[VAL,'MAL']
-ANGIOTENSIN
11
Journal of Medicinal Chemistry, 1971, Vol. 14, No. 6 489
Z-y-tert-Bu-Glu-0-tert-Bu-Tyr-Ala-Gly Pentachlorophenyl Ester (4).-To
a soln of 9.0 g (0.0129 mole) of the fully blocked tetrapeptide 3 in 150 ml of MeOH wm added 13.0 ml of 1 N NaOH and the soln was stirred for 90 min and then concd under rediiced pressure. The residue was flooded with HzO, acidified with 107, citric acid soln, and extd into EtOAc. The EtOAc soln was dried (NazSO4) and concd under reduced pressure to give the tetrapeptide free acid as a solid; yield 8.0 g (9l.yO).To this material in 200 ml of CHzC12 was added 3.1 g (0.0165 mole) of pentachlorophenol and 5.0 g (0.0118 mole) of 1-cyclohexyl-342morpholinoethy1)carbodiimide metho-p-toluenesulfonate. The mixt was shaken for 48 hr a t room temp. The solvent was removed in cucuo and the residue was washed with HzO and crystd from MeOH to yield 5.1 g (47%): mp 187-188', [ c Y ] ~ ~ D 12.9" ( c 2.75, DAIF). Anal. (C41H4L&N4010) C, H, N. r-tert-Bu-Glu-0-tert-Bu-Tyr-Ala-Gly Pentachlorophenyl Ester.HCI (5).-A fine suspension of 4.5 g (0.0048 mole) of the tetrapeptide active ester 4 and 0.5 g of 10% Pd/C in 150 ml of MeOH was treated with 0.173 g (0.0048 mole) of dry HC1 in hIeOH, and the suspension was hydrogenated for 2 hr. The reaction mixt was filtered and the filtrate was concd. The residue was crystd from MeOH-Et20 to give 3.8 g (957,): mp 172', [ a ] %f2.5' '~ (c 4.2, DAIF). Anal. (C33H4~CleN40s) C, H, N. Poly(Gl~-Tyr-AIa-Gly)Gly-l-1~C Et Ester (I).-To a soln of 1.1 mg of glycine-l-14C E t ester HC1 (specific activity 3.15 mCi/ mmole) and 1.82 g (0.018 mole) of Et3N in 5 ml of DMSO was slowly added a soln of 3.0 g (0.00359 mole) of the polymerizing unit 5 in 25.0 ml of DMSO. The transfer vessels were washed with 5.5 ml of I)MSO which was added to the reaction mixt giving a final concn of 100 nimoles/l. The reaction mixt was shaken for 6 days and then centrifuged to yield the product which was washed with three 35-ml portions of HzO, three 35-ml portions of AIeOH, and three 35-ml portions of Et20 and dried to give 0.95 g (49.57,) of the blocked polymer. The protected poiypeptide was dissolved in 50 ml of 90% F&CO?TI and stirred for 50 min, and then concd under reduced presslire to yield the crude polypeptide 1. This material was washed with EtlO, suspended in 20 ml of HzO, and dissolved by the addition of 1 N
NaOH to pH 7.5. The soln was dialyzed against distd HzO for 2 days, then lyophilized to yield the Na salt of the polypeptide. This material was converted into its free acid by acidification with 1 N HCl to pH 2.5. The pptd polypeptide was collected by centrifugation and then lyophilized to yield 0.50 g (32%); radio assay, 33.3 nCi/mg indicates 73.7'3 incorporation of the radioactivelabel. Anal. (C~H~4N407.1.5 HzO)C, HI N. Molecular Weight Determination.-Calibrated columns of Sephadex G-100 (2.5 X 38.5 cm) and of Corning CPG 10-240 glass granules (2.0 X 28 cm) were employed for the molecular weight determination. Using 0.1 M NaC1-0.05 Ji' KHzPO4 corrected to pH 8.0 as eluent, 4 mg of poly(G1y-Tyr-Ala-G1y)Gly1-14C Et ester was passed through each of these columns. The polypeptide was eluted from each column in a vol equiv to that corresponding t o a mol wt of a t least 1 X lo5. Immunochemical Results.-Two rabbits 15 ere treated a t weekly intervals with 500 pg of poly(G1u-Tyr-Ala-G1y)Gly1 - X ' E t ester 1. The first 2 weeks they were injected intradermally using complete Freunds adjuvant as suspemion medium and the 3rd week they were injected sc. The injection on the 4th week was done iv using buffered saline. Bleedings were conducted on the following week and the serum from each animal was not found t o give a precipitin reaction with up to 10,000 pg of the polymer 1. Inhibition Studies.-To 1-ml aliquots of rabbit antisera to p~ly(Tyr-Glu-Ala-Gly)Gly-I-'~CE t ester was added incremental amounts of up to 7000 pg of the polypeptide 1. To each tube was added the equiv point amount of the antigen (30 pg) and the tubes were then incubated a t 37". Each tube showed a precipitin reaction. After standing a t 4" for 48 hr, the ppts were collected, washed twice with HzO, and collected by centrifugation. The amount of protein pptd was estimated by the absorbancy a t 280 mp of n 0.1 LV NaOH soln of the ppts. I t was found that the precipitin reaction between poly(Tyr-Glu-Ala-G1y)Glyl-14C E t ester and its antisera was 50% inhibited by the addition of 6000 pg of p0ly(Glu-Tyr-Ala-Gly)Gly-1-1~C Et ester 1.
Acknowledgments.-This work n as supported by a grant from the National Science Foundation.
Synthesis of [5-Valine,7-N-methylalanine]-angiotensin11, a Hypertensive RUDOLF H. ANDREATTA AND HAROLD A. SCHERAGA* Department of Chemistry, Cornell University, Ithaca, ,Vew York
14850
Received December 17, 19'70
,4 synthesis of the octapeptide H-Asp-Arg-Val-Tyr-Val-His-nIal-Phe-OH ([Val5,A/lal7]-angiotensin11) by the solid phase method is described. This peptide is an analog of [Va15]-angiotensin TI, in which 7-proline is replaced by Mal. The observation that this peptide exhibits marked hypertensive and myotropic activities indicates that the proline residue is not essential for these pharmacological properties; instead the presence of an Kmethylated amino acid in position 7 may be sufficient.
To date, more than 100 analogs4 of angiotensin have been reported5 in addition t o the naturally occurring [Valjl- and [Iles]-angiotensins I and 11. Many of these analogs have been prepared and their biological activity evaluated for the purpose of elucidating the correlation between structural features and biological function. (1) This work was supported by research grants from the National Science Foundation (GB-7571X) a n d from t h e National Heart Institute, National Institutes of Health, U. S.Public Health Service (HE-01662). (2) All amino acid residues are of t h e L variety. T h e following abbreviations are used: Roc = t-butoxycarbonyl; Narg = nitroarginine; Mal = N-methyla1an;ne: T E A = EtaN; T F A = FaCCOrH; AP-R'l = aminopeptidase M.3 (3) G. Pfleiderer, P. G . Celliers, M. Stanulovic, E. D. Wachamuth, H . Determann, a n d G. Braunitzer, Biochem. Z . , 340, 552 (1964). (4) These include analogs with substituted functional groups, with modified sequences, a n d with shorter or extended chain length. ( 5 ) For a survey of t h e literature u p to Oct 1965, see E . Schroder a n d K. Liibke, Peptides, 2 , 52 (1966).
On the basis of these investigations, it has been concludedj that 4 of the 8 amino acids constituting these peptides are essential for biological activity. These are 4-Tyr, 6-His, 7-Pr0, and 8-Phe. I n addition, it has been asserted5 that the C-terminal COzH must not be substituted. I n a recent investigation,6it was shown that the imidazole portion of 6-His is not required for biological function. The conclusion that 'I-Pro is essential for biological activity followed from an investigation of Page and Bumpus17who found that the replacement of 7-Pro in [Ile5]-angiotensinI1 by Ala was accompanied by a total loss of hypertensive activity. Replacement of Pro in angiotensin has been the subject of (6) K Hofmann, R. Andreatta, J. P. Buckley, IF7, E. Hageman, and .\. P. Shapiro, J . Amer. Chem. Soc , 90, 1654 (1968); R . Andreatta and K. Hofmann, zbzd., 90, 7334 (1968). (7) I. H P a g e a n d F M . Bumpus, Physzol. Rev., 41, 331 (1961).
490
Journal of Medicinal Chemistry, 1971, Vol. 14,KO.6
only t u o other studies. I t s exchange for hydroxyproline reduces the biological activitj to 77c,x and its replacement (expansion of ring) by L-pipecolic acid lowered the hypertensive activity to lye of that of the parent c o m p o u ~ i d indicating ,~ again that the Pro is required for biological activity. It may be that the importance of the Pro iide chain lies in the ability of the rigid p) rrolidiiie ring to bring the functionally essential :imino acids 4-Tj r, &His, and 8-Phe into proper relative positionA\la17]-angioten~iii I1 iz A potent hypertensive and n1j otropic agent. The hjliertensive activity of the A h 1 analog n-a- determined in 2 rat preparations againat (16) R . H .lndreatta a n d H. .I,Scheraga, unpublished results. (17) \\-e wish t o express our appreciation t o Dr. Tlierezinha B. Paiva of the Escola Paiilista de AIedicina, Sao Parilo, Brazil, for these results.
Journal of Medicinal Chemistry, 1971, Vol. 14, No. 6 491
[ VAL6,?VI.4L7]-ANGIOTENSIN 11
[Asn1,Va15]-angiotensinIII8 (angiotensinamide, Ciba). I n the pithed rat the Mal analog exhibited 22 f 2y0, and in the nephrectomized rat pretreated with "pentolinium tartrate" 16 f 1% of the potency of the reference standard. Determination of the myotropic activity on the isolated guinea pig ileum showed it to possess 4.3 O.lY0 of the potency of angiotensinamide (Ciba). Hypertensive activities of angiotensin I1 and analogs modified in position 7 are compared in Table 11.
*
TABLE I1 BIOLOGICAL ACTIVITIESO F ANGIOTENSIN A N D -4N.ILOGS Peptidea
Pressor activity'
Residue in position 7
[Valsl- and [Ile5]angiotensin I1 Pro [ Ile5,Hyp7]angiotensin I1 Hydroxyproline [ Ile5,Pip7]angiotensin I1 Pipecolic acid [Ile5,Ala'] angiotensin I1 Ala [Val5,hIal7]angiotensin I1 Mal 11 See text for lit refs to the various peptides. standard [ Asn',VaP] -angiotensin 11.
100
7 1
0
b
22 I n per cent of
Experimental Section A. Materials.-All solvents used were spectral grade. DRIF was dried (BaO) and distd. The Boc-amino acids were prepd according to t,he procedure of Schwyzer, et ~ 1 . ~ 9Purity was checked by tlc. Boc-S"-benzylhistidine was purchased from RIann Research Laboratories. The prepn contained approximately 107, of a ninhydrin-positive impurity (slower moving on tlc) which was removed in the following manner: 2.0 g of the material was treated with boiling dioxane (100 ml), and some iiisol material was removed by filtration. The filtrate was concd in z)acuo to approximately 50 ml, and the desired product crystd by the addition of hexane; white crystals: mp 180-182'; single C1-positive, ninhydrin-negative spot on tlc. The ion-exchange resin AG1-X2 was purchased from Bio-Rad Laboratories, Richmond, Calif. Sephadex G25 superfine was from Pharmacia, Uppsala, Sweden. Cross-linked chloromethylated polystyrene and DCI were from RIann liesearch Laboratories. Aminopeptidase &I was obtained from Ilijhm and Haas GmbH, 61 Darmstadt,,West Germany (distributor: Henley and Co., Inc., New York, N. Y. 10017). B. General Procedures.-hlelting points are Lincorrected. The values reported for [ a ] D were determined on a Cary 60 spectropolarimeter. Elemental analyses were performed by Galbraith Laboratories, Inc., Knoxville, Tenn. Prior t,o analyses the compounds were dried in uacuo over PZOSat 60'. The amino acid composition of acid and enzymic hydrolysates was determined with a Technicon amino acid autoanalyzer. Paper chromatography was performed o n Whatman No. 1 filter paper by the descending technique with the following systems: Rr', n-BuOH-AcOH-HzO, 4: 1:5 (upper phase); 1212, n-BuOHpyridine-AcOEI-HZO, 45: 30: 9:36. Using Merck's silica gel G plates, tlc was performed with the following solvent systems: &I, ~-BL~OH-ACOH-HZO, 60: 20: 20; &I1, ~-BuOH-ACOH-HZO, 40: 30: 30; &Ill, n-BuOH-pyridine-AcOH-Hz0, 30: 20: 6: 24. Acid hydrolysis was carried out in constant boiling HCl, contg l O $ i by vol of a 0.1 111 soln of phenol, a t 105' for 24 hr. AP-?*I digestion was performed as described by Hofmann, et ~ 1 . 2 0 C. Synthetic Procedures. Ts-N-methylalanine.-Methylation of Ts-alaiiinez1 with Ale1 according t o Fischer and Lip(18) Proposed as a n international standard by the Department of Biological Standards, National Institute f o r Medical Research, England, (19) R . Schwyzer, P.Sieber, a n d H. Kappeler, H e l o . Chim. Acta, 42, 2622 (1959). (20) K. Hofmann, F. M . Finn, M. Limetti, J. Montibeller, and G. Zanetti, J. Amer. Chem. Soc., 88,3633 (1966). (21) E.11.. McChesneyand TV, K . Swann, Jr., i b i d . , 69, 1116 (1937).
schitzI2 gave Ts-N-Mal in 947, yield; mp 121-122" (after 2 recrystns from CHCls-hexane); [(Y]DZo -8.8" ( C 2.0, EtOH).22 N-Methylalanine.HBr.-A soln of Ts-N-Mal (5.15 g) and phenol (3.76 g) in glacial AcOH satd with HBr (60 ml) was allowed to stand a t room temp overnight. The mixt was introduced into dry EtzO (400 ml) yielding an oil which crystd upon standing. After 1 hr a t 4' the crystals were collected and washed with EtzO. Recrystn from MeOlI-Et20 gave white, slightly hygroscopic needles; 3.10 g (86%); mp 146-148'; [aI2OD $5.8" (c 2.0, EtOH). Anal. (C4HloBrNO4) C, H, Br, N. N-Methylalanine.23-A soln of N-Mal.HBr (2.76 g) in HzO (15, ml) was passed through a column with anion-exchange resin AGl-X2 (acetate cycle) (dimensions, 2.0 X 10 cm). The column was eluted with water until ninhydrin neg. The solvent was evapd in vacuo, and the solid residue was recrystd from abs EtOH and dried in vacuo a t 60' over PzOF,; yield, 1.30 g (88%); [ a l Z o D +4.8' (C2.0, €110). Boc-N-Mal.-N-Methylalanine was converted into its Boc derivative by the method of Schwyzer, et UZ.,'~ in 87% yield; white needles from EtZO-hexane, mp 91-92'; [ a I z 0 D -31.8' (c 2.0, EtOAc). Anal. (CgH17NO4) C, H, N. OlsenZ4obtained the same product by basic saponification of methyl Boc-h'-methyl+ alaninate; mp 89-01'. Boc-Phe Resin.-A soln of 5.31 g (20 mmoles) of Boc-Phe and 2.8 ml (20 mmoles) of TEA in 100 ml of EtOAc was added to 10 g of chloromethylated poly(styrene-co-2.07, divinylbenzene) which contained 2.07 mmoles of Cl/g. The reaction mixt was stirred a t reflux temp for 24 hr. The esterified resin was collected and washed with EtOAc, hleOH, HzO, and RIeOH, and dried in vucuo; yield, 12.63 g. A sample was hydrolyzed in a sealed tube with 12 A' HC1-dioxane ( 1 : l ) for 24 hr a t 105'. The amino acid content of the hydrolysate rvas determined spectrophotometrically using the ninhydrin test as described by Yemm and Cocking, modified by Rosen.26 The substituted resin was found to contain 1.018 mmoles of Phe/g. Boc-~-benzylaspartylnitroarginylvalyl-O-benzyltyrosylvalyl~~~~-benzylhistidyl-~-methylalanylphenylalanyl Resin.-A portion of the Boc-Phe resin (3.0 g, contg a total of 3.05 mmoles of Boc-Phe) was introduced into a solid phase reaction vessel, and the following steps were used t o introduce each new amino acid iesidue: (1) 3 washings with 40-ml portions of glacial AcOH; HC1 in (2) cleavage of the Boc group by treatment with 1 glacial AcOH (40 ml) for 30 min a t room temp; (3) 3 washings with 40-ml portions of glacial AcOH; (4) 3 washings with 40-ml portions of abs EtOH; (5) 3 washings with 40-ml portions of DMF; (6) neutralization of the hydrochloride with 6 ml of TEA in 40 ml of D N F for 10 min; (7) 3 washings with 40-ml portions of DMF; (8) 3 washings with 40-ml portions of CHZ Clz, (9) addn of 10 mmoles of the appropriate Boc-amino acid in 20 ml of CHZClzand mixing for 10 min; (10) addn of 10 mmoles of DCI in 20 ml of CHZC12, followed by a reaction period of 4 hr a t room temp; (11) 3 washings with 40-ml portions of CHzClz; (12) 3 washings with 40-ml portions of abs EtOH. For coupling of the Boc derivatives of -V"-benzylhistidine and nitroarginine step 8 was deleted, and D M F was used as the solvent in place of CHzClz in steps 9-11. The reaction period for coupling of the Boc-Wm-benzylhistidine was increased to 16 hr. The fully protected polypeptide resin was washed 3 times with 40-ml portions of DMF, followed by 3 washings with 40-ml portions of abs EtOH, and drying in vacuo over KOH pellets; yield, 6.20 g (theor yield: 6.57 g, 3.05 mmoles of peptide resin). Following neutralization of the hydrochloride, the combined TEA s o h (step 6) and D M F washings (step 7 ) of each cycle were evapd to dryness, the residue was dissolved in dil "03, and the C1- content of the soln was detd by the Volhard method. z6, 27 (22) Fischer a n d Lipschitel2 give rnp 121.5-122.5°, [ ~ I Z ~ -6.6' D ( e 2.0, EtOH). (23) Fischer and Lipschitzlz obtained this product by treatment of TsMal with concd HCI a t looo a n d neutralization of the resulting hydrochloride with PbO. They report [ ~ I ~ O +D5 . 5 " ( c 1.5, HzO). (24) R . K . Olsen, J . Org. Chem., 96, 1912 (1970). ( 2 5 ) E. W. Yernrn a n d E. C . Cocking, Analyst, 80, 209 (1955); H . Roeen, Arch. Biochem. Biophys., 67, 10 (1957); see also J. P. Greenstein a n d M. Winita, Chemistry of the Amino ilcids, Vol. 2, Wiley, New York, N . Y., 1961, p 1309. (26) S. Visser, J. Roeloffs, K . E . T. Kerling, a n d E. Havinga, R e d . Trau. Chim. Pays-Bas, 87,559 (1968). (27) L. A. Shchukina, E . P. Semkin, and A . P. Smirnova, K h i m . P r i r . Soedzn., S,358 (1967) [Chem. Abstr., 68,87567(1968)I .
492 Journal of Medicinal Chemistry, 1971, Vol. 14, N o . 6
[ValS,Mal7]-angiotensin 11.-The protected octapeptide polymer was suspended in 40 ml of anhyd TFA, aiid a slow stream of Brz-free HBr was passed through the fritted disk of the renction vessel into the suspension a t room temp, with exclusioii of 1120. After 10 min the suspeiision was filtered, and the reFiii was washed 3 times with 20-ml portions of T were evapd to dryness under reduced pressure a t 25". The product was redimolved in TFA (30 nil), and the soli1 was reevapd. EtzO (250 ml) was added to the syrupy residue, and the ppt was collected, washed with Et&, and dried in. u a c m over KOIT pellets; yellow powder, 1.43 g. This criide, partially protected octapeptide was dissolved in hZeOI~-AcOII-H~C)(5: 1 : 1 ) (200 ml) and hydrogenated a t ordiiiary presslire over freshly prepared l'd black (1 g), After 24 h r an additional 1 g of catalyst v a s added, and the hydrogenation was coiitd for another 24 hr. The catalyst was filtered off with the aid of Celite, and the filtrate n.as coricd t o dryness in zlaczm. The residue was dissolved i n I I . 0 (30 ml), small amounts of insol materid were removed hy filtration, and the s o h was passed through a columi~with aiiioiiexchange resin AGl-X2 (acetate cycle) (dirneiisioiis: 2.0 x 1 0 cm). The coliimn was eluted with € 1 ~ 0until niiihydri1i iieg. The bulk of the solvent was removed in uncuo below 40", and the remainder was lyophilized. After drying i n zucuo over PZO,,, 1.041 g of a crude [Va15,~Ia17]-arigioteiisiii 11 preparitlioii w:is obtained; i t showed two major C1-Po-: impurities 011 tlc, one moving faster, the ot,her slower thaii the main cornponelit. Amino acid ratios of this crude material in acid hydrolysate are: Asp, 1.09; Arg, 0.99; Val, 2.12; Tyr, 1.04; I l k , 0.80; Phe, 0.96." This lyophilized powder was dissolved i n 4.5 1111 of 0.2 LY XcOII and subjected, in 3 approximately equal portiolls, to gel filtmt,ioii on a Sephadex G25 (superfine) column (dimensions: 2.1 X 165 cm) that had been equilibrated with 0.2 iV AcOII, and 150 fractions of 4.0 ml each were collected. h plot of the absorbances a t 280 nm of the various fractions showed 1 major peak with t,he maximum a t fraction 104 (with 2 minor peak,? at fractious 120 aiid 132). Tlc (systems I and 11)of samples showing an ab-orbance of more than 10% of that with maximum ahsorbance, using the Clz test, located practically homogeneous peptide in fractions 99-106, with fractions 90-98 and 107-1 15 containing detect able amounts of the desired peptide besides slower- (fractions 90-99) and faster- (fractions 107-115) moving contaminants. A typical chromatogram, with 379 mg of the crude peptide applied, yielded 178 mg in the central portion. Rechromatography of fraction.; 99-106 in the same way gave a plot with a single symmetrical peak (maximum a t fraction 106), and the homogeiieous fraction, from this second chromatogram (96-1 13) were pooled and lyophilized to give a white powder; yield, 141 mg, total yield 405 rng: (28) This result is in agreement n i t h t h e values of t h e Volhard titrations which also indicate incomplete incorporation of His.
ANDRIZATT.A A N D SCHLIM: I
Discussion The marlied degree of pharmacological activity cxhibited by [Val~,,,lla17]-ungioten~iii 11, which is reported in thi:, investig:itiori, constitutes evidence I1i:it Pro 111 position 7 of angiotensin is not essential for biological function. Our ou 11 result>, together ifith t h o v of prcvious investigationi (compare Table 11),1c:id ~ i tho C O L I clude that an opened pyrrolidine ring of 7-Pro C M I :iIw lead to ph:irmaeologieal activitj provided that I hr chemical nature of the i m d e Y is not altered, :iq it i q c.g , 111 the case of thc biologicnlly inactive 7-Llla :iiialog: 1 e , :dl that seems to be required i n position 7 for hiological activitj I \ :in S-methylated amino acid. N'tiilc S-meth? lation ciin blocl\ :I potenti:rl €1 bonding 4 t c , alter the allou ed conformations of the nearby b:tckbo~ic, or increase t lie lil~elihootlof occurrence of c i ~pcytide bonds, among other tliings, it i s not porsiblc, nt t l i i i time, to determine which of these effect.; is rcsponsibl(~ for the observed biological behavior Khile [YnlJ]- aiid [Val~,~Ia17]-angiotensiri I1 are i : ~ from p1iarm:tcologically equiwlent, coiisideratioii of AIa1 as a tool for exploring the importzince of proline for biological function of certain peptides is nevert helcss suggested Acknowledgment.--The authors express their appreciation to A h . Jane Derbenrfick and to A h . Hua Tjm for skillful technical assistance. 120) Mal, present h u t not determined the ninhrdrin color hield of \Is1 (max 440 nm) l e on11 approximate17 one-third t h a t of P r o a t its inaximuin (440 nm) I t s presence nas further pro7 en b3 tlc of t h e acidic 113d r o h sate (
