Polypeptides. Part XXII. The synthesis of peptides of α-benzylphenyl-. Alanine by the dicyclohexylcarbodi-imide method

摘要

J.C.S. Perkin IPolypeptides. Part XXW The Synthesis of Peptides of a-Benzylphenyl-alanine by the Dicyclohexylcarbodi-imide MethodBy G. C. Barrett, P. M. Hardy, T. A. Harrow, and H. N. Rydon," Department of Chemistry, The University,Exeter EX4 4 0 0Dicyclohexylcarbodi-imide appears to be generally useful for the synthesis of protected dipeptides of a-benzyl-phenylalanine and for adding a-benzylphenylalanyl residues to the N-terminus of larger peptides. It is not, how-ever, generally suitable for adding further amino-acid residues to peptides with an N-terminal a-benzylphenylalanylresidue or for coupling reactions using peptides with a C-terminal a-benzylphenylalanyl residue. Suitable N-protecting groups for a-benzylphenylalanyl peptides are benzyloxycarbonyl and o-nitrophenylsulphenyl.Ethylesters are not suitable for C-protection, since they are difficult to hydrolyse owing to steric hindrance; 2-methyl-thioethyl esters are not subject to this and can be used successfully.Syntheses of two protected peptides corresponding to sequences 6-9 and 1 4 in bradykinin are described.THE work described in this paper had as its ultimateobjective the synthesis of analogues of bradykinin inwhich the phenylalanine residues in positions 5 and 8were replaced, singly or together, by a-benzylphenyl-alanyl -NH*C(CH,Ph),*CO- residues.* Although thisobjective was not realised, the presence of two benzylside-chains gave rise to synthetic difficulties which seemworthy of record and comparison with earlier work onpeptide synthesis with other aa-disubstituted amino-acids.The aa-disubstituted amino-acid most fully studied asa peptide component is a-methylalanine, peptidesyntheses with which were carried out by Kenner 2?3 andFaust 475 and their colleagues.Both groups found thatthe acylation of the amino-group in a-methylalanine andits derivatives is much more affected by steric hindrancethan is acylation by the activated carboxy-group in suchcompounds, but this is not our experience with a-* The abbreviation Bphe is used for this residue. Otherabbreviations are those recommended by I.U.P.A.C. (see Biochem.Part XXI, B. Ridge, H. N. Rydon, and C. R. Snell, J.C.S.M. T. Leplawy, D. S. Jones, G. W. Kenner, and R. C .J., 1972, 126, 773). Nb = P-NO,*C,H,*CH,.Perkin I , 1972, 2041.Sheppard, Tetrahedron, 1960, 11, 39.benzylphenylalanine. Of the commoner N-protectinggroups, both benzyloxycarbonyl 24, and p-tolyl-sulphonyl were used successfully.For carboxy-protection t-butyl esters were favoured,3 althoughmethy12p3 and benzyl* esters were also used. Of thecommoner coupling methods, the dicyclohexylcarbodi-imide,3*4 mixed pivalic anh~dride,~, $-nitrophenylester,' a ~ i d e , ~ . ~ and acid chloride 294 procedures have allbeen used, with varying degrees of success. The presentpaper deals with our experience in applying the dicyclo-hexylcarbodi-imide method to the synthesis of peptidesof a-benzylphenylalanine ; the application of the oxa-zolinone procedure 293 will be reported later.a-Benzylphenylalanine was prepared by the method ofFelkin,s with modifications of the details of the Schmidtreaction on ethyl dibenzylacetoacetate which greatlyimproved the yield of the intermediate N-acetyl-a-3 D.S. Jones, G. W. Kenner, J. Preston, and R. C. Sheppard,J . Chem. SOC., 1965, 6227.4 G. Faust and 13. Lange, J . prakt. Chem., 1960, (4)11, 153.5 G. Faust and M. Kleppel, J. prakt. Chenz., 1960, (4)11, 123.6 J. F. Diehl and E. A. Young, J . Medicin. Chem., 1964, 7 ,7 W. J. McGahreii and M. Goodman, Tetrahedron, 1967, 23,8 H. Felkin, Bull. Soc. chim. France, 1959, 20.820.20171972NPSN P S-OH Hbenzylphenylalanine ethyl ester. No difficulty wasexperienced in the preparation of the N-o-nitrophenyl-sulphenyl derivative by reaction of a-benzylphenyl-alanine with o-nitrophenylsulphenyl chloride in alkalinesolution; this derivative was used extensively incoupling reactions and also as an intermediate in thepreparation of the N-succinimidyl and Z-methylthio-ethyl esters of a-benzylphenylalanine.coupled smoothly with o-nitroarginine $-nitrobenzylester in chloroform solution in the presence of dicyclo-hexylcarbodi-imide to give a 70 yield of the protecteddipeptide (I).The similar coupling of N-o-nitrophenyl-sulp henyl- 01- benzylphenylalanine with L-seryl-L-prolyl-L-phenylalanyl-o-nitro-L-arginine P-nitrobenzyl ester (VI)in acetonitrile likewise gave a 70 yield of the protectedAr-o-Nitrophenylsulphenyl-a-benzylphenylalanineOH H ’ ONbONbONbNO2NO2,NO2I IN ps-Bp he*Arg.ON b Nps*Bphe*Ser*Pro*Phe*Arg. ON bNPSpentapeptide (11).These results indicate that thedicyclohexylcarbodi-imide method is likely to begenerally useful for the introduction of a-benzylphenyl-alanine residues at the N-terminus of the growing peptidechain in stepwise peptide syntheses. However, attemptsto couple the protected dipeptide, N-benzyloxycarbonyl-glycyl-a- benzylphenylalanine with L-seryl-L-prolyl-L-phenylalanine-o-nitro-L-arginine 9-nitrobenzyl ester (VI)using dicyclohexylcarbodi-imide failed, even in thepresence of added 1,2,4-triazole 10 and N-hydroxy-succinimide; l1 the method is thus not generally applic-able to the synthesis of a-benzylphenylalanine peptidesby fragment condensation procedures involving peptidescontaining C-terminal a-benzylphenylalanyl residues.The dicyclohexylcarbodi-imide method is likewise ofonly limited value in coupling reactions involving theaniino-group of a-benzylphenylalanyl residues.Thus,although the coupling by this method of the appropriatecarbosy-components with a-benzylphenylalanine estersgave the protected dipeptides (111; R = Et), (111; R =CH,-CH,-SMe), and (IV) in yields of 75, 90, and 75,respectively, the corresponding reaction between N-o-nitrophenylsulphenyl-L-proline and a-benzylphenyl-alanyl-w-nitro-L-arginine P-nitrobenzyl ester gave a verylow yield of the protected tripeptide (V), the N-acylureaZ*Gly.Bphe*OR Z*Pro*Bphe*OE tNps*Pro*Bphe*Arg( NO,) *ON bderived from the carboxy-component being the mainproduct, and attempts to couple N-benzyloxycarbonyl-and N-o-nitrophenylsulphenyl-glycine with a-benzyl-phenylalanyl-L-seryl-L-prolyl-w-nitro-L-arginyl $-nitro-L.Zervas, D. Borovas, and E. Gazis, J . Amer. Chem. Soc.,1963, 85, 3660.lo H. C. Beyermann and W. Maassen-van der Brink, ActuChiin. Acad. Sci. Hung., 1965, 44, 187.(111) (IV)(V>’ ONbNO2benzyl ester failed completely, even with added 1,2,4-triazole.To summarise, our results indicate that althoughdicyclohexylcarbodi-imide is not generally applicable asa coupling reagent for the synthesis of peptides of a-benzylphenylalanine, it can be used successfully for thesynthesis of dipeptides of a-benzylphenylalanine and oflarger peptides containing N-terminal residues of thisamino-acid.As expected, N-deprotection of a-benzylphenylalaninepeptides is less affected by steric hindrance than isNPSZSer Pro PheNPS OEtOH H OEtOE tOE tSCHEME 1I I z v OEt(m)SCHEME 2C-deprotection.The o-nitrophenylsulphenyl group wasremoved smoothly and in high yield from the peptides(I) and (11) by treatment with hydrogen chloride indioxan and ethyl acetate, respectively, and the benzyl-oxycarbonyl group from (IV) by catalytic hydro-genolysis. The ethyl ester (111; R = Et) resisted allattempts at hydrolysis with either acid l2 or alkali,l1 F. Weygand, D. Hoffmann, and E. Wiinsch, 2. Naturforsch.,1966, 21b, 426.la J. R. Vaughan and J. A. Eichler, J . Amar. Chem. SOC.,1964, 76, 24742636 J.C.S.Perkin Idiffering in this respect from the corresponding a-methyl-alanine derivative; 2*6 that this difficulty is indeed dueto steric hindrance is suggested by the fact that themethiodide of (111; R = CH,-CH,*SMe) readily givesthe free acid (111; R = H) in aqueous acetone at pH10 at room temperature,13 a reaction known to involveattack at a point distant from the a-carbon atom of theamino-acid residue.l*Details of syntheses of the protected tetrapeptides(VI) (Scheme 1) and (VII) (Scheme 2) required for theprojected synthesis of bradykinin analogues are given inthe Experimental section ; dicyclohexylcarbodi-imidewas used in all the coupling reactions.EXPERIMENTALThe purity of all peptides and intermediates was con-firmed by t.1.c.on Kieselgel G. Compounds with freeamino-groups were detected by means of o.1570 ninhydrinin n-butanol at 100"; peptides with N-terminal a-benzyl-phenylalanine residues gave very weak ninhydrin reactions l5and were best detected, as were N-protected peptides, bythe chlorine-starch-iodide method.lsOrganic solutions were dried over magnesium sulphateand concentrated or evaporated under reduced pressure ina rotary evaporator. Light petroleum was the fractionb.p. 40-60" unless otherwise indicated. Optical rotationswere measured with an ETL-NPL Polarimeter, model 143,(path length of 1 cm).a-Benzylphenylalanine and DerivativesThe following procedure gave a much better yield thanthat (ca. 25) obtained by the unmodified method ofFelkin.8 Concentrated sulphuric acid (36.4 ml, 0.68 mol)and sodium azide (34.3 g, 0.48 mol) were added alternately,in small portions, to a stirred solution of recrystallised ethyldibenzylacetoacetate (105 g, 0.34 mol) in redistilled tri-chloroacetic acid (700 g) a t 75-85'.The mixture waskept overnight a t room temperature and then poured intoice-water (1 1). The precipitated solid was dissolved inether and the solution washed with SM-sodium carbonateand water, dried, and concentrated. The product whichcrystallised from the solution (82 g in two crops) was re-crystallised from cyclohexane-ether ; N-acetyl a-benzyl-phenylalanine ethyl ester so obtained (78 g, 75) had m.p.121-123" (lit.,8 124-125") ; the average yield from severalpreparations was 72 ; the quality of the trichloroaceticacid is important.Hydrolysis by the method of Felkin 8gave a-benzylphenylalanine, m.p. 307-308" (Iit.,l7 307-308") (Found: N, 5.4. Calc. for C,,H,,NO,: N, 5.5) in85-95 yield; the hydrochloride, obtained by rapid addi-tion of 611-hydrochloric acid to a hot solution of the acidin 2~-sodium hydroxide had m.p. 314" (lit.,8 314") (Found:N, 4.6. Calc. for Cl,H,,CINO,: N, 4.8).A suspension of the hydrochloride (15 g, 51 mmol) inethanol (300 ml) was saturated with dry hydrogen chlorideand then refluxed for 3 h. Evaporation and two repetitionsof this procedure gave a solid residue, which was extractedwith hot chloroform, leaving undissolved starting material(5.2 g, 35). Addition of ether to the chloroform solution,followed by two recrystallisations of the precipitate froml3 M.J. S. A. Amaral, G. C . Barrett, H. N. Rydon, andJ. H. Willett, J . Chem. SOG. ( C ) , 1966, 807.l4 P. Mamalis and H. N. Rydon, J . Chem. SOL, 1955, 1049.chloroform-ether, gave a-benzylphenylalanine ethyl esterhydrochloride (9.6 g, 58), m.p. 176175" (Found: C, 67.8;H, 6.9; N, 4.4. C1,H,,CINO, requires C, 67-6; H, 6.9; N,4*4y0).o-Nitrophenylsulphenyl chloride ( 10.5 g, 55 mniol) and2~-sodium hydroxide were added alternately in small por-tions over 20 min to a stirred solution of a-benzylphenyl-alanine (12.75 g, 50 mmol) in 2~-sodium hydroxide (25 ml)and dioxan (62.5 ml), the pH being kept at about 8.5 duringthe reaction. The solution was diluted with water (500 ml)and filtered. The filtrate was acidified (Congo Red) with0.5hl-sulphuric acid and the gummy precipitate taken up inethyl acetate, washed with water, and dried.The resultingsolution was concentrated to 250 ml and treated with di-cyclohexylamine (10 ml, 50 mmol). After 18 h a t O", theyellow solid which had separated was filtered off and re-crystallised from ethanol, giving N-o-nitrophenyZsuZphenyZ-a-benzylphenylalanine dicyclohexylammoniuw salt (25.2 g,86), m.p. 198-199" (Found: C, 69.3; H, 7.4; N, 7.5.C,,H,,N,O,S requires C, 69-2; H, 7.4; X, 7.1).The foregoing salt (5.9 g, 10 mmol) was shaken with ethylacetate (30 ml) and 0.25~-sulphuric acid (50 ml). The ethylacetate solution was separated, washed with water, dried,and evaporated.The residue, dried in a vacuum desic-cator, was heated at 70" for 24 h with triethylamine (2.42ml, 17-5 mmol) and 2-chloroethyl methyl sulphide (1-65 g,15 mmol) ; triethylamine (1.38 ml, 10 mmol) and 2-chloro-ethyl methyl sulphide (1.1 g, 10 mmol) were then added andthe mixture was heated for a further 36 h. More amine(0.69 ml, 5 mmol) and sulphide (0.55 g, 5 mmol) were thenadded. After being heated for a further 36 h, the productwas dissolved in ethyl acetate; the solution was washedsuccessively with water, 0.5~-sulphuric acid, saturatedsodium hydrogen carbonate, and water, dried, and evap-orated. The residue (4.0 g), dissolved in a mixture ofanhydrous ethyl acetate (18 ml) and ether (18 ml), wastreated with a freshly prepared solution of anhydroushydrogen chloride (25 mmol) in ethyl acetate (7.5 ml),followed after 2 min by anhydrous ether (180 ml).Evap-oration to dryness, trituration of the residue with ether,and recrystallisation from ethyl acetate gave a-benzyl-flhenylalanine 2-methylthioethyl ester hydrochloride (2.4 g,65y0), m.p. 154-155" (Found: C, 62.1; H, 6-8; N, 3-9.C1,H,4C1N0,S requires C, 62-4; H, 6-6; N, 3.8).N-Hydroxysuccinimide (0.50 g, 4.35 mmol) was added toa solution of N-o-nitrophenylsulphenyl-a-benzylphenyl-alanine from the dicyclohexylammonium salt (2.56 g, 4.35mmol) in dioxan (20 ml). A solution of dicyclohexyl-carbodi-imide (0.90 g, 4.36 mmol) was added and themixture shaken at room temperature for 18 h. Dicyclo-hexylurea was then filtered off, the filtrate evaporated, andthe residue kept overnight at 0" in a little ethyl acetate.Filtration, evaporation of the filtrate, and recrystallisationof the residue from propan-2-01-light petroleum gave N-o-nitrophenylsulphenyl-a-benzylphenylalanine N-succinimidylester (1.90 g, 87), m.p.73-75" (Found: C, 61.1; H, 4.8;N, 7.9. C,,H,,N,O,S requires C, 61.8; H, 4.6; N, 8.3);this ester is dimorphic, the more stable form, m.p. 187O,being obtained by recrystallisation from acetone (Found :C, 61.6; H, 4.6; N, 8.1). This ester (1-67 g, 3.3 mmol)was kept for 30 s with hydrogen chloride (9.9 mmol) inl5 Cf. L. Tailleur and L. Berlinguet, Canad. J . Chenz., 1961,39, 1309; J. Org. Chem., 1962, 27, 653.l6 H. N. Rydon and P. W. G. Smith, Natuve, 1962, 169, 922.l7 L.H. Goodson, I. L. Honigberg, J. J. Lehman, and W. H.Burton, J. Org. Chenz., 1960, 25, 19201972 2637dioxan (23 ml). Ether (100 ml) was then added and theprecipitated solid collected and recrystallised from chloro-form-ether, giving u-benzylphenylalanine N-succinimidylester hydrochloride (1-11 g, 87y0), m.p. 151-152" (Found:C, 58.8; H, 5.3; N, 6.9. C20H,lClN,0,,H,0 requires C ,59.0; H, 5.7; N, 6.9).Peptides of u-Benzylphenylalaninew-Nitro-L-arginine P-nitrobenzyl ester hydrobromide l8(720 mg, 1-65 mmol) and N-o-nitrophenylsulphenyl-u-benzylphenylalanine dicyclohexylammonium salt (975 mg,1-65 mmol) were shaken together in chloroform (60 ml) for2-5 h. Dicyclohexylcarbodi-imide (375 mg, 1-82 mmol) inchloroforni (10 ml) was then added and the mixture shakenovernight. A drop of acetic acid was added and theprecipitated dicyclohexylurea was filtered off.The filtratewas washed successively with water, M-hydrochloric acid,water, saturated sodium hydrogen carbonate solution, andwater, dried, and evaporated. The residue was taken upin a little acetone, kept overnight at O", and filtered from alittle more urea. Evaporation of the filtrate and recrystal-lisation of the residue from ethanol gave N-o-nitrophenyl-sulphenyl-u-benzyl~henylalanyl-o-nitro-L-arginine p-nitro-benzyl ester (I) (870 mg, 71), m.p. 99-102", aIDl9 - 10-0"(c 1.0 in EtOAc) (Found: C, 56-3; H, 5.3; N, 14.9. C,,H,,-N,O,S requires C, 56.4; H, 4.9; N, 15.1y0). This ester(1-4 g, 1.9 niniol) was kept for 1.5 min with hydrogen chloride(5.6 mmol) in dioxan (23 ml).Addition of ether (50 ml),filtration after 30 min at 0", and trituration with ethylacetate and ether, followed by recrystallisation from ethanol-ether gave a-benzylphenylalanyl-o-nitro-L-arginine p-nibro-benzyl ester hydrochloride (760 mg, 64), m.p. 213-215",,1,lB - 16.0" (c 1-0 in HCO-NMe,) (Found: C , 55.7; H, 5.5;N, 15.5. C,gH,4C1N70, requires C, 56.5; H, 5.5; N, 15.6).AT-o-Nitrophenylsulphenyl-u-benzylphenylalanine di-cyclohexylaninionium salt (850 mg, 1-44 mmol) and L-seryl-L-prolyl-L-phenylalanyl-o-nitro-L-arginine p-nitrobenzylester hydrochloride (p. 2638) (1.04 g, 1-44 mmol) were shakenfor 2 h in acetonitrile (20 nil). Dicyclohexylcarbodi-imide(320 nig, 1.51 mmol) in acetonitrile (6 ml) was then addedand shaking was continued overnight.Work-up as usual,with sodium carbonate instead of sodium hydrogen carbon-ate for the alkaline wash, followed by precipitation fromethyl acetate with ether, gave N-o-nitrophenylsulphenyl-u-benry 19 henylalan~~1-~-seryl-~-~rolyl-L-fihenyl~lanyl-o-nitro-~-arginine p-nitrobenzyl ester (11) (1.1 g, 71), m.p. 1 2 A126", u ~ ~ ~ -13.0' (c 1.0 in CHCl,) (Found: C , 57-3; H,5 . 5 ; N, 14.4. C5,H5,Nl1O,,S requires C, 58-0; H, 5.3; N,14.5). This ester (600 mg, 0-56 mmol) was kept for 30 swith hydrogen chloride (1-67 mmol) in ethyl acetate (15 ml).Addition of ether (80 ml) and reprecipitation from ethanol-ethyl acetate with ether gave u-benzylphenylalanyl-~-seryl-~-prolyl-L-phenylalanyl-o-nitro-L-arginine p-nitrobenzyl esterJijtdrochloride (470 mg, 87y0), m.p.155-157", uID25 - 44-6"(c 0-6 in HCO-NMe,) (Found: C, 55.8; H, 5.7; K, 14.4.C4,H55C1N1,011,2H,0 requires C, 55-5; H, 6.0; N, 14.1y0).Dicyclohexylcarbodi-imide (3.45 g, 16.5 mmol) was addedin small quantities to a solution prepared from N-benzyl-oxycarbonylglycine l9 (3.135 g , 15 mmol), triethylamine(2.1 ml, 15 mmol), a-benzylphenylalanine ethyl ester hydro-chloride (4.80 g, 15 mmol), and chloroform (105 ml). Themixture was shaken at room temperature for 24 h and thenworked up in the usual manner. Recrystallisation fromethyl acetate-light petroleum gave N-benzyloxycavbonyl-glycyl-u-benzylphenylala?.tine ethyl ester (I11 ; R = Et) (5.36g, 75y0), m.p.81-82' (Found: C, 70.4; H, 6.5; N, 5.8.CZ8H,,N,O, requires C, 70.9; H, 6.4; N, 5.9). N -Benzyloxycarbonyl-L-prolyl-u-benzylphenylalanine ethylester (IV), prepared similarly in 70 yield as an uncrystal-lisable gum, was hydrogenated for 4 h a t room tempera-ture over 5 palladised charcoal (500 mg) in 95 aqueoust-butyl alcohol (25 mi). Filtration, evaporation, treatmentwith ethereal hydrogen chloride, and recrystallisation fromethanol-ether-light petroleum gave L-prolyl-u-benzylphenyl-alanine ethyl ester hydrochloride (0.4 g, 50), m.p. 176",uID19 +24.0" (c 1.0 in EtOH) (Found: C, 65.9; H, 6.9;N, 6.7. C2,H,,C1N20, requires C, 66.2; H, 7-0; N, 6.7).Dicyclohexylcarbodi-imide (650 mg, 3.15 mmol) in aceto-nitrile (5 ml) was added to a solution prepared from N-benzyloxycarbonylglycine (630 mg, 3.0 mmol) , ct-benzyl-phenylalanine 2-methylthioethyl ester hydrochloride ( 1 100mg, 3.0 mmol), and triethylamine (0.415 ml, 3.0 mmol) inacetonitrile (10 ml).After 18 h a t room temperature themixture was worked up as usual and the product, dissolvedin acetone, was shaken with Zeo-Karb (H+ form; 2 x 3.0 g)for 18 h. Evaporation of the filtrate gave the protectedpeptide (111; R = CH,*CH,*SMe) as a gum (1.41 g, goyo),which could not be induced to crystallise. This was re-fluxed with methyl iodide (2 mi, 32.5 mmol) for 3 h ; moremethyl iodide (2 ml) was then added, followed by a further1 ml after 12 h. After being refluxed for another 3 h, thesolution was evaporated and the residue triturated withether.Recrystallisation from acetone gave N-benzyloxy-carbonylglycyl-u-benzylphenylalanine 2-methylthioethyl estermethiodide (1.25 g, 70), m.p. 104-105" (Found: N, 3.9.C,,H,,IN,O,S requires N, 4.2). The unrecrystallisedmethiodide (5.8 g, 11.1 mmol), dissolved in 25 aqueousacetone (20 ml), was titrated with @5~-sodium hydroxidea t pH 10 for 45 min; consumption of alkali then ceased.Filtration, acidification with O.25~-sulphuric acid, and re-crystallisation of the gummy precipitate from aqueousethanol gave N-benzyloxycarbowylglycyl-a-benzyl~henylalan-ine (111; R = H) (2.73 g, 54 overall), m.p. 171-172'(Found: C, 70.1; H, 6-0; N, 6.2. C2,Hz,N,05 requires C,69.9; H, 5.9; N, 6.3).N-o-Nitrophenylsulphenyl-L-proline dicyclohexylammon-ium salt (1-0 g, 2-22 mmol) and a-benzylphenylalanyl-o-nitro-L-arginine fi-nitrobenzyl ester hydrochloride ( 1-4 g,2-22 mmol) were shaken together in chloroform (30 ml) for1 h.Dicyclohexylcarbodi-imide (0-51 g, 2.44 mmol) inchloroform (10 ml) was added and the mixture shaken for60 h. The precipitated dicyclohexylurea was filtered offand the filtrate evaporated to dryness. The residue wastaken up in ethyl acetate, washed as usual, and concentratedto 10 ml. After 12 h at 5", the crystalline precipitate wasfiltered off and recrystallised from ethanol, affordingNN'-dicyclohexyl-N- (N-o-nitrophenylsulpheny 1) -L-prolylurea(0.46 g, 44), m.p. 193-194" (Found: C, 60.8; H, 7.1;N, 11.8. CZ4H,,N40,S requires C, 60.7; H, 7.2; N, 11-8y0).Addition of ether to the ethyl acetate mother-liquor gavethe protected dipeptide mixed with the original amino-component.Repeated washing with M-sulphuric acid,followed by aqueous 3 sodium carbonate, and recrystal-lisation from ethyl acetate-ether gave N-o-nitrophenyl-sul~henyl-L-prolyl-u-benzyl~henylalanyl-w-nitro-~-arginine p-nitrobenzyl ester (V), m.p. 114-116" (Found: C, 56.7; H,5-4. C4,H,,N,010S requires C, 57-1 ; H, 5.2) in low yield.l8 R. A. Boissonnas, S. Guttmann, and P.-,4. Jaquenoud,Helv. Chim. Acta, 1960, 48, 1349.M. Bergmann and L. Zervas, Bev., 1938, 65, 1192J.C.S. Perkin IPeptides related to BrcsdykininN-o-Nitrophenylsulphenyl-L-phenylalanine dicyclohexyl-ammonium salt (3.95 g, 7-9 mmol) and w-nitro-L-argininep-nitrobenzyl ester hydrobromide l8 (3.43 g, 7.9 mmol) werecoupled in acetonitrile (1 10 ml) with the aid of dicyclohexyl-carbodi-imide (1.78 g, 8.65 mmol).Work-up as usual gaveN-o-nitrophe ny lsulp heny l- L-Phenylalany Z-u- nitro- L-argininep-nilrobenzyl ester (4-4 g, 85) ; a sample recrystallised fromethanol (70 recovery) had m.p. 96-96', aD25 -23.5"(c 1.0 in CHC1,) (Found: C, 51-5; H, 4-9. C2,H,,N,ODSrequires C, 51.4; H, 4.6). The crude ester (8.3 g, 12.7mmol) was kept for 1 min with hydrogen chloride (38 mrnol)in ethyl acetate (30 ml). Addition of ether (200 ml),reprecipitation from ethanol-ethyl acetate with ether, andrecrystallisation from ethanol-ether gave L-Phenylalanyl-w-nitro-L-arginine p-nitrobenzyl ester hydrochZoride (3.97 g,58), m.p.197-198", aD25 -6.0" (c 1.0 in H20) (Found:C, 49.0; H, 5.2; N, 18.2. C2,H2,C1N70, requires C, 49.1;H, 5.3; N, 18.2). Repetition of these procedures withthe appropriate carboxy-components (Scheme 1) gave thefollowing: N-o-nitro~henylsulphenyl-~-~rolyl-L-~henylaZanyl-a-nitro-L-arginine p-nitrobenzyl ester (75), m.p. 112-1 15'(from ethyl acetate-ether), -47.0" (c 1.0 in CHC1,)(Found: N, 16.8. C33H37Ng010S requires M, 17.0) ;L-prolyl-~-~henylalanyl-w-nitro-~-arginirte p-nitrobenzyl esterhydrochloride (70), m.p. 137-139' (from ethanol-ethylacetate), aID26 -17.6" (c 1.0 in HCO*NMe,) (Found: C,50.2; H, 5.4; N, 17.4. C2,H31N80, requires C, 50.4; H,5.6 ; N, 17.7 yo) ; N-o-nitrophenylsulphenyE~-seryl-L-prolyLL-phenylalanyl-o-nitro-L-arginine p-nitrobenzyl ester (50),m.p.104-108" (from chloroform-ether), ,IDz6 - 77.3'(c 1.0 in CHCl,) (Found: C, 50-9; H, 4.9; N, 16.8. C36H42-NlOOl2S requires C, 51.5; H, 5.1; N, 16.7); L-seryl-L-@olyl-L-phenylalanyl-w-nitro-L-arginine p-rtitrobenzyl esterhydrochloride (84y0), m.p. 139-142" (from ethanol-ethylacetate-ether), -33.0" (c 1.0 in HCO-NMe,) (Found:C, 48.9; H, 5.6; N, 17.0. C,,H4,C1N,010,H20 requires C,48.7; H, 5-7; N, 17.0).The following (Scheme 2) were prepared similarly: N-o-nitro~henyZsuZphenyZ-L-pyolyZgZyc~ne ethyl ester (89 ) , m . p.101-103" (from ethyl acetate-light petroleum), 0(ID19-17.0" (c 1.0 in EtOAc) (Found: C, 51.2; H, 5.4; N, 11.9.C,,H,,N,O,S requires C, 51.0; H, 5.4; N, 11.9); L-prolylglycine ethyl ester hydrochloride (90). m.p. 115"(from ethanol-ether), aIDlD -45.8' (c 2-4 in H20) (lit.,,m.p. 1 19-120°, - 39.5') ; N-o-nitrophenylsuZ$henyLL-ProZyl-L-prolyZglycine ethyl ester (go), m.p. 48-51"(from benzenelight petroleum), aIDlg -140" (G 1.0 inEtOAc) (Found: C, 53.8; H, 5.8; N, 12.1. C,OH~~N@,$requires C, 53.3; H, 5-8; N, 12.4) ; ~-p~~ZyZ-~-pr~lyl-glycine ethyl ester hydrochloride (88), m.p. ca. 85" (hygro-scopic) (from ethanol-ether) (Found : N, 12.5. C14HzhClN30, requires N, 12.6) ; N-benzyloxycarbonyl-a-nitro-L-arginyl-~-~rolyZ-~-p-prolylglycine ethyl ester (VII) (45y0), m.p.84" (from ethyl acetate-ethyl-light petroleum), aIDlD-38.5" (G 1.0 in EtOAc) (Found: C, 53.6; H, 6.5; N, 17.5.C2,H4ON8O9 requires C, 53.1; H, 6.4; N, 17.7y0).We thank Messrs. Parke, Davis and Co. Ltd., for a researchstudentship (to T. A. H.).2/1439 Received, 20th June, 1972120 H. N. Rydon and P. W. G. Smith, J . Chem SOL, 1966, 3642.0 Copyright 1972 by The Chemical Societ