proline t-

butyl esterg (X) using N-ethyl 5-phenylisoxazolium 3'-sulfonate. l 1 Compound VI11 was purified by countercurrent distribution in the toluene system. However, all attempts a t crystallization of VI11 Column: CM-cellulose ( 1 . 4 55 ~ cm) failed. 1.6 Material: 164 mg i synthetic nonodecapeptide The carbobenzoxy group was removed from 111 by E catalytic hydrogenolysis in the presence of Pd to yield y 1.2IV. Peptide IV was purified by countercurrent distribution in the toluene system. For the synthesis of .-nz the nonadecapeptide, IV was linked to V by the use of 5 -a 0.8 dicyclohexylcarbodiimide. These reactions are sche.-8 f'OIM NH,;c O.ZM NH.Ac matically presented in Fig. 1. The protected nonadeca\ 1 0" 0.4 peptide VI was isolated by countercurrent distribution in the carbon tetrachloride system followed by thorough washing with methanol t o remove any unreacted IV. 0 VI was obtained in 387, yield. 0 50 100 150 200 250 300 Tube number Compound VI was first treated with trifluoroacetic acid to remove the t-butyloxycarbonyl groups and the Fig. 2.-CMC chromatography of crude CY'-'~-ACTH( V I I ) . The initial buffer was 0.01 M ammonium acetate of p H 4.5, t-butyl ester group. The product was purifiedg by After 3-4 hold-up volumes ( 4 cc./tube) had been collected, a countercurrent distribution in the system l-butanolgradient with respect t o p H and concentration was started by acetic acid-water (4: 1 : 5 by volume) for 100 transfers. introducing 0.2 M ammonia buffer of p H 6 . 7 through a 500-ml. The nonadecapeptide derivative ( K = 3) was isolated mixing flask containing t h e starting buffer. Later, the gradient and the remaining protecting groups were removed by was increased by substituting 0.4 M ammonium acetate of p H 6.7 a s the solution flowing into the mixing flask. reduction with sodium in liquid The nonadecapeptide VI1 was isolated by desalting on (11), with the use of dicyclohexylcarbodiimide.gb IRC-5O resin13afollowed by chromatography on carCompound- I11 could be crystallized in the form of boxymethylcellulose 1 3 b using continuous gradient elution beautiful needles by slow evaporation of a methanol with ammonium acetate. ,4 typical chromatogram solution and was isolated in 78y0yield. The crystalline is shown in Fig. 2 . Rechromatography on C M C tetrapeptide I was prepared b y the reaction of Ne(Fig. 3) yielded VI1 in 43y0 yield; VI1 was found to be carbobenzoxy - N e -t - butyloxycarbonyl - lysine $ -nitrohomogeneous by electrophoresis on paper and on polyphenyl ester with prolyl-valyl-glycine methyl ester8 acrylamide gel.14 followed by saponification of the product. The amino acid composition of a n acid hydrolysate of The nonapeptide derivative in which all the basic VI1 was determined by the chromatographic niethodI5 side chain functions were blocked with the p-toluene(11) R . B. Woodward, R . A . Olofson, and H. Mayer, J Atn. C h r m . S o < , sulfonyl group, namely, N"-carbobenzoxy-Ne-tosyl-ly83, 1010 (1961) (12) V. du Vigneaud and 0. K . Behrens, J Riol C h e m . , 117, 2 i ( 1 9 3 7 ) . syl-pro1yl-valyl- gl ycyl-N'-tosyl- lysyl-P\"-tosyl-lysyl-NG(13) (a) H. B. F. Dixon and M . B. Stack-Ilunne, Biochein. J , 61, 48X tosyl-arginyl-NG-tosyl-arginyl-proline t-butyl ester ( 1 9 5 5 ) ; ( b ) E. A . Peterson and H A Sober. J .An1 Chrriz. .Tor , 1 8 , 7;21

- -

-

(9) (a) C. H. Li, J . Ramachandran. and D . Chung, J . A m . Chem. S O L . , 86, 2711 (19041, ( b ) J. C. Sheehan and G . P . Hess, i b i d . , 1 7 , 1067 (1955). (10) R . Schwyzer and W. Rittel, Heiv. C h i m . A d a , 4 4 , 159 (1961).

(1956). ( 1 4 ) S. Raymond and I-, Weintraub, .Science, 130, 711 ( 1 9 5 9 ) . R Reisfcld, U. J. Lewis, and D. E. Williams, S a l u v e , 196, 281 ( 1 9 6 2 ) .

A

BIOLOGICALLY ACTIVENONADECAPEPTIDE RELATED TO ACTH

July 5 , 1964

TABLE

AMINOACID COMPOSITION OF Ser

Tyr

Met

Theoretical 2 1 Chromatographic 1.81 0.95 Microbiological 1.99 0.98 Determined by a spectrophotometric

Glu

1 1 0.95 1.02 0.94 1.05 method."

1 SYNTHETIC d-lg-ACTH

THE

His

Phe

1 1.06 0.95

1 0.99 1.01

as well as by microbiological Iiieans.l6 The results are shown in Table I and are seen to be in excellent agreement with theory. Since the intermediates were purified by crystallization and the final peptide-forming step involved a glycine residue as the C-terminal, the nonadecapeptide may be considered to be optically homogeneous, as is indeed shown by the microbiological data. Investigation of the various biological activities revealed that VI1 possessed the same potency in several assays as the original nonadecapeptide' within the limits of error of the procedures. Thus the melanocyte-stimulating activity of VI1 was comparable to that of native a,-ACTH as assayed both in vitro18 and in vivo.1g I t may also be seen in Table I1 that the

Arg

3 3.13

+

+

potencies of VI1 in eliciting adrenal response in vitroz0 and in vivoz1 are in agreement with that of the peptide',22synthesized in 1960.

Experimentalz3 Na-Carbobenzoxy-N~-t-butyloxycarbonyl-lysyl-prolyl-valylglycine Methyl Ester .-Prolyl-valyl-glycine methyl esters (1.6 g., 5.6 mmoles) was dissolved in 50 cc. of ethyl acetate and allowed t o react at room temperature with 3.0 g. ( 6 mmoles) of Na-carboben~~xy-h~~-t-butyloxycarbonyl-lysine p-nitrophenyl ester1° for 2 days. The tetrapeptide methyl ester crystallized from t h e reaction mixture. The crystals (2.134 g.) were filtered off, the filtrate was evaporated t o dryness, and t h e residue was (15) D. H. Spackman, W. H. Stein, and S. Moore, Anal. Chem., 80, 1190 (1958). (16) T h e microbiological assay was carried out by the Shankrnan Laboratories, Los Angeles, Calif. (17) T . W. Goodwin and K . A. Morton, Biochem. J . , 40, 628 (19460: (18) K. Shizume, A. B. Lerner, and T. B. Fitzpatrick, Endocrinology, 64, 553 (1954). (19) L. T . Hogben and D . Slome, Proc. Roy. Soc. (London), B108, 10 (1931). (20) M . Saffran and A. V. Schally, Endocrinology, 66, 523 (19551; C . Rerup, Acla Ewdocvinoi., 29, 83 (1958). (21) H . S. Lipscomb and D. H. Nelson, Endocrinology, 71, 13 (1962). (22) C. H. Li, Recent Progr. Hormone Res., 18, 1 (1962). (23) All melting points were determined on a Fisher-Johns block and are uncorrected. Microanalyses were performed by the Microanalytical Laboratory of the Department of Chemistry, University of California, Berkeley. All samples for microanalysis were dried in a n Abderhalden pistol with P i 0 1a t 77' for 16 hr. a t 3 mm. pressure. Paper chromatography was performed on Whatman No. 1 filter paper a t room temperature. The solvents employed were 1-butanol-acetic acid-water ( 4 : 1 : 1, BAW) and 2-butanol-107, ammonia (85: 15, SBA), both by volume. Peptide spots were detected by ninhydrin reagent and by the chlorine method.24 Countercurrent distribution was carried out using the toluene system (toluene-chloroform-methanol-water, 5 : 5 : 8: 2) or the carbon tetrachloride system (carbon tetrachloride-chloroform-methanol-0.01 M ammonium acetate, 1 : 3 : 3 : l ) , both by volume. ( 2 4 ) H. Zahn and E . Rexroth, Z . A n a l . Chem., 148, 181 (1955).

..

3

2.12 2.10

2.95 2.95

Pro

Val

2 2.01 2.02

1 0.91 0.93

Na-Carbobenzoxy-N~-t-butyloxycarbonyl-lysyl-prolyl-valyl-

-

1.6

1963 prepn.

*

.

Lys

2

glycine (I).-The tetrapeptide methyl ester described above (2.23 g . , 3.45 mmoles) was dissolved in 30 cc. of acetone and cooled t o 0'. A solution of 1 N NaOH ( 7 cc., 7 mmoles) was slowly added and the mixture was then stirred at room temperature for 1 h r . T h e solution was poured into 150 cc. of ice water and the p H adjusted t o 3 with ice-cold 10% citric acid. T h e acetone was removed i n vacuo and the aqueous phase was ex-

-

1. In vitro steroidogenesis, I.U./mg. 40 33 2. In vitro M S H activity," units/g. 1.4 X lo7 7 X lo7 3. In viao MSH activity,b units/g. 0.1 0.1 a T h e same preparation of o,-ACTH was assayed i n vitro for M S H activity in 1960 'and 1963 and found, respectively, t o possess 1.4 X lo7 and 6 X lo7 units/g. The dose produces a change in melanophore index in hypophysectomized Rana pipiens of from 1 to 3 within an hour.

1.00"

3.11

TABLE I1 1960 prepn.

Gly

Try

1

crystallized from warm ethyl acetate t o yield an additional 0.633 g. of the tetrapeptide; total yield 2.767 g . (76%), m.p. 137-138'. Recrystallization from ethyl acetate did not alter the melting point, [ a l Z s D -86.6" ( c 0.5, methanol). The tetrapeptide methyl ester was found t o be homogeneous in paper A chromatography in two solvents, Rt BAW 0.93 and Rf ~ B 0.94. Anal: Calcd. for C32H49X;509(647.8): C, 59.3; H , 7.62; N, 10.8. Found: C , 59.2; H, 7.83; N, 10.6.

BIOLOGICAL ACTIVITIESOF T w o SYNTHETIC CY~-~~-.~CTH Biological activity

2717

Column: CM;cellulore (1.4 x 55 cm) Material: 85 mg synthetic

n

y 0

.-5

1.2

-

C

-.-8 -0

0.8 -

8 -

-.

0.4

0 AM " . A t

L I

0

30

100 150 Tube number,

Fig. 3.-Rechromatographyof

200

J

250

VI1 on C M C ; conditions a s Fig. 2.

tracted twice with ethyl acetate. The ethyl acetate extracts were pooled, washed with water until neutral, and dried over anhydrous sodium sulfate. The solvent was removed i n vacuo to yield a residue which was homogeneous in paper chromatography in two solvents, Rt BAW 0.83 and R f~ B 0.51. A The residue was triturated with ethyl acetate t o yield 2.13 g. (98%) of I in the form of fine platelets, m.p. 117-118", [ ~ ] * S D -72.8" ( c 1, methanol). Anal. Calcd. for Cs1H4,NaOs (633.7): C, 88.8; H , 7.48; N, 11.1. Found: C, 58.6; H , 7.34; N, 11.1.

NQ-Carbobenzoxy-N~-t-butyloxycarbonyl-lysyl-proly~-valy~glycyl-Ne-t-butyloxycarbonyl-lysyl - Ne - t - butyloxycarbonyl-lysyl~G-tosyl-arginyl-NG-tosyl-arginyl-proline t-Butyl Ester (III).Compound I (0.79 g., 1.24 mmoles) and II@ 01.55 g., 1.24 mmoles) were dissolved in a mixture of 25 cc. of ethyl acetate and 3 cc. of dimethylformamide. The solution was cooled t o 0" and 0.256 g. (1.24 mmoles) of dicyclochexylcarbodiimide~~ was added, the mixture stirred at 0' for 2 hr., and stored a t 4' for 24 hr. Glacial acetic acid ( 1 cc.) was added and after 2 hr. the solvent was removed i n vacuo. The residue was dissolved in ethyl acetate and the insoluble dicyclohexylurea was removed by filtration. The ethyl acetate solution was washed with icecold 5% citric acid, water, 5% sodium bicarbonate, and water. The organic phase was dried over anhydrous sodium sulfate, the solvent removed zn varuo, and the residue redissolved in methanol. Slow evaporation of the methanol resulted in the formation of long needles of crystalline 111, 1.80 g. (787;), m.p. 120-125'. A sample was recrystallized from methanol, m.p. 123-125', [ a I z 5 D -48.0" ( c 1, methanol). Compound 111

2718

COMMUNICATIONS TO THE EDITOR

I'-ol. 8ti

was found to be honiogeGeous in paper chromatography in two XI, m . p . 120-130" [ a ] * h ~-37.1" ((. 1, methanol). I'eptidc X I solvents, RfB A W 0.81 and RfS B A 0.81. was found t o be homogeneous in paper chromatography, Ri Anal. Calcd. for C S E H I Y E N I E(1864): S ? ~ ~ ~ C , 56.7; H, 7.46; BAT%-0.86 and Ri m . 4 0.88. S , 13.5. Found: C, 56.4; H , 7.62; N, 13.3. Anal. Calcd. for CEGHI?OIYI~S~O?J (1895): C, 54.5; H , 6.86; Ne -t-Butyloxycarbonyl-lysyl-prolyl-valyl-glycyl-Nf-t-butyloxy-S , 13.3. Found: C, 54.1; H , 6.51; N , 13.1. carbonyl-lysyl-N%butyloxycarbonyl-lysyl- NG-tosyl- arginyl- NGCarbobenzoxy- seryl-tyrosyl-seryl-methionyl-; - benzyl-glutosyl-arginyl-proline &Butyl Ester (IV).-Compound 111, ( 1.86 tamyl-histidyl-phenylalanylN" - tosyl - arginyl - tryptophyl- glycylg . , 1 mtnole) was dissolved in 40 cc. of methanol and submitted Nf -1-butyloxycarbonyl-lysyl-prolyl-valyl-glycyl-Ne - t - butyloxycarto catalytic hydrogenolysis using a Vibro-mixerz6 in the presbonyl-lysyl-N~-t-butyloxycarbonyl-lysyl-N"-tosyl-arginylN'I- tosence of Pd freshly prepared from 1 g. of PdC12 for 8 hr. The yl-arginyl-proline t-Butyl Ester (VI).-The decapeptideg 1catalyst was filtered off and the filtrate and washings were (0.336 g . , 0.2 nimole) and 11. (0.346 g., 0.2 mmole) were disevaporated to dryness. The residue was purified by countersolved in 2 cc. of dimethylformamide and cooled to 0'. Dicycurrent distribution in the toluene system for 100 transfers. clohexylcarbodiimide (0.046 g . , 0.22 mmole) was added and A single peak with K = 1 was seen and isolated to yield 1.4 g. the mixture was stirred a t 0" for 1 hr. and kept a t 4 " for (81cl';) of I\., m . p . 120-130', [ O ] * ~ D-42.7' ( c 1, methanol). 3 days. Glacial acetic acid (0.5 cc.) was added, and after 2 Compound 11' was found t o be homogeneous in paper chromahr. the solution was evaporated t o dryness in C Q C Z ~ . The residue tography, RfB . ~ \ v 0.76 and RfS B A 0.81. was submitted t o countercurrent distribution i n the carbon .-lnnl. Calcd. for C s ~ H l s ~ X l ~ S(1730): ?020 C, 55.6; H , 7.70; tetrachloride system for 80 transfers t o remove urireacted 1' ( K S,14.6. Found: C, 55.9; S , 7.59; N , 14.4. = 1 ) . The material from tubes 0 to 14 was isolated and washed Na-Carbobenzoxy-Nf-tosyl-1ysyl-prolyl-valyl-glyc yl-Ne-tosylexhaustively with water followed by methanol t o remove u n lysyl-Ne-tosyl-lysyl-N"-tosyl-arginyl.N"-tosyl- arginyl - proline t reacted IV, Peptide 1.1 was obtained in 38C; yield (0.255 g . ) . Butyl Ester ( VIII).-Nm-Carbobenzoxy-N~-tosyl-lysyl-prolylSeryl-tyrosyl-seryl-methionyl-glutamyl-histidyl-phenylalanylvalyl-glycine' ( I X , 0.69 g., 1 nimole) was dissolved in 30 arginyl-tryptophyl-glycyl-lysyl-prolyl - valyl - glycyl - lysyl-lysyl- arcc. of acetonitrile. The solution was cooled t o 0" and 0.14 cc. ginyl-arginyl-proline (VII).-Compound VI (0.362 g . , 0.107 ( 1 nimole) of triethylamine and 0.254 g. (1 mmole) of X-ethyl-5mmole) was dissolved in 25 cc. of trifluoroacetic acid and stirred a t room temperature for 2 hr. The solvent was removed phenylisoxazolium 3'-sulfonate1' were added. The mixture was stirred a t 0' for 1 hr., 1.36 g . [ l mmole) of Sf-tosyl-lysyl-Sfzn vacuo and the residue purified by countercurrent distribution tosyl-lysyl-N'~-tosyl-arginyl-S':-tos~-l-argiiiyl-proline t-butyl esfor 100 transfers in the system 1-butanol-acetic acid-water terg ( X ) was added and the stirring continued a t room teinpera( 4 : 1 : 5 by volume). The peak with K = 3 (ultraviolet arid nintiire for 36 hr. The solvent was removed i n z'acuo and the residue hydrin positive) was isolated to yield 0.205 g. ( 6 ; j r ; ) of the submitted to countercurrent distribution in the toluene system for partially protected nonadecapeptide. This material was dis100 transfers. h large peak with K = 0.22 corresponding t o the solved in 150 cc. of freshly distilled liquid ammonia and treated 'desired nonadecapeptide and two small peaks, K = 1.22 (unrewith small pieces of sodium until the blue color persisted for Hcted S )and K = 4.56 (unreacted I X , were observed and were 30-40 min. The ammonia was then allowed t o evaporate and the residue dried over concentrated H2S04and Pt-Oi. The resiwell separated from one another. The major peak ( K = 0.22) was pooled, evaporated t o dryness, the residue dissolved in methanol, due was dissolved in 20 cc. of 0.1 .Y acetic acid and desalted on IRC-50 resin as described earlier.g The crude nonadecapeptide filtered free of traces of insoluble material, and evaporated t o dryness to yield 1.45 g. (71.jr;) of the protected nonapeptide was eluted with pyridine-acetic acid-water ( 3 0 :4 : 66 by volume) 1.111. The peptide 1'111 was found to be homogeneous in paper and isolated by lyophilization t o yield 0.164 g . of material. chroinatography, RfB.LM 0.91 and Ri S B A 0.82, [ a 1 2 ' D -45.9" This was applied on a carboxymethylcellulose column a n d chromatographed using continuous gradient elution with arn( ( 1, methanol), 1n.p. 115-120°. A n a l . Calcd. for C94H1d5SlgSjO?2 (2030): C , 55.7; H, 6.56; monium acetate (Fig. 2 ) . The major peak was isolated and S ,12.4. Found: C , 55.5; H , 6.41; S,12.6. rechromatographed on C M C (Fig. 3 ) . Peptide 1.11 was then N~-Tosyl-lysyl-prolyl-valyl-glycyl-N~-tosyl-lysyl-N~-tosyl-lysylisolated by lyophilization ( 3 X ) in a yield of 43C, (0.085 g . ; peptide N'j-tosyl-arginyl-N';-tosyl-arginyl-proline t-Butyl Ester (XI).content based on ultraviolet, 82";). Peptide VI1 was found to be homogeneous by electrophoresis on paper (mobility relative Compound 1'111 (1.4 g., 0.69 mmole) was dissolved in 30 cc. of methanol and decarbobenzoxylated by catalytic hydrogenolysis t o lysine, 0.81; p H 3.7, 400 volts, 4 h r . ) and polyacrylamide gel. i n the presence of I'd freshly prepared from 1 g. of PdC12. After The amino acid composition of i.11 as determined by the cliromatographic and microbiological procedures was found t o be in CO. evolution had stopped ( 8 hr.), the catalyst was filtered off, waslied with methanol, and the filtrate and washings evaporated excellent agreement with theoretically expected values (Table I ) ; [ a I z 5 D -84.9" (c 0.5, 0.1 M acetic acid). to dryness in caruo. The residue was purified by countercurrent distribution in the toluene system for 100 transfers. A single Acknowledgment.-The authors wish to thank D r . syininetrical peak with K = 0.76 was seen. The material in the peak was pooled, evaporated t o dryness, redissolved in methanol, A . Tanaka, C. LT.Jordan, Jr., and R u t h Johnson for and precipitated from anhydrous ether t o yield 1.04 g. (80:;) of bioassay data. This work was supported in part by __ ~~~~~

( 2 3 ) Yibro-mixer, A . G Fucr Chemic-Apparatebau, Zurich, Model E l .

the United States Public Health Service (GAT-2907).

C O M M U N I C A T I O N S T O THE EDITOR Ion Pairs in Reactions of Trityl Benzoate' Sir: While R f X - ion pairs have been invoked in discussions of solvolysis and exchange reactions of trityl chloride,* no actual attempt was made until very recently to estimate for any trityl system the importance (1) Research sponsoi-ed by the Sational Science Foundation. ( 2 ) (a) K F. Hudson and B.Saville, Chem. I n d , (London), 1423 (1954); ( b ) C . G. Swain and if, AI. Kreevoy, J . A , ~Chem. so6., 77, 1122 (1955); (c) E I ) . Hughes. C . K . I n y o l d , el ai., J . Chem. Soc., 1265 (1957); (d) C. G. Swain and E E. Pegues, J . A m . Chem. Soc., 8 0 , 812 (1958).

of ion pair return under any particular set of conditions. For carbonyl-lYO-labeledtrityl benzoate (RX) in dry acetone a t BO0, Swain and Tsuchihashi3 recently reported a first-order rate constant of 3.8 X lop6sec.-' for l80equilibration, this equilibration being completely suppressed initially in the presence of LiN3. Firstorder rate constants (1 Ofik)from following disappearance of azide ion were 4.4 f 0.4 and 6.3 f 0.6 for 0.006 and 0.010 -21 LiN3, respectively. X nearly quantitative (3) C. G. Swain and G. Tsuchihashi, i b i d . , 84, 2021 (1962).