Sept. 5 , 1963
DECA4PEPTIDEDERIVATIVES OF
was removed b y filtration, and the filtrate was adjusted to p H 7.0. T h e neutral solution was steam distilled, and seven 100-ml. fractions were collected. T h e first fraction (A,,, 227 mp, u 394) was mixed with 1.5 1. of Brady reagent and allowed t o stand at room temperature for 12 hr. The precipitate was isolated b y filtration and recrystallized once from acetic acid and a second time from benzene-petroleum ether (60-70'); m.p. 184-186' (lit. for crotonaldehyde 2,4-dinitrophenylhydrazone, 187-188'). The infrared spectrum was identical with t h a t of an authentic sample of crotonaldehyde 2,4-dinitrophenylhydrazone. The fourth fraction from the steam distillation was combined with 500 ml. of Brady reagent and allowed t o stand a t room temperature for 12 h r . The precipitate was isolated by filtration and recrystallized from ethanol, m.p. 164-166' (lit. for formaldehyde 2,4-dinitrophenylhydrazone, ;64-166'). The infrared spectrum was identical with t h a t of an authentic sample of formaldehyde 2,4-dinitrophenylhydrazone. Methyl Actinospectoic Acid Methyl Ester (XXII).-Ten grams of actinospectinoic acid was added to a mixture of 95 ml. of acetyl chloride and 600 ml. of methanol, and the solution was allowed t o stand a t room temperature for 48 hr. The precipitated actinamiae dihydrochloride ( w t . 8.05 g.) was removed by filtration. The filtrate was mixed with an equal volume of ether and allowed t o stand a t room temperature for 24 hr. A second precipitate had formed and was removed b y filtration. T h e filtrate was concentrated t o a volume of 340 ml. under reduced pressure and adjusted t o a p H of 8.4 with methanolic sodium hydroxide prepared b y dissolving 12 g. of sodium hydroxide in 400 ml. of absolute methanol. After the precipitate had been removed by filtration, the filtrate was concentrated to 450 ml. and again filtered. This filtrate was concentrated t o 130 ml., and 500 nil. of ether and 300 ml. of acetone were added. The mixture was allowed to stand a t room temperature for 12 hr.
[CONTRIBUTION F R O M T H E
BIOCHEMISTRY DEPARTMENT, UNIVERSITY
2659
INSULIN
and filtered. The organic solvents were removed from the filtrate by evaporation under reduced pressure, leaving 6.0 g. of viscous liquid. T h e liquid was fractionated, collecting the material boiling at 51-54" at 0.05 m m . T h e infrared spectrum of the distillate had absorption bands at 3450 and 1750-li40 c m . ? and [ c Y ] ~ ~ -65.5" D (c 0.998, 95% ethanol). Anal. Calcd. for CsHlaOs: C, 50.57; H , 7.43; 0, 42.11; CHJC(3), 7.85; C H 3 0 ( 2 ) , 32.6. Found: C, 49.49; H, 7.75; 0 , 4 3 . 3 1 ; CHaC, 6.8; CHsO,26.62. N,N'-Bis-(ethylcarbamoy1)-actinospectinoic Acid (XIX).--A solution of -5.0 g. of S,?;'-bis-( ethylcarbamoy1)-actinospectacin in 25 ml. of 0.1 N b a r i u m hydroxide was allowed to stand a t room temperature for 5 hr. Sufficient 0.1 A' sulfuric acid was added t o remove barium ion, and the precipitate was removed by centrifugation. The supernatant was filtered and freeze-dried. The residue was distributed through 500 transfers in a countercurrent distribution apparatus using a 1-butanol-watcr system. Tubes 180-260 were combined and evaporated to dryness under reduced pressure. The residue was dissolved in 50 ml. of water, and the solution was freeze-dried giving 1.35 g. of amorphous solid, m . p . 16.5-171' dec., [aIi5D -63" ( c 1, I-120). The pKa' of this material was 3.57. The infrared spectrum had bands a t 3390, 1720, 1606, 1535, 1245, 1190, 1055, and 1023 ern.-'. Anal. Calcd. for C Z O H ~ ~ N ~ OC, ~ C48.77; ,: H, 7.37; S,11.40; mol. w t . , 474. Found: C, 49.22; H , 7.34; N,11.06; mol. w t . (elect. titr.), 481. Periodate Titration of N,N'-Bis-(ethylcarbamoy1)-actinospectinoic Acid.--This was run by the Fleury-Lange procedure'B using 492 mg. (1.04 mmoles) dissolved in 30 ml. of 0.1 ,\if sodium periodate solution. There was no periodate consumption in 3.5 hr.
OF PITTSBURGH SCHOOL OF
MEDICINE, PITTSBURGH 13,
P E N N A .]
Insulin Peptides. VII. The Synthesis of Two Decapeptide Derivatives Containing the C-Terminal Sequence of the B-Chain of Insulin' BY PANAYOTIS G . KATSOYANNIS AND (IN PART)I'ENJI SUZUKI RECEIVED APRIL8, 1963 The partially protected decapeptide ~-methyl-~-glutamyl-~-arginylglycyl-~-phenylalanyl-~-phenylalanyl-~tyrosyl-~-threonyl-~-prolyl-N'-tosyl-~-lysyl-~-alan~ne methyl ester dihydrochloride and the fully protected decapeptide N-carbobenzoxy-~-benzyl-~-glutamyl-Nw-tosyl-~-arginylglycyl-~-phenylalanyl-~-phenylalanyl-~-tyrosyl-~-threonyl-~-prolyl-Ir;~-tosyl-~-lysyl-~-alanine methyl ester have been synthesized and their chemical and stereochemical homogeneity has been established These decapeptide derivatives contain amino acid sequences found in the C-terminal portion of the R-chain of insulin.
Studies are under way in this laboratory directed toward the synthesis of the A- and B-chains of insulin and eventually to the total synthesis of this protein.2 T o this end several peptide derivatives embodying within their structures amino acid sequences found in the insulin chains have been prepared and the detailed synthesis of a number of these derivatives has been already As a step toward the synthesis of the B-chain of insulin the preparation of certain derivatives of the decapeptide L-glutamyl-L-arginylglycyl-L-phenylalanyl-Lphenylalanyl-L-tyrosyl - L - threonyl- L - prolyl - L - lysyl - Lalanine was desired. This decapeptide contains the C-terminal sequence of the B-chain of insulin. Peptide derivatives containing some of the amino acid (1) This work was supported b y a Research Career Development Award (GM-K3-151.51) from t h e Public Health Service a n d a g r a n t (A-3067) from t h e S a t i o n a l I n s t i t u t e of Arthritis a n d Metabolic Diseases, Public Health Service, f o r which we wish t o express our appreciation. ( 2 ) A preliminary report of t h e work described in this paper a n d of t h e work carried out t h u s far in our laboratory which is directed toward t h e synthesis of insulin has been presented ( P . G . K . ) in t h e Eighth S a t i o n a l Medicinal Chemistry Symposium of t h e American Chemical Society held in Boulder, Colo., J u n e 18-20, 1962. (3) P. G . Katsoyannis, J . A m . Chem. Soc., 83, 4063 (1901). (4) P G . Katsoyannis a n d K . Suzuki, i b i d . , 83, 1057 (1961). ( 5 ) P G. Katsoyannis a n d K . Suzuki, i b i d . , 84, 1120 (1962). (6) P G . Katsoyannis, K . Suzuki, and A. Tometsko, ibid., 86, 1139 (1963). ( 7 ) P . G . Katsoyannis a n d K . Suzuki, ibid., 86, 1679 ( 1 9 6 3 ) . (8) P. G. Katsoyannis, K . F u k u d a , and A . Tometsko, i b i d . , 8 6 , 1681 (1963).
sequences of the above decapeptide have been prepared by other investigators.Y-" In the present communication the synthesis of the partially protected decapeptide y-methyl-L-glutarnyl-L-arginylglycyl-Lphenylalanyl - L - phenylalanyl - L - tyrosyl - L - threonyl - Lprolyl-Ne-tosyl-L-lysyl-L-alanine methyl ester dihydrochloride (V) and of the fully protected decapeptide Ncarbobenzoxy-.y-benzyl-L-glutamyl-Nw - tosyl-1, - arginylglycyl-L-phenylalanyl-L-phenylalanyl-Ltyrosyl- L - threonyl-L-prolyl-N'-tosyl-L-lysyl-L-alaninemethyl ester (VII) is reported. The key intermediate in the synthesis of these derivatives vvas the protected heptapeptide N-carbobenzoxy - I - phenylalanyl - L - phenylalanyl - L - tyrosyl - Lthreonyl- I,-prolyl-N'- tosyl-L-lysyl-L-alanine methyl ester, whotie preparation has been reported p r e v i o ~ s l y . ~ Removal OF the carbobenzoxy group from the protected heptapeptide b y catalytic hydrogenation and coupling of the resulting product with the N-carbobenzoxyglycine p-nitrophenyl esterI2 afforded the protected octapeptide N-carbobenzoxyglycyl-L-phenylalanyl-Lprolyl- N'- tosyl - Lphenylalanyl-L-tyrosyl-L-threonyl-Llysyl-L-alanine methyl ester (I) in 80% yield. The (9) J . E . Shields a n d F. H . C a r p e n t e r , ibid., 83, 3000 (1961). (10) L.-T. K e , Y . - T . K u n g , W . - T . Chi, and C . - I . Niu, S c i . Sinirn, 11, 337 (1902). (11) J. K u n d e and H. Zahn, A n n . , 646, 137 (1961). (12) B. Jselin, W . R i t t e l , P. Sieber, a n d R . Schwyzer, H e h C h i n . Acin 40, 373 (1957).
2660
PANAYOTIS KATSOYANNIS AND KENJISIJZUKI
Vol. s5
same peptide derivative was prepared by Kunde and L-lysyl-L-alanine methyl ester dihydrochloride (V) . This Zahn" in 31y0 yield by the condensation of N-carbopeptide derivative gave the correct elemental analysis and benzoxyglycyl- L - phenylalanyl-L-phenylalanyl - L - tyroproduced a single spot, ninhydrin and Sakaguchi positive, sine with L-threonyl-L-prolyl-Ne-tosyl-L-lysyl-L-alanine on paper chromatography in two solvent systems. methyl ester by the carbodiimide13 method and by Ke Amino acid analysis of an acid hydrolysate showed et d., l U by coupling t-prolyl-Ne-tosyl-L-lysyl-L-alanine the expected composition with an average amino acid methyl ester with N-carbobenzoxyglycyl-L-phenylrecovery of 93y0 of theory. The decapeptide derivaalanyl-L-phenylalanyl-L-tyrosyl-L-threonine by the cartive was completely digestible by LAP. This was bodiimide or the azide method in 42 and 53yoyield, ascertained by amino acid analysis of the digest by an respectively. automatic analyzer. The constituent amino acids, The chemical homogeneity of the protected octawith the exception of glutamic acid, were obtained peptide I was ascertained by elemental analysis and in the stoichiometrically correct proportions with an paper chromatography of the decarbobenzoxylated average recovery of amino acids of S970. As can be derivative in two solvent systems. In both systems seen, from the amino acid ratios reported (Experimenthe chromatograms exhibited sharp single spots indicatal section) only traces of glutamic acid were obtained. tive of the presence of a single component. Hofmann, et n1.,19 observed a similar situation with glutamine during LAP digestion of glutarnine-contairiCatalytic hydrogenation of the protected octapeping peptides. In that case quantitative determinatide resulted in the removal of the carbobenzoxy group. tion of glutamine by the ninhydrin technique was not The resulting product was condensed with tricarbofeasible because of pyrrolidonecarboxylic acid formaberizoxy-~-arginine'~,~~ by the carbodiimide method to give tricarbobenzoxy-L-arginylglycyl-L-phenylalanyl-tion. I t then appears possible that during LAP digestion of the partially protected decapeptide and the L-phenylalanyl-L-tyrosyl-L-threonylL - prolyl- N'- tosylsubsequent amino acid analysis of the digest by the L-lysyl-L-alanine methyl ester (11) in 44% yield. automatic analyzer the y-methylglutamic acid residue Elemental analysis and paper chromatography of the was converted to a large extent to pyrrolidonecarboxylic decarbobenzoxylated derivative in two solvent systems acid and hence did not give a ninhydrin reaction. This established the chemical purity of this peptide derivainterpretation is supported by the finding t h a t acid tive. Decarbobenzoxylation of the protected nonahydrolysis of the decapeptide yields glutamic acid in peptide by catalytic hydrogenation in the presence of the expected amounts. HC1 resulted in the formation of L-arginylglycyl-LFor the synthesis of the fully protected nonapeptide phenylalanyl - L .- phenylalanyl - L - tyrosyl - L - threonyl - Ln'"-carbobenzoxy-Nw-tosyl-L-arginylglycyl-L-phenylalaprolyl-N'-tosyl-L-lysyl-L-alanine methyl ester dihydronyl-L-phenylalanyl -L- tyrosyl- L - threonyl- L - prolyl- Nechloride (111) in 98y0 yield. The chemical purity of tosyl-L-lysyl-L-alanine methyl ester (VI) the product rethis partially protected nonapeptide was established sulting by hydrogenolysis of N-carbobenzoxyglycyl-1,by elemental analysis, paper chromatography in two phenylalanyl - L - phenylalanyl- L - tyrosyl- L - threonyl- Lsolvent systems, and amino acid analysis of an acid prolyl-Ne-tosyl-t-lysyl-L-alaninemethyl ester ( I ) wag hydrolysate. I n the latter case the constituent amino condensed with N"-carbobenzoxy-N"-tosyl-I,-ar~~itiine."' acids were obtained in the expected ratio and in an Activation of the latter compound was accomplished average recovery of 88% of theory. For ascertaining ' by the use of 2-ethyl-5-phenyloxazolium-:3'-sulfonate. its stereochemical homogeneity the partially protected The protected nonapeptide VI was thus obtained in nonapeptide 111 was digested with leucine aminopepSi?; yield and in analytically pure form. Elemental tidase (LAP) and the digest was analyzed by an autoanalysis, paper chromatography of the decarbohenzmatic analyzer. The containing amino acids were oxylated derivative in two solvent systems, and obtained in the expected ratios and in 72% average amino acid analysis of an acid hydrolysate served as recovery, indicating that the digestion was complete. criteria of the chemical homogeneity of this peptide This suggests16 t h a t no racemization of the constituent derivative. amino acids had occurred during the synthetic processes From the results obtained by paper chromatography leading to the preparation of the nonapeptide derivative. and quantitative amino acid analysis, with the use of Interaction of the partially protected nonapeptide an automatic analyzer, of the acid hydrolysate of the I11 with N-carbobenzoxy-y-methyl-L-glutamic acid protected nonapeptide (Experimental section) it is p-nitrophenyl ester",'* afforded the decapeptide hydroN-carbobenzoxy---methyl-L-glutamyl-L-apparent that the tosyl group was split almost quantichloride tatively from lysines and arginine during acid hydrolyarginylglycyl-L-phenylalanyl-L-phenylalanylL - tyrosylsis. L-threonyl-L-prolyl-N'-tosyl-L-lysyl-L-alanine methyl Exposure of the fully protected nonapeptide VI to ester (IV) in 60.5Yc yield. The chemical homogeneity HBr in acetic acid and coupling of the ensuing decarof this decapeptide was established by elemental bobenzoxylated derivative with N-carbobenzoxy-yanalysis and paper chromatography of the decarbobenzyl-L-glutamic acid p-nitrophenyl ester" afforded benzoxylated derivative. Chromatograms obtained the protected decapeptide N-carbobenzoxy-y-benzylin two solvent systems exhibited single ninhydrin and L - glutamyl- Nw - tosyl-L-arginylglycyl-L-phenylalanyl-1,Sakaguchi positive spots. Catalytic hydrogenation phenylalanyl- L - tyrosyl-L - threonyl-L-prolyl-N'-tosyl-r,of the protected decapeptide in aqueous acetic acid lysyl-L-alanine methyl ester (VII) in iO% yield. The containing HC1, followed by lyophilization, yielded chemical purity of this product was established by elealmost quantitatively the partially protected decapepmental analysis, paper chromatography of the decarbotide y-niethyl-~-glutamyl-arginylglycyl-~-phenylalanylbenzoxylated derivative in two solvent systerns. and L-phenylalanyl-L-tyr~syl-L-threonylL - prolyl- N'- tosylamino acid analysis of an acid hydrolysate. For evalua(1.3) J C . Sheehan and G P. Hess, J . A m Chem Soc., 7 7 , 1087 (1955). tion of stereochemical homogeneity, the protected deca( 1 4 ) I. Zervas. .\.I. W i n i t z , a n d J . P. Greenstein. J . Org Chem.. 2 2 , 1515 peptide was decarbobenzoxylated on exposure to HBr in (19.77). ( I ? ) I,. Zervas, T. T. Otani, Sf Winitz, and J P. Greenstein, J . A m . C h e w SOL..8 1 , 2878 (1939) ( I f , ) K H o f m a n n . . 4 n ~.\- Y . Acail. S c i , 88, ti89 (1960). (17) G 1,osse. J. Jrschkeit, and W. Lagenbeck, Z . Chem., 1, 279 (1961). ( I S ) SI Goodman, E. E. Schmitt. a n d D . A Yphantis. J . A m C h e m S o c . . 84, 128:3 (1962).
(I!?) K . Hofmann, T A. Thompson, H Yajirna, E 'I' Schwartz, and H Inouye, ibtd , 82, 3715 (1980) (20) J Rarnachandran and C. H 1.i. J O i g . ( ' h e m , 27, -1000 ( 1 9 0 2 ) . ( 2 1 ) R . B. Woodward, R A Olofson. and H Mayer. J A m C'hrm .?or BS, 1010 (1961) ( 2 2 ) RI. Goodman and K C . Stueben, : b i d , 8 1 , .?!JXO ff!l.i
Sept. 5 , 1963
DECAPEPTIDE DERIVATIVES OF INSULIN
acetic acid and the product incubated with LAP. The high insolubility of the deblocked peptidederivativein the incubation medium made impossible the digestion of the entire incubated sample and hence it was impractical to express the results of the digestion in quantitative terms. However, amino acid analysis of the digest by the automatic analyzer and by paper chromatography provided convincing evidence regarding the optical purity of the decapeptide. The chromatogram from the analyzer exhibited peaksz3 corresponding to N'-tosyllysine, Nu-tosylarginine, threonine, glutamic acid, glycine, alanine, tyrosine, phenylalanine, and proline only. Similarly, the paper chromatogram of the digest in the Partridge systemz4exhibited only ninhydrin positive spots with Ri's corresponding to the same amino acid residues. I t is therefore concluded that the digestion of the decapeptide derivative by L.4P is complete and this is indicative of its optical purity. It may be noted t h a t whereas LAP digestion of the y-methyl derivative of the decapeptide V followed by amino acid analysis gave only traces of glutamic acid, the y-benzyl derivative of the decapeptide under similar conditions produced glutamic acid in amounts corresponding to t h a t of the other constituent amino acids. The possibility then appeared t h a t y-benzyl-L-glutamic acid is hydrolyzed during incubation with L.4P. This was confirmed by incubating y-benzyl-L-glutamic acid with LAP, followed by paper chromatography of the digest. The chromatogram developed in the Partridge system exhibited only a single ninhydrin positive spot with Rf identical with that of glutamic acid. Experimental
2601
for the dihydrate'" is 190-193" and for the anhydrous" compound is 195--199"); [ a ]z 8 -25.8' ~ (c 0.21, dimethylfortnamide). For paper chromatography a sample was decarbobenzoxylated by catalytic hydrogenation in the presence of HCI. Ri' 0.88, Riz 6.1 X his, single sharp ninhydrin positive spot. Anal. Calcd. for CssH,iSsOlsS.HzO: C, 60.8; H , 6.32; N > 10.0. Found: C , 6 0 . 6 ; H , 6 . 2 9 ; K,9.7. Tricarbobenzoxy-L-arginylglycyl-L-phenylalanyl-L-phenylalanyl-L-tyrosyl-L-threonyl-L-prolylN e -tosyl- L -1ysyl- L - alanine Methyl Ester 0.5 Hydrate iII).--N-Carbobenzoxygl) ylalanyl-L-phenylalanyl-L-tyrosyl-L-threonyl-~-prolyl-Sf-t~~syl-~-
lysyl-~-alaninemethyl ester ( 2 g ) was dissolved in methanol ( 2 5 ml.) containing 1 N HCI ( 2 ml.) and hydrogeriated for 6 hr. i n t h e presence of 10';; palladium-charcoal catalyst (0.8 g . ) . The catalyst was filtered off and the filtrate was concentrated to dryness in vatuo. Tracrs of HC1 were eliminated from the retnaining product by the addition of methanol followed by evaporation. T h e residue was dissolved in diniethylforniatnide ( 15 nil.) containing triethylamine (0.24 nil.) and cooled a t 0" ; tricarhoheiizoxy-L-arginine (0.95 g.) was added to this solution followed by N,S'-dicyclohexylcarbodiimide ( 0 . 4 5 g . ) , T h e reaction mixture was stirred a t 5" for 24 h r . and acidified with drops of acetic acid. The precipitated N , S ' dicyclohexylurea was filtered off and the filtrate mixed with cold water (I50 nil.) containing HCI (1 nil.). The precipitated product was collected by filtration, washed with water, and dried, Fur purification this crude product was dissolved by slight warrning in diniethylformarnide (12 ml.)and the solution cooled to 0'. A second crop of N,N'-dicvclohexylurea was precipitated and removed by filtration. The filtrate was mixed with tetrahydrofuran (10 ml.) and ether (80 m l . ) . The precipitated white solid was isolated by filtration and was further purified b y reprecipitation from aqueous acetic acid; wt. 1.2 g. (44';.',), m . p . 188- t90", [ a ]z 8 -37.5' ~ ( c 0.22, dimethylformamide). For paper chrornatography a sample was decarbobenzoxylated by catalytic hydrogenation in the presence of HC1; Ril 0.80, Rt28.35 X his, single ninhydrin positive spot. Anal. Calcd. for C,iH,~,S130&0.5 H20: C, 61.3; I € , 6.16; N, 10.9. Found: C , 60.7; H , 6.03; N , 11.3.
L-Arginylglycyl-L-phenylalanyl-L-phenylalanyl-~-tyrosyl-~~threonyl-L-prolyl-Ne-tosyl-L-lysyl-L-alanine Methyl Ester Dihy-
Capillary melting points were determined for all compounds and are uncorrected. For paper chromatography the protected peptides were deblocked either with H B r in acetic acid or by catalytic hydrogenation and chromatographed on Whatman S o . 1 filter paper a t room temperature. Ril values refer to t h e Partridge systemz4; Rrz values refer t o t h e systemZ5 1-butanol-pyridirie-acetic acidwater. 30:20 : 6 : 24, and are expressed as a multiple of the distance traveled by a histidine marker. The enzymatic analyses ( L h P ) were performed according the procedure described by Hofmann et ~ 1 . ~ T h~ e ~amino ~ ' acid analyses of acid and LAP hydrolysates were carried out with a Beckman-Spinco amino acid analyzer, Model 120, according t o t h e method of Spackman, Stein, and Moore.z8
drochloride Hydrate (III).--Tricarbohenzoxy-L-arginylglycyl-Lphenylalanyl-L-phenylalanyl-L-tyrosyl-L - threonpl - L - prolyl - N 'tosyl-I~-lysyl-L-alanine methyl ester (1.15 g . ) ~ : i dissolved s in a mixture of acetic acid (32 m l , ) , water (18 rnl.), and 2 .V NCI ( 1 . 1
respectively, (21) S . M . Partridge, Riochem. J . , 43, 238 (1948). (251 S. G. Waley a n d G. Watson, ibrd., 611, 328 (1953). ( 2 6 ) K Hofmann a n d H . Yajima, J . A m . Chein. S o t , 83, 2289 (1901) (27) K. H o f m a n n , H Yajima, T -Y I i u , N . Yanaihara, C Yanaihara, a n d J. I, Humes, ibrd , 84, 4.181 (1902) ( 2 8 ) See reference in footnote 2 3
~-Methyl-~-glutamyl-~-arginylglycyl-~-phenylalanyl-~-phenylalanyl-L-tyrosyl-L-threonyl-L-prolyl-Netosyl - L - lysyl- L alanine Methyl Ester 3 Hydrate (V).~-C.lrhobenzox?--y-methyl-L-glutarn_____
m l . ) and hydrogenated for G hr. in the presence of IO(,; palladiun-charcoal catalyst (0.8g.). The catalyst was removed by filtration and the filtrate was lyophilized. The white solid thus in uacuo: wt. 1 .lit g. ( 9 8 L I ) ; obtained was further dried over P205 [ a ]Z R -25 ~ 6" ( c 0.23, 50';; aqueous acetic acid); Ri' 0.80, K i 2 8.35 X his, single spot ninhydrin atid Sakaguchi positive. Anal. Calcd. for CsiHaaSiaO11SCIr,H10: C, 54.1; H , 6.51; N , 13.5. Found: C, 53.9; H , 6.46; S, 1?,,1, Amino acid analysis of an acid hydrolysate showed the expected composition expressed in mol:u ratios: arg, .opglyI N-Carbobenzoxyglycyl-~-phenylalanyl-~-phenylalanyl-i~-tyroplie~.ortyru.~xthru.y~prol.~B1ysO.ylaIan.~~. The average amino acid syl-~-threonyl-~-prolyl-N'-tosyl-~-lysyl-~-alanine Methyl Ester recovery was 88!),; of theory. Hydrate (I).-S-Carbobenzoxy-~-phenylalanyl-~-phenylalanyl.A sample of t h e product was digested with L?.P and t h e digest ~-tyrosyl-~-threonyl-~-prolyl-r\;(-tosyl-~-lysyl-~-alanitie methyl was analyzed with the automatic amino acid analyzer. The ester ( 5 g . J was hydrogenated for 8 hr. over 1052 palladium-charfollowing ratios were obtainedz9: argl .ooglyo.yrpli coal catalyst (1.5 g.) in methanol (80 ml.) containing 1 N thrl prol S~-tosyllysu.y,alao.y~. Average recovery 72' HC1 ( 7 ml.). The catalyst was filtered off and the filN-Carbobenzoxy-y-methyl-L-glutamyl-L-arginylglycyl-~,-phentrate evaporated t o dryness i n z'acuo. The remaining prodylalanyl-~-phenylalanyl-~-tyrosyl-~-threonyl-~-prolylN'- tosyl- Luct was dried by the addition of methanol followed by lysyl-L-alanine Methyl Ester Hydrochloride 0.5 Hydrate fIV) evaporation under reduced pressure. The residue was dissolved T o a solution of L-arginylglyc~~l-L-pher!ylalanyl-L-phenyl:~lanylin dirnethylformamide (40 ml.) containing triethylamine ( 1 nil.). methyl L-tyrosyl-L-threonyl-L-prolpl-SC-tosyl-~~-lysyl-i~-ala~~ine T o this solution N-carbobenzoxyglycine p-nitrophenyl ester (1.8 ester dihydrochloride (1 g. j in diinethylforrnaliiide ( 1 % n i l , ) , trig.) was added. After 24 h r . the reaction mixture was diluted ethylamine (0.l l ml.) was added followed by S-c~rrbobetizoxy-ywith 1 N NHlOH ( 3 ml.), stirred 1 hr., and subsequently mixed methyl-L-glutamic acid p-nitrophenyl ester 10.32 g.). The rewith ethyl acetate (500 m l . ) and water (200 ml.). The organic action mixture was allowed t o stand a t room temperature for layer was washed with 1 N ",OH, water, 1 N HCI, and water 36 hr., diluted with 1 N KHC03 ( 1 n i l . ) , stirred for 1 h r . , and again. ( I n order t o prevent precipitation of the product during poured into cold 1 iV KHCOI (60 ml.). The precipitated prodthe washing, methanol was added t o the ethyl acetate solution.) uct was isolated by filtration and washed successively with 1 S Concentration of the solvent to a small volume resulted in the KH,OH, water, 1 iV HCI, water, and dried. On reprecipitation precipitation of the product, which was isolated by filtration and froni methanol-ether, 0.71 g. ( 6 0 . 5 5 i ) of product was obtained, was further purified by reprecipitation from aqueous methanol; m.p. 161-163', [ a ]2 8 -33.8" ~ ( c 0 . 2 , dirnethylformarnide). wt. 4.2 g. (80G/1), m.p. 182-185" (the reported melting point A n d . Calcd. for CisHss?\'isO~ySCI.0.5Hp0: C, 57.1 ; H , (23) Nu-Tosylarginine and Ne-tosyllysine are eluted from t h e short col6.38; S, 12.1. Found: C, s6.6; H , 6.11; S ,12.7. u m n (15 cm.) of t h e Beckman-Spinco analyzer, which is routinely used for For paper chromatography 2 sample was decarb[)betizos~lated the determination of the basic amino acids [S Moore, D. H . Spackman, and by catalytic hydrogenation in t h e presence of HCI; K i ' 0.78, W H . Stein, A n a l . Chem., 30, 1185 (19.58)1 a f t e r 40 ml. and RO ml. of effluent, Riz 8.Fi3 X his, single spot ninhydrin and Sakaguchi positive.
-
(29) T h e reported value for arginine is in reality the sum of arginine and ornithine which was produced from arginine during the 1.AP digestion of the peptide [ K Hofmann. e l a i , J A m C h e m . S o [ , 83, 22!94 I l ! ~ t ~ 11 1
COMMUNICATIONS TO THE EDITOR
2662
yl-L-arginylglycyl-L- phenylalanyl - L - phenylalanyl- L - tyrosyl- Lthreonyl-L-prolyl-S'-tosyl-L-lysyl-L-alaninemethyl ester hydrochloride 0.5 hydrate (92 mg.) was dissolved in a mixture of acetic acid ( 6 ml.) water ( 2 ml.), and 1 N HCI (0.2 ml.) and hydrogenated for 6 hr. in the presence of 10:; palladium-charcoal catalyst (0.1 g.). The catalyst was filtered off and t h e filtrate lyophilized. T h e resulting product was dried over P 2 0 jin uacuo; wt. 84 mg. (987,); [ a I z 6-33.6' ~ ( c 0.19,50%',aqueousaceticacidj; Rr'0.78, RrZ8.53 X his, single spot ninhydrin and Sakaguchi positive. Anal. Calcd. for C6,Hg4~~,01iSC1~.3H~O: C, 52.7; H, 6.60; E,12.9. Found: C, 52.2; H , 6 . 8 8 ; S , 12.9.
VOl. 85
The protected nonapeptide was decarbobenzoxylated on exposure t o HBr in acetic acid and subjected t o paper chromatography; Rr' 0.92, Rr2 6.8 X his, single ninhydrin positive spot. A sample of the decarbobenzoxylated nonapeptide was subjected to acid hydrolysis. Paper chromatography of t h e hydrolysate showed the presence of ninhydrin-positive spots with R+'s 0.14, 0.16, 0.22, 0.27, 0.33, 0.37, 0.46, and 0.63, identical with the Ri's,of authentic samples of lysine, arginine, glycine, threonine, alanine, proline, tyrosine, and phenylalanine, respectively. The chromatogram exhibited also traces of two ninhydrin-positjve spots with Ril s O.TO and 0.76 corresponding to the hydrochlorides of Sw-tosylarginine and Sc-tosyllysine, respectively. Amino acid analysis of an acid hydrolysate of the decarbo.. benzoxylated nonapeptide by an autoniatic amino acid analyzer showed the ratios: lyso.grarg,.~2thro.gspro1.o6gty1.1oata~,.oaphe~.,~~; average amino acid recovery was 87% of theory.
Amino analysis of t h e decapeptide dihydrochloride by an automatic analyzer after ,acid hydrolysis showed the expected composition expressed in molar ratios: lyso.srarg,.gothrl.orglui . ~ ~ p r o ~ .oralal . ~ ~ g.ortyro.grphei ly~ .96. The average amino acid recovery was 93% of theory. N-Carbobenzoxy-~~-benzyl-L-glutamyl-N"-tosyl-L-arginylglycyl-~-phenylalanyl-~-phenylalanyl-~-tyrosyl-~-threonyl-~ -proAmino acid analysis of an LAP digest showed the ratioszg: lyl-N'-tosyl-L-lysyl-rd-alanine Methyl Ester (VII).-A suspension Nf-tosyllys~.~iarg~.~~thrl.~~pro~.~~gly~.~oala~.~~tyr~.~~phe~.~~glu~.~s; avphenylalanyl - L of Sa-carbobenzoxy-NTW-tosyl-L-arginylglycyl-Lerage recovery 89% of theory. tosyl - L - lysyl -LN"-Carbobenzoxy-N"-tosyl-~-arginylglycyl-~-~henylalanyl-~- phenylalanyl-L-tyrosyl-L-threonyl-L - prolyl -ITfalanine methyl ester (4.5 g. j in acetic acid (18 nil.) was treated phenylalanyl-~-tyrosyl-~-threonyl-~-prolyl-N~-tosyl-~-lysyl-~ - alawith 4 &'V HBr in acetic acid (18 ml.). After 1 hr. a t room t e n nine Methyl Ester 1.5 Hydrate (VI).--S-Carbobenzoxyglycyl-Lperature, anhydrous ether (300 ml.) was added and the precipiphenylalanyl-L-phenylalanyl-L-tyrosylL - threonyl- I. -prolyl - N'tated product was isolated b y filtration, washed with ether, and tosyl-L-lysyl-L-alanine methyl ester hydrate (4.05 g.) was disdried over KOH in Z ~ C U O . T o a solution of this product in solved in methanol (60 ml.) containing 2 S HCI ( 2 ml.) and dimethylformamide (30 ml.), triethylamine (1.2 ml.) was added hydrogenated for 0 hr. over lo!& palladium-charcoal catalyst followed by- N-carbobenzoxy-y-benzyl-L-glutamic acid p-nitro(1.6 g.). The catalyst was filtered off and the filtrate concenphenyl ester (1.42 g.). -1fter 24 lir. the reaction mixture was trated to dryness in oncuo. The solid residue of octapeptide ester diluted with 1 iV KHCO, 13 ml.), stirred for 30 min., and poured hydrochloride was dried by the addition of methanol followed by into ice-cold 0.5 N S H I O H (200 ml.). T h e precipitated product evaporation. T h e residue was then used directly for condensation was filtered off, washed successively with 1 N ",OH, water, 1 with S"-carbobenzoxy-X"-tosyl-L-arginine. HCI, and water, and dried. On reprecipitation from methanol, X solution of ?;a-carbobenzoxy-Sw-tosyl-L-arginine ( 1.48 g.) in 3.77 g. (70%) of product was obtained, m.p. 197-200", [ U ] ~ ' D acetonitrile (40 m1.j was cooled t o 0' and triethylamine (0.46 ml.) -38.7' ( c 0.20, dimethylformamide). was added followed b y 2-ethyl-5-phenyloxazolium-3'-sulfonate (0.84 g.). After 1 hr. a t 0' the reaction mixture was diluted Anal. Calcd. for CssHlosNlaOzlSs: C, 60.0; H, 6.17; S , with a solution of the octapeptide ester in dimethylformamide and 11.1. Found: C , 59.7; H, 5.99; X, 10.7. For paper chroacetonitrile prepared as noted: the hydrochloride salt, which had matography a sample was decarbobenzoxylated on exposure t o been made as described previously, was dissolved in a mixture of HBr in acetic acid in t h e usual manner; Rr' 0.91, Rr2 8.93 X his, dimethylformarnide (15 ml.) and acetonitrile (15 m1.j containing single ninhydrin positive spot. triethylamine (0.46 ml.), stirred 5 niin., and then added to the Amino acid analysis of an acid hydrolysate of the decarboreaction mivture prepared as described above. After 24 hr. at benzoxylatecl peptide showed the expected composition exroom temperature the reaction mixture was diluted with 0 . 5 LV pressed i n molar ratios: lysi.nsargo.sjthrn.ssgluI.nproi.,oglyl.ozS a H C 0 3 (250 ml.). The precipitated product was collected b y alal.ojtyrn.jophe2.lo; average recovery 76'x of theory. filtration, washed successively with water, 1 N HCI, and water The deblocked peptide was digested with LAP. The results of again, On reprecipitation from methanol-ether, 4.29 g. ( 8 i % j the digestion were discussed previously. of product was obtained, m.p. undetermined; t h e peptide sinters Acknowledgments.-The authors wish to thank Mrs. a t 125" and eventually it is converted to a liquid a t 160"; [ a ]2 7 ~ Ursula Huppertz for the enzymetic analyses and Mrs. - 2 3 . 5 " ( c 0.17, dimethylformamide).
.Anal. Calcd. for C;6H9j?;130~sS2,1.5t120: c , 57.5; 6.28; S,11.6. Found: C, 57.4; H , 6.40; N , 11.4.
H,
Jemele Hudson for the amino acid analyses reported in this work.
COMMUNICATIONS T O THE EDITOR The Free Radical-Catalyzed Disproportionation of Arylsilanes. A New Homolytic Aromatic Displacement Reaction
Sir :
Diphenylsilane undergoes extensive disproportionation on exposure to ultraviolet radiation at 70 to 130' or on warming to 130' with a variety of peroxy or azo initiators. PhzSiH?
hlthough the acid1- and base2-catalyzed and thermal3 redistribution of substituents on a silicon are well known, no example involving radical intermediates has been reported. l y e wish to report t h a t a rapid redistribution of phenyl and hydrogen on silicon occurs in a variety of phenylsilanes in the presence of conventional free radical sources.
*
PhSk
+ €€Si=
R ---f
PhSi-
+ H S* i E
This reaction is a surprisingly efficient homolytic aromatic displacement reaction with silyl groups serving as both the attacking and leaving radicals. (1) G h Russell, J A m Chern Soc , 81,481.5 (1959). (21 J. W. R y a n , ibid , 84, 4730 (1962). ( 3 ) H Gilman and D. H . hIiles, J . Org. Chem , 93, 320 (1038)
Ph4Si
+ PhsSiH + PhSiHs + SiHa
No reaction occurs a t these temperatures in the absence of the initiator. T h e data in Table I give the extent of this reaction with some representative initiators. The dialkylperoxides appear to be the most efficient catal y s t ~with ~ minimum kinetic chain lengths of over 60.5 As seen from Table 11, a variety of phenylalkylsilanes undergo this radical-catalyzed reaction. No evidence for the redistribution of alkyl groups was observed. (4) As the d a t a in Table 11 show increasiug chain length with higher temperature, the higher decompusition temperature of the dialkylperuxides is a n advantage. These peroxides are a!so normally less susceptible t o induced decomposition which could be a serious side reaction uf the electrophilic silyl radical. (3) T h e moles of PhlSiHZ consumed per possible initiating radical from Table I This calculation ignores the reverse reaction (PhaSiH f PhSiHR 2PhSSiH2) and is thus a minimum figure
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