POLYPEPTIDES.
Journal of Medicinal Chemistry, 1970, Vol. 13, S o . 6 839
XLVI
Studies on Polypeptides. XLVI. Synthesis of a Stably Labeled, Biologically Active Adrenocorticotropin Fragment'-3 LUISMORODER A N D KLAUSHOFMANN 17 ' olc L I L R~seurch Ltcboratory, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania
1681;
Received April 16, 1970
A synthesis of [I4C]phenylalanine7,glutaminej-P-corticotropin~~~o amide is described. The synthetic peptide exhibited lS070 of the in vivo rat steroidogenic activity of corticotropin A, on a weight basis. It is concluded that the -,-carboxyl group of glutamic acid6 in P-corticotropini-zo amide is not essential for biological function.
The labeling of ACTH with radioiodine for immunological studies has been reported by several invest i g a t o r ~ ~but - ~ the effect of such labeling on the adrenocorticotropic activity of the hormone has not been assessed adequately. Since the 39 amino acid residue ACTH molecule contains various positions (Tyr-2 and -23, His-6, and Trp- 9), which are susceptible to iodination it is difficult to pinpoint the exact position of the iodine in the labeled molecules. Moreover, the relatively short half-life of the availableiodine isotopes necessitates frequent relabeling and the labeling pattern of the ensuing products may well vary from batch to batch. In connection with biochemical studies we required a stably labeled peptide of established structure possessing adrenocorticotropic activity. The synthesis of such a material, i.e., [ 14C]Phe7,Gln5-/3-corticotropin~-~o amide is described in this communication. Preparative Aspe~ts.-[1~C]Phe~,Gln5 - p - corticotropinl-ao amide (I) n-as constructed via the route shown on Chart I. The Phe residue was selected for labeling since it is located in the very section (active site) of the ACTH molecule which appears to be connected intimately with biological function.8 The synthesis is patterned according to a scheme which was employed for the preparation of Abut4,Gln5-/3-corticotropin~-zo amideg but incorporates several improvements. Steps involving radioactive intermediates were perfected by the use of corresponding unlabeled compounds. The pentapeptide histidylphenylalanylarginyltryptophylglycine (VI) a structural element which is common to a-lISH, @->ISH,and ACTH from various species1° has been the subject of a number of synthetic
(1) See Ii. Hofmann, R . Andreatta, H. Bohn, and L. hloroder, J . X e d . Chem., 13, 339 (1970) for paper S L Y in this series. ( 2 ) Supported by grants from the U. S. Public Health Service and the Hoffmann-La Roche Foundation. ( 3 ) The amino acid residues except glycine are of the L configuration. .ibbreviations used are: Boc = t-butoxycarbonyl: Z = benzyloxycarbonyl; O N H S = A'-hydroxysuccinimido: TEA = EtrN: T F A = FaCCO2H; CM-cellulose = carboxymethylcellulose; Bio-Rex 70 = cation-exchange resin (Bio-Rad Co., Richmond, Calif.; Amberlite IRA-400 = anionexchange resin (Rohm and Haas Co.. Philadelphia, P a . ) ; P P O = 2,5benzene; diphenyloxazole; P O P O P = 1,4-bis [2-(4-methyl-5-phenyloxaaolyl)l AP-Lf = aminopeptidase hl. [G. Pfleiderer, P . G. Celliers, &I. Stanulovic, E. D. Wachsmuth, H. Determann, and G. Braunitzer, Biochem. Z., 340, 552 (1964) I ; Corticotropin 4 1 is column-purified porcine ACTH, which exhibits i n uiuo steroidogenic actirity of approximately 100U/mg [H. B. F. Dixon a n d hI. P. S t a c k - h n n e , Biorliem. .I., 61, 483 (1955)I. (4) kV. .\I. Hunter a n d 1'.C. Greenwood, .Vafure(London),194, 495 (1962). ( 5 ) J. P. Felber, Erperientia, 19, 227 (1963). (6) R.S. Yalow, S. M . Glick, J. Roth, and S. A. Berson, ,J. Clin. E n d o crinol. Metab., 24, 1219 (1964). (7) S. A . Rerson and R.S. Yalow, .I. Clin. I n a e s t . , 47, 2T25 (1908). (8) K . Hofmann, Brookhuuen Sump. B i d . , 13, 184 (1960). (9) K. Hofmann, J. Rosenthaler, R. D. Wells, and H. Yajima, J . Amer. Chem. Soc., 86, 4991 (1964). (10) F o r a review see K. Hofmann, Annu. Reu. Biochem., 31, 213 (1962).
studies."-'j Since previous routes to this peptide involved steps which proceeded in poor yields, or approacheh in which strong acids were employed to remove protecting groups from intermediates containing the acid-sensitive Trp residue we have now devised an alternative method. Methyl tryptophylglycinate was acylated with a mixed anhydride of benzyloxycarbonylnitroarginine by the method of AndersonIG to give methyl benzyloxycarbonylnitroarginyltryptophylglycinate, which was saponified. The ensuing product I1 was hydrogenated to afford 111. Randomly labeled [14C]Phe was carbobenzoxylated and the benzyloxycarbonyl derivative converted into the N-hydroxysuccinimido ester (IV) .l 7 This ester was used to acylate I11 to give the labeled peptide derivative V in excellent yield. The benzyloxycarbonyl group was removed by hydrogenolysis and the ensuing free tetrapeptide coupled with benzyloxycarbonylhistidine azide.' Hydrogenation of the protected pentapeptide intermediate gave VI. The labeled protected decapeptide VI11 resulted from the reaction of the azide VIIS with VI. In order to have available the labeled N-terminal decapeptide section of the ACTH molecule an aliquot of VI11 was deprotected. Our original synthesis18 of the tetrapeptide amide XI has now been replaced by a modified route which eliminates several steps and proceeds in better yields. For the preparation of valine amide the N-hydroxysuccinimido ester of benzyloxycarbonylvaline17 was treated with KH3 in CHC13 and the ensuing benzyloxycarbonylvaline amide was deblockcd by hydrogenation. The valine amide was treated with N-hydroxysuccinimidobenzyloxycarbonylprolinate17to afford benzyloxycarbonylprolylvaline amide, which was converted into IX as described.lg For the preparation of X benzyloxycarbonylnitroarginylprolylvaline amide (IX) was deblocked with HBr in AcOH and the ensuing peptide amide coupled x i t h a mixed anhydride of benzyloxycarbonylnitroarginine. l.l6 The arginylarginylprolylvaline amide XI which resulted when X was subjected to hydrogenolysis was used without purification. The coupling of XI (11) K. Hofmann, RI. E. Woolner, G. Spiihler, and E. T. Schxartz, J . Amer. Chem. Soc., 80, 1486 (1958). (12) K. Hofmann and S. Lande, ibid., 83, 2286 (1961). (13) R . Schwyaer a n d C. H. Li, Xature ( L o n d o n ) , 182, 1669 (1958). (14) R. Geiger, K. Sturm, a n d U ' . Siedel, Chem. Ber.. 96, 1080 (1963). (15) K. Inouye, Bull. Chem. Soc. J a p . , 88, 1148 (1965). (16) G. W. Anderson, F. M. Callahan, a n d J. E. Zimmerman, J. Amer. Chum. Soc., 89, 178 ( l 9 6 i ) . ( 1 7 ) G . W. Anderson, J. E. Zimmerman, and F. hI. Callahan, ibid., 86, 1839 (1964). (18) K. Hofmann, H. Yajima, T-Y. Liu, N. Tanaihara, C. 1-anaihara, and J. L. Humes, ibid., 84, 4481 (1962).
c l l . ~ l l lI
2 N A R G PRC VAL N t i p
2 N A R G T R P GLY O H
in)
[E! t
1
(IPI
.
I
--
2 N A R G . N A R G PRO V A L N H Z
H A R G T R P GLY O H
ZI4C P H E ONHS
ImI
--'
(XI Boc
i
B O C BOG
f I I I Z LYS P R O V A L GLY LYS LYS CON3 t H ARG ARG P R O V A L N H 2
Z14C P H E , A R G . T R P G L Y . O H
(?a)
(PI
B O G SER TYR SER M E T G L N CON3 t H H I S I 4 C (YE) i -,_-_!
'
B O C SER T Y R SER M E T G L N H I S
1 PHE ARG
I
-J
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i BOC
BOC B O C
I
I
1
2 L Y S P R O V A L GLY L Y S L Y S ARG ARG PRO V A L N H 2
R P GLY O H
(Xml
(IT)
t BOG 14
C
PHE ARG
R P GLY O H
BOC BOG
I I / H L Y S P R O V A L GLY L Y S L Y S A R G ARG PRO VAL NH,
t
IXIII!
t
Bot B O G I I I B O G S E R TYR S E R . M E T G L N . H I S I 4 C P H E A R G T R P GLY L Y S P R O V A L G L Y L Y S L Y S A R G A R G P R O V A L N H 2 BOG
(3x1
1 H
SER TYR SER M E T G L N H 1 S I 4 C P H E A R G T R P GLY L Y S P R O V A L G L Y L Y S L Y S A R G A R G P R O V A L N H 2
(1)
arid X11 and the partial deprotection of the resulting XI11 to give XIV is d e ~ c r i b e d . ~ The time-consuming removal of SH&3;2c from V l I-cellulose chromatographed peptides by lyophilization t'o constant' weight was replaced by desalting with I3o-Rex 70.'9 The tosylate salts of VI11 and XIV were coupled by the DCI procedure,lqLOarid the ensuing protected peptide XV purified on ClI-cellulose. The homogeneous protected peptide XV was deblocked with 90%, trifluoroacetic acid, trifluoroscetic acid ions werr exchanged for AcO-, and the peptide was exposed t o thioglycolic acid to reduce fully the methionine residue. The :Imino acid ratios in acid hydrolysates of the fin:tl product agreed with t'hose expected by theory.
Experimental Section General Procedures.--hleltiti~ points are uncorrected. Hotau-ere determined with a Zeiss precision polarimeter. hLIeasiu'emerits were carried oiit, with ail Ilg lamp at 546 atid 578 ~iip :tiid extrapolated t o the .iSY-mp Sa liiie. Elemental atialyses were performed tiy Hchwarzkopf .\Iicroanalytird L:tt)or:ttory, \I'orid4e, N. Y-.: 0 valiies were' :tct,ually detei,niitied tind l i c i t computed by difference. The tmiiio acid compositioil of acid atid enzymic hydrolysates wan determined with a BeckmarinSpinco Model 120 amino acid atial>-zer.21 The figures in parentheses are average recoveries of amino acids. Nle and a-amino~-guatiidopropioriicacid were iised as internal standards. Acid Ii>-drolysates were perfomietl iii c'oiiatnrit boiling point €IC1 t t t 110" for 24 Iir in evacuated tubes; values are 1101,corrected for :miino acid destruction. AP-RI digestions and thioglycolate wduct'ions were carried out as described.2z See Hofmann, rt r 1 1 . , ~ 3 1ions
.
J . A m r r . Chem. Soc., 88, 5914 (1966). Scliiryzer a n d H. Kappeler. Helv. Chim.Acta, 46, 1550 (1963). ( 2 1 ) S. Moore, D. H. Spackman, and W.Ff. Stein, A d . Chem., SO, 1185 ( I !lrS8). ( 2 2 ) li. llufiiiann. I,'. A l . 1"inn. A l . l , i i i i t . t t i , .I. llontibellel, and C.Z u n r t t i , .1., l r w r . Clirrn. S o c . , 88, 883.3 (19681. (231 I'anailiara, ihid., 87, 611 (1Y65). (19) K. Hofmann a n d €1. Bohn,
do) 11.
e m s f o r t l r chloni:ttciK'"iiis. 'I'h tlesignatioii of the solvent tems lined f ( J I ' L ~ L 'chromatogrnphy ,f paper XIA\: iii thih series' is inill the E;xperimeiit:il Secti rorrert and should read a': folloM-s: AJir = Z?r)I; IZr"' = alld R f l Y = R f X . lladioiictivit,y was Iiieairired with :t 1':tc:liard XIotlel 574 licliiitl scintillation spectrometer it1 a sciiit,illation medium composed of PPO ('3.8 g), POPOP (0.2 g ) in toluene (2000 ml), mid et glycol niotioniethyl ether (1200 nil). A total of ,5000 roui collect'ed for each sample and converted into dpm in the usual manner, using [14C]tolueneas iiiterrial standard. Steroidogenic activity was determined in hypophysectoriiizetl nr Dexamet,hasone-blockedrata.24 .Y-Hydroxysuccinimidobenzyloxycarbony 1 [I4C]phenyialaninate (IV).---Phenylalanine (0.6 g; 3.63 mmoles) and randomly labeled [ W ] P h e obt,ained from Itit,ernational Chemical and Nuclear Chrp. (0.5 mCi in 0.01 S IlC1) were dissolved in l X N a O H t :1.7 nil) arid carbobenzoxylated.2b The oily benayloxycarhonyl derivative (1.03 g; 3.44 mmoles) was then converted into the A;tiydroxysucciriimido ester." The ester was crystallized to c m ioactivity from CHC1,--EtzO; yield 1.02 g 1"; lit." rnp 14O,.i--14l0; specific radioactivity for desigiiatirJii of sulveiit
Benzyloxycarbonylvaline Amide.- -J$--Hydro ~!.lr)s?-carbonvlvalinate17(20 g ) wa3 dissolved :it111 :L s l i ~ wst,reani trf SI-13 w w ~ ~ a s i et hrough d t h e w l i i t l i o t t for 2 11r :ti 0'. The solveiit v - u ttvnpor:tttd itlid the pro(iricq W : I ~ tllisetl. three tinies froin 3IeOlI: yield 11.S6 g. 'rhv roiillot her liquors were evapor x a s tlist,ributed between H20 arid EtOAc. The EtOAr: l : i y ( ~ ~ w w e evaporated aiid the residue yield 1.36 g : total yield 13.22 g +21.4" (c 1.6, DalF); firvT 0 . +22' (c 2.0, 1>3IF). BenzyloxycarbonylprolylvalineAmide: line amide (11.0 g; 43.95 mmoles) was hy IIeOH (120 ml) and 203: AcOH (13.5 ml). The erisiiing valine :imide acetate was dissolved in 1)3\IF (150 ml), N-hydroxysuc~rhon yl jiroliiI :t t ( 15.22 g ; 4:3,!)4 nini ole.: ) ..
( 2 4 ) 11. I:. I,eI,o\-it~ nnil 1:. I.. 1:nael. L'ndurrinoloyy, 76, 8:iI i l \ I M ) . 12.5) \V. Grassrnann and E. \\'Unscti. C h r m . R e v . , 91, 4W2 (1958). ,261 hI. Bodanszky and N. .I. \t-illiuns, J . i l m r r . Chem. Soc., 89, ( i X i ilY6i).
Journal of Medicinal Chemistry, 1970, Vol. I S , No. 6 841
POLYPEPTIDES. XLVI and TEA (6.27 ml)were added and the mixture was kept at room temperature for 12 hr. The solution was evaporated to dryness and the residue was crystallized first from 1LleOH-H20 then from PrleOH-EtzO: yield 11.78 g (77.17,); mp 205-207'; [ Q ~ ] ~ ~ -80.4' (c 0.964, AcOH); RfVI 0.8; lit.11 mp 201-203'; [aI23~ -80.2' (C 1.14, AcOH). Benzyloxycarbonylnitroarginylnitroarginylprolylvaline Amide MeOH Solvate (X).-Benzyloxycarbonylnitroarginylprolylvaline amide's (IX) (2.3 g; 4.19 mmoles) was dissolved in AcOH (6 ml), HBr in AcOH (3.3 A') (20 ml) was added, and thesolution was kept a t room temperature for 1 hr. The bulk of the solvent m?as removed, the product was precipitated by addition of E t 2 0 and was reprecipitated three times from NeOH with Et,O and dried over KOH. The hydrobromide was dissolved in D M F (25 ml) and TEA was added to a p H of 8 (approximately 0.74 ml required). To this solution was added a t 0' a solution containing a mixed anhydride prepared from benzyloxycarbonylnitroarginine (2.04 g; 5.77 mmoles) in T H F (40 ml) with N methylmorpholine (0.644 ml; 5.77 mmoles) and isobutyl chloroformate (0.68 ml; 5.19 mmoles).'e The reaction mixture was stirred for 10 min a t O', the temperature was raised slowly t o 45", then the bulk of the solvents was evaporated, and the product precipitated by addition of EtOH. For purification the material was distributed between n-BuOH and 10% AcOH, the n-BuOH layers were washed with 5% ?iaHC03 and H20 and were concentrated to a small volume. The product was precipitated by addition of EtOAc; yield 2.71 g (837,); a sample for analysis was precipitated once from MeOH with EtOAc and once from MeOH with EtlO: mp 128-130'; [a]"D -31.3' (C 1.09, DLIF); [ a ] " D -54.6" (C 1.0 MeOH). Anal. ( C ~ Q H ~ ~ N ~ ~ O ~ ~ C, . C H, H ~N,O 0. H): Arginylarginylprolylvaline Amide Triacetate Tetrahydrate (XI). -Compound X (3.25 g ) was hydrogenated for 24 hr over P d in JleOH (50 ml) and 10% AcOH (100 ml). The product was lyophilized several times from dioxane-HzO (1: 1): yield 3.13 g (97%); [ a ] 2 s D , -44.2' ( C 1.0, 1070 ACOH); lit.18 [aIs1D -40.2' (c 1.1, 10% -4cOH); origin; 0.4; amino acid ratios in AP-M digest, Argl.gProl.lVall.o(88%). Benzyloxycarbonylnitroarginyltryptophylglycine (II).-Methyl tryptophylglycinate hydrochloride hemihydrate12 (2.12 g; 6.8 mmoles) in T H F (120 ml) and TEA (0.95 ml; 6.81 mmoles) nws acylated with a mixed anhydride prepared from benzyloxycarbonylnitroarginine (4.24 g; 12 mmoles).'6 The reaction mixture was evaporated to dryness, the residue was dissolved in a mixture of EtOAc (450 ml) and MeOH (50 ml), and the solution was washed in the usual manner with 1 S HC1-saturated KaHC03 and H20. The dried organic phases were evaporated and the residue was triturated first with EtnO then with boiling EtOAc: yield 3.9 g (947,); mp 117-118'; [ O ~ ] ~ O D -20.5" (c 1.33, MeOH); RfvI 0.8; 0.8; lit.'4 mp 126-128'; [ ( Y ] ~ ~-25.8' D (c 1.3, MeOH). This material (3.4 g; 5.57 mmoles) was dissolved in dioxane (20 ml) and 1N NaOH (6.5 ml; 6.5 mmoles) was added. The mixture was kept a t room temperature for 45 min, the organic solvent was evaporated, 1.5% N H 4 0 g (80 ml) was added to the residue, and the solution was extracted with EtOAc. The organic layers were washed with three 40-ml portions of 37, NHdOH and the pooled aqueous phases were cooled a t 0' and acidified with 1 S HC1. The precipitate was collected, washed with HpO, and dried over PZOS and KOH: yield 2.83 g (857,); mp 211-213'; [ a ] " D -13.2' (C 1.5, DMF); Rf'I 0.7; Rf'" 0.6. Anal. ( C Z ~ H S Z NC, ~O H,~N, ) 0. Arginyltryptophylglycine (III).-Benzyloxycarbonylnitroarginyltryptophylglycine (11)(3.2 g) was hydrogenated for 24 hr over Pd in NeOH (80 ml) and 107, AcOH (80 ml). The crude material was applied t o a CM-cellulose column (3 X 25 cm) in 500 ml of H 2 0 and the column was eluted with pH 6.8 NH40Ac,0.005 M (500 ml) and 0.015 Jf (3000 ml). Sakaguchi-positive 0.015 3-f eluates were pooled, the solution was concentrated to approximately 300 ml, and Bio-Rex 70 ( H c form) was added until the supernatant was Sakaguchi negative. The resin was collected and was washed with HgO until the washings failed t o give a Nessler test. The washed resin was then suspended in ice-cold, freshly prepared 4 N NHaOH (150 ml) and the suspension was stirred for 30 min. The resin was collected and eluted in the same manner with three additional portions of NH40H. The combined eluates were evaporated t o a small volume, and the s o h tion was lyophilized: yield 1.99 g (89%); [(Y]%D $3.9" (c 1.01, HzO); RfV10.1; RrvlI 0.4 single C1-, Sakaguchi-, Ehrlich-, and ninhydrin-positive spot; amino acid ratios in AP-M digest, Argl.oTrpl.lGlyl.o(95%). A sample was converted into the ace-
.
tate by lyophilization from 5y0 AcOH: [ a I z 9 +26.3' ~, (c 0.98,) Hz0). Benzyloxycarbonyl [W]phenylalanylarginyltryptophylglycine DMonohydrate (V).-Compound IV (1.014 g; 2.56 mmoles) was added t o a solution of arginyltryptophylglycine (1.175 g; 2.81 mmoles) in D M F (40 ml), HZO (2 ml), and TEA (0.36 ml; 2.56 mmoles) and the mixture w&s kept a t room temperature for 12 hr. The bulk of the solvent was removed and the product was precipitated by addition of H20. For purification the material wai twice precipitated from DAlF with HlO: yield 1.7 g (84%); [cY]'?D -12.9' (c 0.924, DLIF); RfVI 0.5; RfJII 0.7. Anal. ( C ~ S H ~ Z N S O ~ C, . HH, Z ON, ) 0. Histidyl [ 14C]phenylalanylarginyltryptophylglycine Diacetate Dihydrate (VI).-The monohydrate V (1.695 g; 2.364 mmoles) was hydrogenated for 24 hr over P d in DMF (100 ml) and the product was isolated in the usual manner and precipitated from MeOH with E t 2 0 : [ a I z 8 -4.0' ~, (c 1.5, 1,\' HCl); [alz8~, -4.1" (c 1.0, 1 N HC1). A solution of this product in D l I F (30 ml) was added a t -60" t o an azide solution prepared from benzyloxycarbonylhistidine hydrazide2?(1.434 g; 4.728 mmoles) in the manner described previously.' The reaction mixture w&s stirred a t 5" for 24 hr, the bulk of the solvent was evaporated, and the product was precipitated by addition of H20 saturated with EtOAc. The protected peptide was decarbobenzoxylated by hydrogenation over Pd in 50% aq MeOH (100 ml) and D M F (20 ml) and the product was lyophilized from 10% AcOH: yield 1.92 g (94%); [ a ] " ~-12.0' (c 0.855, 1 i v HC1); lit.12 [ a ] 2 8 D -12.0' (C 1.0, 1M HCl); Rr" 0.1; Rfv" 0.4. N-t-Butoxycarbonylseryltyrosylserylmethionylglutaminylhistidy1[ W]phenylalanylarginyltryptophylglycine Acetate Dihydrate (VlII).-To a stirred solution cooled a t -10" of N-tbutoxycarbonylseryltyrosylserylmethionylglutamine hydrazide monohydrate' (1.324 g; 1.773 mmoles) in DhfF (9 ml) and Hz0 (0.75 ml) 1 S HC1 (3.6 ml) was added followed by NaSOz (0.135 g; 1.96 mmoles) in 1.5 ml of HzO. The solution was stirred a t - 10" for 30 min, then TEA (107, in D M F 2.5 ml; 1.80 mmoles) was added. To this solution was added a solution, precooled a t OD, of VI (0.949 g; 1.110 mmoles) in D J I F (10 ml), HzO (2 ml), and TE.4 (10% in D M F 1.55 ml; 1.18 mmoles). The mixture was stirred a t 5' for 72 hr, the pH being maintained a t 7.5-8.0 by addition of TEA. Hz0 (150 ml) was added and the mixture was kept in a refrigerator for 12 hr. The gelatinous precipitate was collected and dissolved in 20'7, AcOH and the solution was ~ ( c 0.6, AcOH); lyophilized: yield 1.36 g (82%); [ a I z 8-14.5' ~ (e 0.321, AcOH); R f V I 0.3; RfV" 0.6; amino lit." [ a I 2 5-21.8' acid ratios in acid hydrolysate: Ser~.gTyrl.ohIetl.oGlul.lHisl.oPhel.oArgl.oGly,.o(97%). Anal. (C&87N17017.AcOH. 2H20 C, H, N. Tosylate Salt.-To an ice-cold solution of VI11 (106 mg; 7 1 pmoles) in 50% aq pyridine (4 ml) Ts monohydrate (14 mg; 71 pmoles) in HzO (0.5 ml)was added. The solution was lyophilized and the residue dried over KOH. Seryltyrosylserylmethionylglutaminylhistidyl[ W]phenylalanylarginylttyptophylglycineAcetate Hydrate.-Compound VI11 (68 mg) was dissolved in 90% TFA (2 ml) and the solution was kept a t room temperature for 30 min. The bulk of the TFA was evaporated, He0 ( 5 ml) was added, and the solution was lyophilized. TFA ions were exchanged for AcO- on a column (1 X 5 cm) of IRA400 and the eluate was lyophilized. The resulting fluffy colorless material dissolved in H20 (100 ml) was added to a CM-cellulose column (1 X 10 cm) which was eluted with 60 ml of HzO and the following NHaOAc solutions: 0.01 Af (50 ml); 0.025 Jf (50 ml); 0.035 M (50 ml); and 0.05 M (50 ml). Bbsorbancy measurements a t 280 mp located the desired compound in the 0.05 31 eluates. These fractions were pooled and added to a column (1 X 2 cm) of Bio-Rex 70. The column was washed with HzO (100 ml), then the desired material was eluted with 307, AcOH. Radioactive fractions were pooled, evaporated to a small volume and lyophilized; yield 37 mg: [a]"D -229.3" (c 0.27, 1 7, AcOH); RfvI 0.2; Rf\" 0.5; amino acid ratios in acid hydrolysate Ser~.~Tyr~l.~lLIetl.~Glu~.oHis~.oPhel.oArg,.oGlyl.o(81%); specific radioactivity 0.117 mCi/mmole. Seryltyrosylserylmethionylglutaminylhistidyl [ 14C]phenylalanylarginyltryptophylglycylprolylvalylglycy~~ysy~~ysylarginylarginylprolylvaline Amide Acetate Hydrate (I).-The dried tosylate salts derived from VI11 (500 mg; 0.31 mmole) and (XIV)S (550 mg; 0.27 mmole) were dissolved in D M F (24 ml), pyridine (12 (27)
(28)
R. W. Holley and E. Sondheimer, J . Amer. Chsm SOC.,76, 1326 (1954). R. Schwyaer and H. Kappeler. Helu. Chin. A d a , 44, 1997 (1961).
‘JT:.4 iii T)l\IF (0.447 rill) :itid J>(!I ( f t J l l 1 j;$O-ing : r t i i l c ~ t i ovtti’ a prrioii of 4 X hi. :it rooiii 1 r ~ r i i l ) f ~ i ’ ~ t i i r i ~ . P;tOAcb (500 ml) was added arid the precipit,ate was collected, washed wit,h Et,OAc, arid dried. The solid was dissolved in 50“, 3ZeOI-T (XI mi) and the solution was pasaed through a coluinir ( 2 x 3 m i ) of IRA-400 which was u-ashed with 50?: I l e O H i i i i t il the eluate was free of radioactivity iapprosirnately 250 ml recluiredj. D l I F (15 ml) was added, arid the solution was (-(inwitrated t o ti volume of approximately 150 r i d . This .soliitii)n w:ts added t o a Chl-cellulose colunin ( 2 . 5 X 1.i rm which K : ~ C eliited with lO
