15 AMINO-ACIDS AND PEPTIDES By P. M.Hardy (Department of Chemistry University of Exeter Stocker Road Exeter) Last year’s Report took stock of the methods currently in general use for amino- acid protection formation of the peptide bond and structural elucidation and outlined present ideas on racemisation and peptide conformations. Methods and ideas do not change rapidly enough to warrant such an approach more than once in about every three years. This year’s Report then concentrates on innovations in the field and shows the advances made during 1968to update last year’s more general review. Publications this year include an introductory monograph on peptides and proteins,’ the Proceedings of the Ninth European Peptide Symposium,2 and I.U.P.A.C. “Tentative Rules” for the abbreviated nomenclature of synthetic polypeptides3 and a one-letter notation for amino- acid sequence^.^ Amino-acids.-Several new unsaturated amino-acids have been isolated.From the seeds of Aesculus cali$ornica 2-amino-4-methylhex-4-enoicacid (1;R1 = H R2 = H R3 = Me) 2-amino-6-hydroxy-4-methylhex-4-enoic acid (1; R’ = OH R2 = H R3 = Me) the dipeptide y-glutamyl-2-amino-4- methylhex-4-enoic acid and 0-(methylenecyclopropy1)-P-methylalanine (2; R = Me) together with 2-amino-4-methylhexanoicacid have been character- ised.’ L-( -)-2-Amino-5-methylhex-4-enoic acid (1;R’ = H R2 = Me R3= H) D. T. Elmore ‘Peptides and Proteins’ Cambridge University Press 1968. Peptides 1968 Proceedings of the Ninth European Peptide Symposium Orsay France April 1968 ed.E. Bricas North Holland Publishing Co.;(a)P. Sieber and B. Iselin p. 85 ;(b) B. Rzeszotarska and S. Wiejak p. 86; (c) E. Schnabel H. Herzog P. Hoffman E. Klauke and I. Ugi p. 91; (d)P. G. Pietta F. Chillemi and A. Corbellini,p. 104;(e) L. Birkhofer and F. Miiller p. 151 ;(f)R. Hirschmann p. 139; (4) R. B. Merrifield and V. Littau p. 179; (h) A. Loffett and J. Close p. 189; (i) K. Brunfeldt J. Halstrom and P. Roepstorff p. 194; (j)G. W. H. A. Mansfeld H. Hindriks and H. C. Beyerman p. 197; (k) R. Garner D. J. Schafer W. B. Watkins and G. T. Young p. 145; (I) E. Bayer H. Hagen- maier G. Jung and W. Konig p. 162; (m) E. Wunsch G. Wendlberger,E. Jaeger and R. Scharf p. 229 ; (n)E. Scoffone F. Marchiori L. Moroder R Rocchi and A.Scatturin p. 325; F. M. Finn J. P. Visser and K. Hofmann p. 330; (0)A Anastasi L. Bernadi G. Bosiso R De Castiglione 0.Goffredo G. Bertaccini V. Erspamer and M. Impicciatore p. 247; (p)S. Bajusz Z Paulay Z Lhng K. Medzihadszky L. Kisfaludy and M. Low p. 237; (4)V. K. Antonov L. I. Andreeva and M. M.Shemyakin p. 78; (r) M. Bodansky J. Izdebski I. Muramatsu and k Bodansky p. 306; (s) R G. Hiskey R L. Smith A. M. Thomas J. T. Sparrow and W. C. Jones jun.,p. 209; (t) H. Zahn W. Danho G. Schmidt J. Dahlman A. Costopanagiotis and E. Engels p. 222; (u)G. Gawne G. W. Kenner N. H. Rogers R C. Sheppard and K. Titlestad p. 28. I.U.P.A.C. Combined Commission on Biochemical Nomenclature Tentative Rules for Abbreviated Nomenclature of Synthetic Polypeptides Biochemistry 1968 7 483 Arch.Biochem. Biophys. 1968,123,633; J. Bid. Chem 1968,243,2451. I.U.P.A.C. Combined Commission on Biochemical Nomenclature Tentative Rules for a One-letter Notation for Amino-acid Sequences Biochemistry 1968,7,2703 ; Arch. Biochem Biophys. 1968,125 i-v; J. Biol. Chem. 1968,243 3557. L. A. Fowden and k Smith Phytochemistry 1968,7,809; D. S Millington and R C. Sheppard ibid. p. 1027. 510 P.M. Hardy has been found in the mushroom Leucocortinurius bulbiger,6 while L-2-amino- 3-formylpent-3-enoic acid (3 ;R = CHO) and~-2-amino-3-hydroxymethylpent-3-enoic acid (3;R = CH,*OH) have been isolated from another mushroom Bankera fuligineoalba.’ Degradation of hypoglycin A (2; R = H) to (+)-3- methylpentanoic acid of known S configuration now establishes the stereo- chemistry as ( +)-(2S,4S)-2-amino-4,5-methanohex-5-enoic acid.’ Conversion of allenic aldehydes into allenic amino-acids has been found useful only for the synthesis of 2-amino-3,3-dimethylalka-4,5-dienoic acids but a more general route involving the reaction of 1-bromoalkadienes with diethyl formylamino- malonate has been established.’ In this way hypoglycin A has been synthesised in only three stages.8 R I ,NH2 MeCH=C-CH ‘CO,H A survey of the amino-acids present in the seeds of forty species of the genus Acacia has been made and L-P-N-acetyl-ap-diaminopropionicacid isolated for the first time.lo E-N-methyl- and E-NN-dimethyl-lysines have previously been shown to occur in histones; c-N-trimethyl-lysine has now been isolated from the same source.A hepatotoxic amino-acid L-a-amino-samidino- caproic acid (indospicine) has been identified in Indigofera spicata.l2 The first example of a naturally occurring pyridinium containing amino-acid has been found in tobacco leaves. The diester of this amino-acid N-(3-amino-3-carboxy- propy1)-P-carboxypyridinium betaine (4),was synthesised from methyl-L-a- amino-y-iodobutyrate hydrochloride (derived from homoserine) by heating with ethyl nicotinate ; acid hydrolysis liberated material identical with the natural product. Other new naturally occurring amino-acids include rn-hydr~xyphenylglycine,~~ 3,5-dihydroxyphenylglycine,’ and p-hydroxymethyl- L-phenylalanine.15 Fusarinines A and B have been found to contain two and three residues respectively of 6-N-(cis-5-hydroxy-3-methylpent-2-enoyl)-6-N-hydroxyornithine (5) previously found in ferrirhodin.Labile ester bonds join the carboxy-group of one unit with the 6-N-hydroxy-group of another. l6 The asparagine residue of bacitracin has been converted into a 2,4-diaminobutyric G. Dardenne and J. Casimir Phytochemistry 1968,7 1401. ’ R. P. Doyle and B. Levenberg Biochemistry 1968,7 2457. D. K. Black and S. R. Landor J. Chem. SOC. (C) 1968,281,283. D. K. Black and S. R. Landor J. Chem. SOC.(C),1968,288. lo A. S. Seneviratne and L. Fowden Phytochemistry 1968,7 1039. K. Hempel H. W. Lange and L. Birkhofer Naturwiss. 1968,55 37. M. P. Hegarty and A. W. Pound Nature 1968,217 354. l3 M. Noguchi H.Sakuma and E. Tamaki Arch. Biochem. Biophys. 1968,125,1017. l4 L. P. Miiller and H. R. Schiitte Z. Naturforsch. 1968,23b 659. l5 N. H. Sloane and S. C. Smith Biochim. Biophys. Acta 1968,158,394. l6 J. M. Sayer and T. F. Emery Biochemistry 1968,7 184. Amino-acid and Peptides 51 1 unit by dehydration of the side-chain amide to a nitrile and subsequent reduction. After acid hydrolysis of the modified antibiotic the liberated 2,4-diaminobutyric acid was found to have the L-configuration showing that it must be the aspartic acid residue of bacitracin which has the D-configuration. l7 N-Acetylcysta-thionine has been isolated from the urine of cases of congenital cystathioninuria,' and the rather unstable mixed disulphide of P-mercaptolactate and cysteine has been identified in the urine of mentally defective patients.lg OH I r r2 (5) HO*CH2*CH2* -CH.C0.N.CH2*CH2.CH2* H.CO2H A new general synthesis of amino-acids from glycine involves alkylation of the NN-bis(trimethylsily1) ethyl ester after conversion into the sodio-derivative with sodium bis(trimethylsily1)amide.All the Si-N bonds are then cleaved rapidly by dilute aqueous hydrochloric acid to liberate the new amino-acid.20 The glycine residues in protected dipeptides can be alkylated photochemically in an acetone-initiated reaction ; isobutene but-1-ene and toluene produce leucine norleucine and phenylalanine respectively. 21 An interesting asym- metric synthesis of L-aspartic acid has been described. L-erythro-Diphenyl-1 ,2-ethanolamine (6),prepared from L-glutamic acid gives (7) on condensation with (6) (7) (8) C1 H,N- -H I kH,CO,Me E.Ratti C. Lauinger and C. Ressler J. Org. Chem. 1968,33 1309. T. Perry S. Hansen D. Love and C. A. Finch Nature 1968,218,178. M. Ampola E M. Bixby M. F. Efron R Parker W. Seddon and E. P. Young,Biochem J. 1968 107,16P. K. Ruhlmann and G. Kuhrt Angew. Chem Internat. Edn. 1968,7,809. D. Elad and J. Sperling Chem. Comm. 1968 655. 512 P.M. Hardy dimethyl acetylenedicarboxylate. Catalytic hydrogenation using Raney nickel stereospecifically generates (8) with the ester group axial. Hydrogenolysis of (8)with palladium as the catalyst followed by the addition of hydrogen chloride gives the hydrochloride of optically pure a-methyl L-aspartate (9).22D-( -)-a-Aminophenylacetic acid has been prepared by the hydrolysis of D-( -)-a-aminophenylacetonitrile ( + )-hemitartrate with aqueous hydrochloric acid.This seems to be the first preparation of an optically active amino-acid from the corresponding optically active a-ami~~o-nitrile.~~ N-Acetyl-N-(3-amino-2,4,6-tri-iodophenyl)-~-aminoisobutyric acid has been synthesised and found to be an excellent oral cholecystographic agent of low cis-Glycolation of 3,4-dehydroproline is described in the first part of a project to synthesise all the isomers of the hitherto unknown 3,4-dihydroxy- prolines.25 Racemisation during the conversion of cystine dimethyl ester into a-benzyloxycarbonyldiaminopropionicacid frustrated the synthesis of optically active material.26 N(2)-Hydroxyasparagine was also found to racemise during preparati~n.~~ Recent synthetic studies on amino-acids include the preparation of 2,5-dihydr0phenylalanine,~y6 b’-trihydroxy-~-leucine,~~ hexafluoroleucine [2-amino-4,4-bis(trifluoromethyl)butyricacid],30 the leucine antagonists DL-cyclopent-2-enyl and cyclohex- 1-enyl-alanine~,~’ and tri-N-methylhi~tidine.~~ DL-Alanine benzenesulphonate may be resolved by seeding a supersaturated solution in 97 % aqueous acetone with one optical antipode. A 91 % yield of almost optically pure material can be obtained. The mother liquor yields the other enantiomorph. 33 Resolution by stereoselective interaction of basic amino-acids (including arginine) and metal complexes has been described.For example when DL-histidine is added to DL-[CO(EDTA)] -ion in acidic aqueous ethanol the optically pure diastereoisomer [~-hisH,]( +)s46 1 [Co(EDTA)], 2H20 crystallises first.34 Lysine can also be resolved by adding a solution of + dodecatungstophosphoric acid slowly to a concentrated solution of K ( -)546 [Co(EDTA)] -(1rnol.) and DL-lysine hydrochloride (2mol.). The initial precipi- tate is the tungstophosphate of D-lysine. If sufficient is added then the L-lysine salt eventually precipitate^.^^ In the cobalt complex of diglycine (10)the protons of the C-terminal residue (H;) exchange with deuterium in alkaline deuterium oxide but the (H;) protons do not. In the corresponding complex of glycyl-L- alanine no change occurs in the c.d.spectrum during exchange so the reaction ’’ J. P. Vigneron H. Kagan and A. Horeay Tetrahedron Letters 1968 5681. 23 J. Schawartz and G. Eibel Chem and Ind. 1968 1698. 24 J. A. Korver Rec. Trau. chim. 1968,87 308. ” C. B. Hudson A. V. Robertson and W. R J. Simpson,Austral. J. Chem. 1968,21 769 26 L. Benoiton Canad. J. Chem. 1968,46 1549. ’’ E. Falco and G. B. Brown J. Medicin Chem. 1968 11 142. M. L. Snow C. Lauinger and C. Ressler J. Org. Chem. 1968,33 1774. ’’ F. Weygand and F. Mayer Chem Ber. 1968,101 2065. ’O J. Lazar and W. A. Sheppard J. Medicin Chem. 1968,11,138. 31 T. H. Porter R M. Gipson and W. Shive J. Medicin Chem. 1968,11,263. ’’ V. N. Reinhold Y. Ishikawa and D. B. Melville J. Medicin Chem. 1968 11 258. ’’ I. Chibata S. Yamada M. Yamamoto and M.Wade Experientia 1968,24,638. 34 R D. Gillard P. R Mitchell and H. L. Roberts Nature 1968 217 949. Amino-acids and Peptides 513 seems to be stereospecific. 3s 0.r.d. studies on N-dimed~nyl~~ and N-(2-pyridyl N-o~ide)~amino-acids suggest correlations with absolute configuration. ' The optical activity of the disulphide bond in L-cystine and some of its deriva- tives has been studied in 0 I-\ I=/"CHR'Co2H (R = amino-acid side chain) (R = amino-acid side chain) (13) L-and ~-3,5-Dicklorotyrosines have been made by the chlorination of tyrosine in propionic acid at 0-5". The product can be salted out. L-Amino- acid oxidase and catalase will attack the L-derivative. 39 Benzyloxycarbonyl-L-asparagine reacts with phosgene to give benzyloxycarbonyl-P-cyano-L-alanine.L-Asparagine itself reacts with phosgene to give P-cyano-N-carboxy-L- alanine anhydride which forms poly-( P-cyano-L-alanine) on treatment with t~iethylamine.~~ N-a-Formyl-L-tryptophyl peptides have been found to cyclise in trifluoroacetic acid to 3,4-dihydro-~-carboline-3-carboxamides (11); this may be useful for the synthesis of alkaloids derived from P-~arboline.~' Amino-acids will react with S-methylisothiosemicarbazide hydriodide in aqueous ethanol to produce N-aminoguanyl amino-acids (12). These deriva- tives have been condensed into peptide analogues.42 a-Amino-acids or their esters also react with 2,5-diethoxytetrahydrofuran in acetic acid to give a-pyrrolo-acids and esters (13). Regeneration of the amino-acid is not possible 35 R.D. Gillard P. R Mitchell and N. c. Payne Chem Comm. 1968 1150. 3b P. Crabbe B. Halpern and E. Santos Tetrahedron 1968,4299,4315. 37 V.Tortorella and G. Bettani Cazzetta 1968 % 316. D. Coleman and E.R Blout J. Amer. Chem SOC. 1968,90 2405. 39 K. R Brady and R P. Spencer J. Org. Chem. 1968,33 1665. 40 M. Wilchek S. Ariely and k Patchornik,J. Org. Chem. 1968,33 1258. 41 k Previero,MA Coletti-Previero and Lb-G. Barry Canad. J. Chem. 1968,46,3404. 42 J. Gante Chem Ber. 1968,101 1195. 514 P.M. Hardy and optical homogeneity has not been e~tablished.~~ Progress has been made in g.1.c. techniques enabling all the protein amino-acids to be separated on a single run.44 Prebiotic Studies.-There is much interest in models of prebiological systems.The thermal synthesis of polyamino-acids (‘pr~teinoids.)~~ and their MSH46 and glutamic acid oxyreductase4’ activities have been examined. The high- temperature synthesis of amino-acids from acetylene carbon dioxide and ammonia has been shown not to be a Strecker reaction4* U.V. irradiation of ammonium thiocyanate has produced methionine ; the sulphur-containing amino-acids have previously resisted synthesis under possible prebiotic condition^.^^ Experiments with oligomers of hydrogen cyanide such as diaminomaleonitrile have shown that when heated with water alone they will give rise to peptides which contain up to eleven types of amino-a~id.~’ Peptides containing up to five types of amino-acid have been prepared by irradiation of hydroxylamine and ethylene glycol mixtures at 185-235 nm.5 Montmoril- lonite gel (a clay of silicon and aluminium oxide layers) in water at pH 5 will convert alanine adenylate into homopeptides of D.P.10-12 in 16% yield.52 Aspartic acid copper complex is partially resolved when a supersaturated solution is seeded with wool or cotton,53 while D-tyrosine decomposes in solution more quickly than L-tyrosine when bombarded with polarised p-particles from 90Sr;54 these observations are of interest in connection with the natural predominance of L-amino-acids. L-Amino-acids have also been shown to stabilise nucleic acid helices more effectively than D-aminoacids. Peptides :Structural Elucidation.-The Edman degradation has been carried out with a copolymer of acrylic acid and styryl isothiocyanate.’ Fluorescein isothiocyanate can also be used in the Edman method; its fluorescence enables the thiohydantoin to be detected easily.57 An isotope dilution technique has also been described in which phenyl [35Slisothiocyanate is used.58 2-Fluoro- 43 J.Gloede K. PoduSka H. Gross and J. Rudinger Coll. Czech. Chem. Comm. 1968,33 1307. 44 A. Darbre and A. Islam Biochem J.,1968,106,923;C. W. Gehrke R W. Zumwalt and L. L. Wall J. Chromatog. 1968,37 398; See also G. E. Pollack and A. H. Kawauchi Analyt. Chem. 1968 40,1356;B. A. Halpern V. A. Close A. Wegmann and T. W. Westley Tetrahedron Letters 1968 31 19; F.Shakrokhiv and C. W. Gehrke J. Chromatog.,1968,36,31; J. R Coulter and C. S. Hann ibid.p. 42. 45 S. W. Fox and T. V. Waekneldt Biochim Biophys. Acta. 1968,160 246. 46 S.W. Fox and W. Ching-Tso Science 1968,160 3827. 47 G. Krampitz W. Haas and S. Baars-Diehl,Nntunviss. 1968 55 345. 48 K.Samochocka,A. L. Kawezynski and M. Taube Angew. Chem Znternat. Edn. 1968,7 392. 49 G. Steinman k E. Smith and J. J. Silver Science 1968 159 1108. 50 R E.Moser and C. N. Matthews Experientia 1968,24,658;R E.Moser A. R Claggett and C. N. Matthews Tetrahedron Letters 1968 1599. 51 A. Zamoram C. Lanzarinii and A. Rura Gazzetta 1968,9?3,210 214. ” M. Paecht-Horowitz I.U.P.A.C. 5th International Symposium on the Chemistry of Natural Products London July 8-13tk 1968,Abstract D24,p. 232 53 K. Harada Nature 1968,218 199. 54 A S.Goray Nature 1968,219 338.” G. Manecke and G. Giinzel Naturwiss. 1968,55,84. ’‘ E.J. Gabbay R Kleinman and S. R Shimshak J. Amer. Chem SOC.,1968 90,927. 57 H. Maeda and H. Kawauchi Biochem Biophys. Rex Comm. 1968,31 188. G. L. Callewaert and C. A. Vernon Biochem J. 1968,107 728. Amino-acids and Peptides pyridine N-oxide has been suggested for stepwise N-terminal analysis but it can also be used for carboxy-group activation giving rise to N-acyloxy-2- pyridones.59 7-Chloro-4-nitro-2,1,3-benzo-oxa-diazole is a new fluorogenic reagent for amino-acids. Derivatives are excited by visible light and the reagent is more stable to moisture and more soluble in aqueous solutions than dansyl chloride.60 Reduction of 3,5-dinitro-2-pyridyl derivatives of peptides with sodium borohydride at pH 8-9 gives dihydro- or tetrahydro- pyridine products with enhanced nitrogen basicity.Cleavage of the N-terminal amino-acid from such reduced peptide derivatives occurs at pH 5-6 over 12 hr at 40-50" or in 5 min. at 100". Other amide bonds and disulphide bridges are untouched.61 Tetrafluorosuccinic anhydride can be used for the reversible protection of protein amino-groups ; the derivative is cleaved by trifluoro- acetic acid.62 A six-stage sequential degradation of amino-acids from the C-terminus of a peptide has been carried out in a favourable case by use of ammonium thiocyanate and acetic anhydride. The peptidyl thiohydantoins formed are cleaved with acetohydroxamic acid under mild condition^.^ A method for selective cleavage of peptides at serine and threonine residues has shown reasonable results in preliminary tests on dipeptide derivatives.The side-chain hydroxy-groups react with phosgene to give O-chlorocarbonyl derivatives (14) which cyclise to oxazolidones (15) when heated under reflux in xylene. Mild alkaline treatment then liberates the carboxylic acid derived from the N-terminal amino-acid and a 2-0x0-oxazolidine-4-carboxylic derivative (16).64 Lysine residues can be converted into NN-dimethyl-lysine by a reductive alkylation method under conditions where disulphide bridges are not attacked.65 Modification of tryptophan and cysteine residues with (14)-NH*CHR*CONH-CH*CO-(15)-NH*CHR.CON-CH.C0-I I I kH2 k0 CH ClCO~O/ 'O/ (16) -NH*CHR.CO,H + N-CH.CO-I I 59 D.Sarantakis J. K Sutherland C. Tortorella and D. Tortorella J. Chem Soc. (C) 1968 72. 6o P. B. Ghosh and M W. Whitehouse Biochem J. 1968,108,155. 61 A. Signor and E.Bordignon Tetrahedron 1968,24,6995. 62 G. Braunitzer K Bayreuther H. Fujiki and B. Schrank Z. physiol. Chem. 1968,349 265. 63 G. R Stark Biochemistry 1968,7 1796. 64 T. Kaneko S. Kusomoto T. Inui and T. Shiba Bull. Chem Soc.Japan 1968,41,2155; T. Kaneko I. Takeuchi and T. Inui ibid. p. 974. 65 G. E. Means and R E Feeney Biochemistry 1968,7 2192 Diacetyl 6-Deoxy talose 0 p I OMe CH,Ph CHMe Mo % I *CH*C04NH*CH.C0,NH*&-I*C0,NH*CH*CH,0 Tri-o-met hyl % Me[CH,],,CH==CH-CH.CH,*CO,NHI I rhamnose r; i Phe i AlloThr i AlA i Alaninol @ Amino-acids and Peptides 517 sulphenyl halides has been further explored,66 and azobenzene-2-sulphenyl bromide has been found to be a selective reagent for cysteine thiol modifica- tion being unreactive towards amino-groups and tryptophan.This reagent has the advantages of high solubility and great stability in water since its true structure is 2-phenyl-1,2,3-benzo-thia-diazoliumbromide (17).67 Amino-terminal tyrosine residues can be determined spectrophotometrically after conversion into 5,7-dibromo-6-hydroxyindole-2-carboxamide with N-bromo- succinimide.6 Mass spectrometry. Techniques using N-methylation of amide groups to increase the volatility of peptides continue to be explored. Methylation of peptides by silver oxide-methyl iodide-dimethylformamide mixtures is not straightforward for methionine glutamic acid and aspartic acid residue^.^' It has been found difficult in some cases to determine the C-terminal residues of N-methylated large peptides as they are too weak to be seen by mass spectro- metr~.~' The structure of mycoside, (18)has been determined by mass spectro- metry of a derivative prepared by Hakamori methylation.This method of methylation originally developed for use with glycolipids involves the reaction of the methylsulphinyl carbanion in dimethyl sulphoxide with the peptide prior to treatment with methyl iodide. After 12 hr. amide NH methylation was complete. The mass spectra of N-acyl-N-methyl oligopeptide methyl esters has been discussed,72 and the mass spectrometry of amino-acids and peptides in general has been reviewed.73 Extensive studies on the modes of fragmentation of peptides containing aromatic and heterocyclic residues have been rep~rted.'~ Peptide Synthesis-Protecting groups.A study has been made of a series of aralkyloxycarbonyl amino-protecting groups and the 1-@biphenyl)-1-methylethoxycarbonyl residue has been proposed as especially suitable for use in peptide synthesis. This group is cleaved with dilute acetic acid 3000 times faster than is the t-butoxycarbonyl group which permits its selective removal in the presence of the latter. No effects due to steric hindrance are observed,in contrast to the trityl re~idue.'~*~" An alternative route to t-butoxy- carbonyl and t-pentyloxycarbonyl amino-acids by use of t-butyl and t-pentyl 66 E.Scoffone A. Fontana and R Rocchi. Biochemistry. 1968,7.971. 67 A. Fontana F. M. Veronese and E. Scoffone Biochemistry 1968 7 3901; A. Buroway F. Liversedge and C. E. Vellins J. Chem SOC., 1954 4481. M. Wilchek T.Spande and B. Witkop Biochemistry 1968,7 1787. 69 K. L. Agarwal R A. W. Johnstone G.W. Kenner D. S. Millington and R C. Sheppard Nature 1968,219,498. 70 D. W. Thomas B. C. Das S. D. GCro and E. Lederer ref. 52 Abstract A24 p. 38. E. Vilkas and E Lederer Tetrahedron Letters 1968 3089. 72 B. C. Das S. D. GCro and E. Lederer Nature 1968,217 547. 73 J. H. Jones Quart. Rev. 1968,22 302. 74 E. 1. Vinogradova V. M. Lipkin Y. B. Alakhov M. Yu Feigina Yu. B. Schvetsov Zhur. obshchei Khim. 1968,38,777; E. I. Vinogradova V. M. Lipkin Y.B. Alakhov and Yu B.S'chvetsov ibid. p. 787; M. M. Shemyakin Yu A. Ovchinnikov A. A. Kiryushin,E.I. Vinogradova Yu R Alakhov V. M. Lipkin and B. V. Rozynov ibid. p. 798. '' P. Sieber and B. Iselin Helu. Chim Acra 1968 51 614. 518 P.M. Hardy quinolin-8-yl carbonates is now available.2b,76 t-Butyl fluoroformate has also been used to introduce the t-butoxycarbonyl group; in contrast to t-butyl chloroformate it is stable at 0” for several months but the required carbonyl chlorofluoride is not readily 77 The useful derivatives N(a)-t- butoxycarbonyl-N(o)-t-butoxycarbonyl-L-arginine, some of its active esters and the t-butyl ester of N(o)-t-butoxycarbonyl-L-argininehave now been prepared. Catalytic hydrogenation of benzyloxycarbonyl amino-acid and peptide p-nitrophenyl esters in the presence of hydrochloric acid (1 equiv.) is reported to remove the amino-protecting group without appreciable reduction of the nitro-group.When prehydrogenated solvent and catalyst are used reaction is complete in 5 min.79 It has also been found possible to hydrogenolyse benzyloxycarbonyl groups quantitatively from peptides containing methionine in the presence of boron trifluoride+ther in anhydrous methanol. Under these conditions free carboxy-groups are esterified. This can be avoided by use of t-butyl alcohol or acetic acid as solvent but reaction times are much prolonged.8o Schiff bases derived from benzoylacetone have been proposed for amino-protection. Yields are high and the protected amino-acids can be coupled through mixed anhydrides or uia cyanomethyl or phenacyl ester derivatives.Recrystallisation of cyanomethyl esters of this type is however accompanied by racemisation. 81 Hydroxylaminolysis can be used to remove the phthaloyl amino-protecting group from peptides under mild conditions. The sodium salt of N-hydroxyphthalimide can be filtered off from the methanolic solution after 15 min. and the product can then be isolated.82 p-Nitrophenyl acetate and other esters except formates have been found to acylate homolysine lysine and ornithine exclusively at the o-ami9o-group at pH 11. At less alkaline pH values the reaction is preferential but not selective. ap-Diamino-propionic and butyric acids are not attacked selectively at any pH.83 Pentamethylbenzyl ethers of a-hydroxy-acids have been used successfully in depsipeptide synthesis.These esters are crystalline when the corresponding benzyl and t-butyl esters are oils and can be removed with cold trifluoroacetic acid.84 An alternative procedure to treatment with methyl iodide for sensitising C-protecting p-methylthioethyl ester groups towards alkali has been developed. Oxidation of a relatively alkali-sensitive series of y-benzylglutamyl peptides with a-C- terminal P-methylthioethyl esters by use of hydrogen peroxide in the presence 76 B. Rzeszotarska and S. Wiejak Angew. Chem Internat. Edn 1968,7 379; Annalen 1968,716 216. 77 E.Schnabel H. Herzog P. Hoffman E Klauke and I. Ugi Angew. Chem Internat. Edn. 1968 7,380;Annalen 1968,716 165. ” H.Arold and S. Reissmann Z. Chem. 1968,3 107. 79 J. Kovacs and R L. Rodin J. Org. Chem. 1968,33 2418. H. Yajima K. Kamasaki Y. Kinomura T. Oshima S. Kimoto and M.Okamoto Chem and Pharm Bull (Japan) 1968,16,1342 D.Breazu A. Balog C Daicoviciu and E Varga ref. 52 Abstract D22 p. 228. 82 0.Neunhoeffer G. Lehmann D.Haberer and G.Steinle Annalen 1968,712 208. 83 J. LeClerc and L.Benoiton Cad J. Chem. 1968,46,1047. 84 F.H. C. Stewart Austral. J. Chem. 1968,21,1327. Amino-acids and Peptides 519 of ammonium molybdate produced the P-methylsulphonylethyl esters which lost no y-ester on fission of the a-ester with alkali8’ The similarly alkali- labile 2-p-tolylsulphonylethyl esters have also been advocated for C-protec- tion. 86 4-(Methy1thio)phenyl esters can be used for C-protection during peptide synthesis and converted when required into an active ester by oxidation to the 4-(methylsulphony1)phenyl ester ;87 this active ester was originally introduced for peptide cyclisation.88 A preliminary study indicates that NN-dimethylaminoethyl esters of N-protected amino-acids are rapidly hydrolysed by 2-4 % sodium hydrogen carbonate in aqueous dimethylform- amide but are resistant to hydrogenolysis or acid cleavage. 89 Two new S-protecting groups for cysteine are proposed. The l-phenyl- cyclohexyl group emerged as the most promising from a study of eleven types of S-derivative. S-( 1-Phenylcyclohexy1)cysteine can be prepared by a boron trifluoride-ther-catalysed condensation of cysteine hydrochloride with 1-phenylcyclohexanol.The group is cleaved by trifluoroacetic acid.” S-Acet-amidomethylcysteine can be prepared from cysteine and acetamidomethanol in strongly acid solution. This protecting group is stable both to acid and to alkali including trifluoroacetic acid and liquid hydrogen fluoride at O” but can be removed by Hg’ + at pH 4.91 Peptide synthesis using O-~arbamoyl~~ and 0-alkoxycarbonylg3 tyrosine protection has been explored. The 2,2,2- trifluoro-N-benzyloxycarbonylaminoethylresidue has been suggested for the protection of serine and threonine hydroxy-groups. It can be introduced with N-benzyloxycarbonyl- l-chloro-2,2,2-trifluoroethylamine(19 ; R’ = C1 R2= PhCH,) in the presence of triethylamine and removed when required either by catalytic hydrogenolysis or by fission with hydrogen fluoride.94 The closely similar 1-butoxycarbonylamino-2,2,2-trifluoroethylgroup has been used to protect the iminazole NH of histidine; it is inserted by the reaction of (19; R’ = OAc or OBz R2= CMe,) with N-t-butoxycarbonylhistidine methyl ester.” The piperidino-oxycarbonyl group has also been proposed for histidine imino-group prote~tion.~~ An improved synthesis of O-benzyl- threonine without concomitant racemisation has enabled O-benzyl-N-t-butoxycarbonyl-L-threonine to be conveniently ~repared.’~ N(a)-(2-Nitro- phenylsulpheny1)-0-t-butyl-L-threonine, serine hydroxyproline and tyrosine P.M. Hardy H. N. Rydon and R C. Thompson Tetrahedron Letters 1968 2525. 86 A. W. Miller and C. J. M. Stirling J.Chem SOC. (C) 1968 2612 13’ 3.J. Johnson and P. M. Jacobs Chem Comm. 1968 73. ‘I3 R Schwyzer and P. Sieber Helu. Chim Acta. 1958 41 2190. A. E. Greben V. F. Martynov and M. A. Titov Zhur. obschei Khim. 1968,38,664. W. Konig R Geiger and W. Siedel Chem Ber. 1968,101 681. 91 D. F. Veber J. D. Milkowski R. G. Denkewalter and R. Hirschmann Tetrahedron Letters 1968,3057. 92 G. Jager R Geiger and W. Siedel Chem. Ber. 1968,101,2762. 93 R Geiger G. Jager and W. Siedel Chem Ber. 196% 101 2189. 94 F. Weygand W. Steglich F. Fraunberger P. Pietta and J. Schmid Chem Ber. 1968 101,923. 95 F. Weygand W. Steglich A. Maienhofer and k Bauer Chem Ber. 1968 101 1894. 96 G. Jager R Geiger and W. Siedel Chem. Ber. 1968 101 3537. 91 T. Mizoguchi G. Levin P.M. Woolley. and J. M. Stewart. J. Org. Chem. 1968 33 903. 520 P. M. Hardy have been obtained as solid dicyclohexylammonium salts and several active esters of each have been prepared.98 The 2,4-dimethoxybenzyl and 2,4,6- trimethoxybenzyl groups show great promise for the protection of asparagine and glutamine amide groups. They improve the solubility in organic solvents of peptides containing these residues and prevent dehydration to the nitrile during some coupling reactions. The o-protected amides are prepared by coupling the appropriate methoxybenzylamine with dicyclohexylcarbodi-imide to the free p-and y-carboxy-groups of a-protected aspartic or glutamic acids. The groups are stable to hydrogenolysis and to hydrogen chloride in methanol but treatment with trifluoroacetic acid at room temperature over 25-30 hr.liberates the free o-amides.2d* 99 OEt (2 1) Formation of the peptide bond. Several ingenious new methods of coupling amino-acids have been described. 2-Ethoxy-M-ethoxycarbonyl-1,2-dihydro-quinoline (20) does not easily react with amines but with N-protected amino- acids mixed anhydrides are formed presumably uia (21) liberating quinoline. To prepare peptides the mixed anhydride is formed in the presence of an amino-acid or peptide ester. Under these conditions racemisation was shown to be absent by the Young test probably because the slow formation but relatively rapid consumption of the mixed anhydride minimises its accumula- tion. O0 A method of peptide synthesis involving oxidation-reduction con-densation has been developed.The cupric salt of an N-protected amino-acid couples with an o-nitrophenylsulphenyl amino-acid ethyl ester in the presence of triphenyl phosphine to give the N-protected dipeptide ethyl ester triphenyl- phosphine oxide and cupric o-nitrophenylsulphenate. A further example is illustrated in (22).lo1 N-Protected amino-acid or peptide active esters can be coupled with free amino-acids or peptides in the presence of N-trimethyl- silylacetamide and a trace of sulphuric acid under anhydrous conditions without apparent racemisation. Reaction goes presumably through the amino-acid trimethylsilyl ester ; the resulting peptide ester is hydrolysed by 98 E. Wiinsch and F. Angelo Chem. Ber. 1968,101 323.99 F. Weygand W. Steglich T. Bjornason R Aktar and N. Chytil Chem. Ber. 1968 101 3623 3642; P. Pietta F. Chillemi and k Corbellini ibid. p. 3649. loo k Belleau and G.Mnlek J. Amer. Chem Soc. 1968,90 1651. T. Makaiyuma M. Veki M. Maruyama and R Matsueda. J. Amer. Chem. SOC..1968,W.4490. Amino-acids and Peptides 521 the sodium hydrogen carbonate wash during work-up.2e Peptide bond forma- tion has been found to occur in surprisingly high yields on a column of Dowex 50 (H') ion-exchange resin on which an amino-acid is held when a solution of a second amino-acid is simply passed down the column. Yields of up to 50% of dipeptide have been obtained.lo2 Synthesis of peptides by oxidation of N-acyl-a-amino-acid phenylhydrazides has been studied. The phenyl- hydrazides were prepared by a papain-catalysed condensation.After oxidation with N-bromosuccinimide or lead tetra-acetate the unstable but often crystal- line phenyldi-imide can be isolated and stored at -70" until coupled with the amino-component. Some racemisation has been found to occur. lo3 Use of thiazolidine-2,5-diones instead of N-carboxy-anhydrides for the synthesis of peptides in aqueous medium by reaction with free amino-acids at carefully controlled pH values as developed by Hirschmann '04 gives higher yields but racemisation occurs. Glycine however has less tendency to undergo isocyanate formation and histidine less tendency to undergo other side reac- tions than if the N-carboxy-anhydrides were used.2f '05 Further details and studies on o-hydroxyphenyl 4,5-dichloro-2-hydroxyphenyl,'06and piperidino active ester^''^ are reported.Their resistance to racemisation during coupling is ascribed to their possession of a neighbouring group capable of hydrogen bonding to the incoming amine and accepting a proton from it thus accelerat- ing aminolysis. N-t-Butyl-5-methylisoxazoliumperchlorate will form enol esters with N-protected amino-acids. These esters are stable enough to be stored but so far only coupling with benzylamine has been reported.''* (22) Z-L-Phe-OH + H-Gly-OEt + HgCl + (O-NO~*C~H~*S)~ + 2NEt3 + Ph,P + Z-L-Phe-Gly-OEt (89%) + 2NEt3H'Cl-+ H~(o-NO~.C~H~*S)~ + Ph3PO Solid-phase methods of peptide synthesis continue to receive much attention both from the point of view of improvement of materials and yields on coupling and also in their increasing routine application.Autoradiographs of solid- phase resin beads bound to peptides containing tritium-labelled proline show clearly that the peptide chains are located quite uniformly throughout the polymeric support and not just near the surface.2g Two papers on polymers derived from phenol for use in solid-phase synthesis have appeared. After methylation of phenol-formaldehyde resin with diazomethane it can be S. Yamashita and N. Ishikawa Experientia 1968,24 1079. H. B. Milne and C. F. Most jun.,J. Org. Chem. 1968,33 169. lo4 R Hirschmann R G. Strachan H. Schwam E. F. Schoen~waldt,H. Joshua H. Barkemeyer D. F. Veber W. J. Paleveda T. A. Jacob T. E. Beesley and R. G. Denkewalter J.Org. Chem. 1967 32 3415. lo' R S. Dewey E. F. Schoenewaldt H. Joshua W. J. Paleveda jun. H. Schwan H. Barkemeyer B. H. Anson D. F. Veber R G. Denkewalter and R. Hirschmann,J. Amer. Chem SOC.,1968,W. 3254. '06 J. H. Jones and G. T. Young J. Chem. SOC.(C) 1968,436. lo' J. H. Jones and G. T. Young,J. Chem. SOC.(C) 1968,53. lo* R. B. Woodward and D. J. Woodman J. Amer. Chem. SOC.,1968,90,1371. 522 P.M. Hardy chloromethylated in the usual way. Its utility is demonstrated by the synthesis of a hexapeptide.Iog A polyphenol resin prepared from phenol and s-trioxan in bis-(2-ethoxyethyl) ether has been used for a synthesis of oxytocin and fragments of physalaemin. ' Two new groups for attaching amino-acids to polystyrene resins have been explored (23 ;R = benzene ring of polystyrene).These can be esterified by N-protected amino-acids by use of carbonyl(di- imidazole) and are compatible with N-benzyloxycarbonyl protection. '' (23) RCO. [CHZI3- OH and RCH,*NAc. [CH2In. OH Peptides can be removed from polymer supports if they are present as sub- stituted benzyl esters by transesterification with an anion exchange resin. a-Functions of aspartic and glutamic acids can also however be trans- esterified during this process. Attempted preparation by the Merrifield method of hexapeptides containing the sequence Asp-Gly gave predominantly the succinimido-derivatives. This occurred during conventional synthesis only when the P-carboxy-group of the aspartic acid was esterified.I13 Three more types of automated solid-phase apparatus have been described.2~-i Two new polymeric hydroxy-compounds for activating N-acyl amino-acids upon esterification have been applied. Poly-(8-hydroxy-5-vinylquinoline) is a copolymer prepared from 8-benzyloxy-5-vinylquinolineand divinyl- benzene. Condensation of copoly(ethy1ene-maleic anhydride) with hy- droxylamine gives copoly(ethy1ene-N-hydroxymaleimide) a polymeric analogue of N-hydroxysuccinimide. 11' A novel way of utilising polymers for peptide synthesis has been described. The 4-picolyl ester of an amino-acid is coupled to an N-protected amino-acid by dicyclohexylcarbodi-imide in solution. After filtration from urea the dipeptide is absorbed on Sulphoethyl Sephadex through its basic ester group and excess of acylating agent and the co-products can be washed away leaving the pure dipeptide held to the Sephadex.The peptide can be eluted from the polymer by 2% triethylamine in aqueous tetrahydrofuran the amino-protecting group split off and the cycle is repeated until the desired peptide is built 'I6 Esters of p-dimethyl- aminoazobenzyl alcohol can be used in the same way; they have the added advantage of being easily followed on a Sephadex column by their dark red colour. ' '09 G. Losse C. Madlung and P. Lorenz Chem. Ber. 1968,101 1257. N. Inukai K. Nakano and M. Murakami Bull. Chem. SOC. Japan 1968,41,182. ''I M. A. Tilak and C. S. Hollinden Tetrahedron Letters 1968 1297. B. Halpern L. Chew V. Close and W. Patton Tetrahedron Letters 1968,5163. '13 M.A. Ondetti A. Deer J. T. Sheehan J. Pluscec and D. Kocy Biochemistry 1968,7 4069. l4 Van G. Manecke and E. Haake Naturwiss. 1968,55,343. '15 D. Laufer T. M. Chapman D. I. Marlborough V. M. Naidya and E. R Blout J. Amer. Chem. Soc. 1968,90,2696. R. Camble R Gamer and G. T. Young Nature 1968,217,247. T. Wieland and W. Racky Chimia (Switz.) 1968,22 375. Amino-acih and Peptides Racemisation. A method for determining racemisation in the C-terminal amino-acid of a benzyloxycarbonyl dipeptide after coupling with an amino-acid t-butyl ester has been evolved. After removal of the protecting groups the N-terminal amino-acid is degraded by the Edman technique and any resulting diastereoisomeric dipeptides are separated by paper chromatography."* The extent of racemisation of an amino-acid can be estimated by conversion to a dipeptide with L-leucine N-carboxy-anhydride and running the product on an amino-acid analyser to separate the diastereoisomeric dipeptides.By use of pmole samples one part of D in 10oO parts of L can be detected. Basic amino-acids are treated with L-glutamic acid N-carboxyanhydride to give dipeptides with favourable elution positions.' Racemisation of amino-acids can also be detected by the g.1.c. of N-(-)-menthyloxycarbonyl'20 or ( +)-2-butyl esterI2' derivatives. Diastereoisomeric dioxopiperazines can also be separated by g.l.c.'22 Use of a I3C-H satellite peak of the methyl group doublet in the n.m.r. spectrum of alanine dipeptides is claimed to improve the sensitivity of detection of racemisation tenfold over a previously published method.72 The ready racemisation of S-benzyl-L-cysteine has received further study.The pentachlorophenyl ester of S-benzyl-N-benzyl- oxycarbonyl-L-cysteine on incubation with [35S]-a-toluenethiol in the presence of triethylamine racemised without incorporation of radioactivity ruling out a mechanism involving p-elimination and re-addition. Under these conditions the corresponding p-nitrophenyl ester does incorporate radioactivity but this is thought to be due to conversion into the thiobenzyl ester since the radioactivity is lost again on hydrazinolysis of the product. S-Benzyl-L- cysteine active esters also show no incorporation of deuterium after treatment with alkaline deuteriomethanol seemingly also ruling out racemisation by direct ionisation of an a-hydrogen atom.123 Synthesis of Natural Peptide.-The outstanding achievement of the year was the synthesis of a peptide with the amino-acid sequence of C.pasteurianum ferredoxin which contains fifty-five amino-acid residues by the solid-phase technique. Amino-acids were coupled as their t-butoxycarbonyl derivatives by use of dicyclohexylcarbodi-imide in dichloromethane. After fifty-four coupling steps 38 % of free amino-group (relative to the amino-group liberated by the first t-butoxycarbonyl splitting) was available. This corresponds to an average yield of 98.9 % for the two steps in each synthetic cycle. A 51"/,crude yield of S-benzyl peptide was obtained. After removal of the benzyl groups the peptide was purified as the S-sulphonate.The yield of product from the E. Taschner,L. Lubiewska M. Smulkowski and H. Wajciekowska Experimeniu 1968,24 521. J. M. Manning and S. Moore J. Biol. Chem. 1968 243 5591. J. W. Westley and B. Halpern J. Org. Chem. 1968,33 3978. G.E.Pollack and A. H. Kawauchi Analyt. Chem. 1968,40 1356. "'J. W. Westley V. A. Close D. E. Nitecki and B. Halpern Analyt. Chem. 1968,40 1888; A.B. Mauger J. Chromatog. 1968,37,315. J Kovacs G. L. Mayers R H. Johnson. and U. R. Ghatak. Chem Comm.. 1968 1066. 524 P.M. Hardy thiol peptide was similar to the 15% of S-sulphonate obtained from natural ferredoxin under these conditions.26*'24 Five groups of workers have been studying porcine thyrocalcitonin the thyroid hormone which lowers plasma calcium by direct inhibition of bone breakdown.The amino-acid sequence of this dotriacontapeptide (24a) has been completely established by three groups working independently,'25* and partially elucidated by a fourth group.'" Two syntheses of material possessing full biological activity have appeared.'28*lZ9 The CIBA group link protected sequences 10-24 and 25-32 and finally add sequence 1-9 by a dicyclohexylcarbodi-imide coupling in the presence of N-hydroxysuccinimide as the preformed heterodetic cyclic peptide.lz8 The Sandoz group link sequences 10-19 and 20-32 before similarly adding sequences 1-9 already containing the disulphide bridge. The sequence 10-32 differs in the two cases in the degree of side-chain protection; in one peptide there is none,lz9 and in the other five t-butyl groups are present.l2 * Human calcitonin has been isolated from thyroid tumour tissue as a mixture of monomer and dimer. The dimer reverts to the monomer on treatment with ammonium hydroxide. The molecule is the same size as the porcine variety and has a similar disulphide bridge but differs in sequence at eighteen posi- tions (24b).I3' Synthesis has also been reported. 13' The pharmacology of thyrocalcitonin has been reviewed. 13' s---S (24) I I [a) H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala-Tyr-TrpArgAsn 12 3 4 5 6 7 8 9 101112131415 (b) GlY Met-Gly-Thr-Thr Gln-Asp. ~ Leu-Am-AspPhe-His-Arg-Phe-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-NH 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Phe-Lys-Thr -Pro Gln-Thr-Ala-Ile Val-GI y-Ala-~ 124 E.Bayer G. Jung and H. Hagenmaier Tetrahedron 1968,24,4853. It' F. W. Kahnt B. Riniker I. MacIntyre and R Neher Helu. Chim Act4 1968,51,214; R Neher B. Riniker H. Zuber W. Rittel and F. W. Kahnt ibid. p. 917; P. H. Bell W. F. Barg Jr. D. F. Colucci M. C. Davies C. Dziobnowski M. E. Englert E. Heyder R Paul and E. Snedeker,J.Amer. Chem SOC. 1968,W. 2704. Iz6 H. B. Brewer H. T. Keutmann J. T. Potts,jun. R. A. Rensfeld R. Schlueter and P. L. Munson J. Biol. Chem 1968,243,5739; J. T. Potts jun. H. D. Niall H. T. Keutmann H. B. Brewer jun. and L. J. Deftos Proc. Nat. Acad. Sci. U.S.A 1968,59 321. 127 T. E. Beesley R. E. Harmon,T. A. Jacob C. F.Hammick D. F. Veber F.J. Wolf R.Hirschmann and R. G. Denkewalter J. Amer. Chem. SOC.,1968,W 3255. 12' W. Rittel M. Brugger B. Kamber B. Riniker and P. Sieber Helu. Chim Acta 1968 51 924. 129 St Guttman J. Pless Ed Sandrin P-A Jacquenoud H. Bossert and H. Willems Helv. Chim. Acta 1968,51,1155. lJo B. Riniker R Neher R Maier F. W. Kahnt P. G. H. Byfield T. V. Gudmundson L. Galante and I. MacIntyre Helu. Chim Acta 1968 51 1738; R Neher B. Riniker R Maier P. G. H. Byfield T. V.Gudmundson and I. MacIntyre Nature 1968,220,984; R Neher B. Riniker W. Rittel and H. Zuber Helu. Chim Acta 1968 51 1900. P. Sieber M. Brugger B. Kamber B. Riniker and W. Rittel Helv. Chim Acta 1968,51 2057. A. Tenenhouse H. Rasmussen and C. D. Hawker Ann Rev. Pharmacol. 1968,8 319. Amino-acih and Peptides 525 A cyclic decapeptide antitoxin antamanide has been isolated from Amanita phalloides.If administered before or simultaneously with the toxins phalloidin or a-amanatine it counteracts their lethal action. The sequence (25) was determined by a combination of gas chromatography and mass spectrometry. (25) Pro -+ Phe - Phe - Val - Pro t 1 Pro tPhe tPhe tVal + Pro Antamanide has been synthesised from the linear decapeptide by a mixed anhydride cyclisation in 30% yield.'33 Further details of the synthesis of glucagon have appeared.2m- 134 The 0.r.d. of aqueous solutions of glucagon indicate that it is largely a random coil with perhaps one turn of an a-helix. There can be no steric objection to helix formation as it is largely helical in 2-chloroethanol.When the compound is left in acid solid possessing the antiparallel p-conformation is precipitated. 35 A second synthesis of secretin by use of a fragment condensation approach136 complements the original stepwise preparation. Peptides corresponding to two proposed sequences 42-46 of tobacco mosaic virus protein have been synthesised and the sequence Thr-Val-Val-Glu-Arg has been found to correspond to a tryptic peptide obtained from the protein. 38 The isolation structure and synthesis of the identical ovine and bovine gastrins (ValSAla'o-porcine gastrin I) has been reported. 39 The same group has prepared Leu" and Leu'.Le~'~ porcine gastrin I analogues.140 An exten- sive series of modifications of the C-terminal tetrapeptide sequence of gastrin has been detailed.I4' Bradykinin has been synthesised by a solid-phase method142 and also by an improved conventional method in which the N(o)-133 Tk Wieland G.Liiben H. Ottenheym J. Faesel J. Y. De Vries A. Prox and J. Schmid Angew. Chem Internat. Edn 1968,7 204. 134 E. Wunsch A. Zwick and A. Fontana Chem Ber. 1968,101 326; E. Wiinsch A. Zwick and E. Jaeger ibid. p. 336; E. Wunsch and G. Wendlberger ibid.,pp. 3418 and 3659; E. Wiinsch E. Jaeger and R Scharf ibid. p. 3664. 135 W. B. Gratzer C. H. Beavan H. W. E. Rattle and E M. Bradbury European J. Biochem. 1968 3 276. M. A. Ondetti V. L. Narayanan M. von Satza J. T. Sheehan F. F. Sabo and M. Bodansky J. Amer. Chem Soc. 1968,90,4711. 13' M. Bodansky M. A. Ondetti S. D. Levine and N. J.Williams J. Amer. Chem SOC.,1967 SS 6753. J. D. Young C. Y.Leung and W. A. Rombauts Biochemistry 1968,7,2475. 139 K. L. Agarwal J. Beacham P. H. Bentley R A. Gregory G. W. Kenner R C. Sheppard and H. J. Tracy Nature 1968,219 614. 140 G. W. Kenner J. J. Mendive and R C.Sheppard J. Chem SOC.(C) 1968,761. 14' K. L. Agarwal G. W. Kenner and R C. Sheppard J. Chem SOC.(C), 1968,1384; H. Gregory A. H. Laird J. S. Morley and J. M. Smith ibid. p. 522; H Gregory D. S. Jones and J. S. Morley ibid. p. 531; H. Gregory J. S.Morley J. M. Smith and M. J. Smithen ibid. p. 715; J. S. Morley and J. M. Smith ibid. p. 726; H. Gregory and J. S. Morley ibid. p. 910. 14' M. Fridkin A. Patchornik and E. Katchalski J. Amer. Chem SOC. 1968 90 2953. 526 P.M. Hardy tosyl group used for arginine protection is removed rapidly and cleanly by liquid hydrogen fluoride at despite a previous report of its stability to this reagent.144 N(o)-Nitro protecting groups can also be cleaved from bradykinin by hydrogen fluoride in the presence of anisole. Bradykinin and the hitherto unknown Val' Thr6 analogue have been found in the skin of Rana nigromaculata Hall0we11.~~~ Structure-function relationships in some partly synthetic modified ribonucleases continue to be explored. 2n* 14' Full details of the isolation and structural determination of caerulein have now appeared the synthesis of eighteen analogues is reported.20 Details of the synthesis of the sequence 1-20 of melittin are also available.'" ACTH and MSH. Monkey P-MSH (Arg6-bovine P-MSH) has been synthe- sised.The &-amino-group of the single lysine residue was protected with a formyl group which was eventually removed with hydrazine acetate. ' ' A synthetic stereoisomer of a-MSH containing four D residues has been prepared but is much less active than u-MSH.'~~ Human ACTH has now been synthe- sised,2r and a 6-aminovaleric acid analogue of the sequence 11-19 of ACTH has been reported. Three analogues of porcine corticotropin with high steroidogenic activity in uiuo have been isolated from pituitary sources. They are similar in size to ACTH but differ in amino-acid comp~sition.'~~ A new pituitary peptide H-Arg-Trp-Asp-Arg-Phe-Trp-OH has been identified but has no vasopressin- or corticotropin-releasing activity.Oxytocin and vasopressin. Isomeric dimers of oxytocin probably parallel and antiparallel dimers have been isolated as by-products in the oxidation of oxytoceine to oxytocin. They can also be prepared from oxytocin by di- sulphide interchange in the presence of triethylamine. Both compounds have low oxytocic activity. '56 Acetone inactivation of oxytocin has been found to be due to the formation of a substituted 2,2-dimethyl-imidazolidin-4-one derivative of the N-terminal Cys-Tyr sequence (26),in which the isopropylidene group from acetone forms a bridge between the N-terminal amino-group and the NH of the adjacent peptide bond. Treatment with 0.25% acetic acid at 143 R H. Mazur and G. Plume Experientia 1968,24 661. 144 S. Sakakibara Y. Shimonishi Y.Kishida M. Okada and H. Sugihara Bull. Chem SOC.Japan 1967,40,2167. 14' S. Sakakibara H. Nakamizy Y. Kishida and S. Yoshimura Bull. Chem SOC.Japan 1968,41 1477. 14' T. Nakajima Chem and Pharm Bull. (Japan) 1968,16 769. 14' R Rocchi L. Moroder F. Marchiori E. Ferranese and E. Scoffone J. Amer. Chem SOC., 1968 90,5885. 14' A. Anastasi V. Erspamer and R Endean Arch. Biochem Biophys. 1968,125 57. 149 A. Anastasi,L. Bernardi G. Bertaccini G. Bosiso R DeCastiglione,V. Erspamer 0.Goffredo and M. Impicciatore Experientia 1968,24 771. lSo E. Schroder Annalen 1968,711,227. H. Yajima Y. Okada Y. Kinomura and H. Minami J. Amer. Chem SOC., 1968,90 527. lS2 H. Yajima and K Kawasaki Chem and Phann Bull. (Japan) 1968,16 1387. lS3 W. Oelofson and C.H. Li J. Org. Chem. 1968,33 1581. 154 S. Lande M. Sribuey R K McDonald and W.Boxt Biochem Biophys Actu 1968,154,429. lS5 A. V. Schally and J. F. Barrett Biochem Biophys. Acta 1968,154 595. D. Yamashiro D. B. Hope and V. du Vigneaud J. Amer. Chem. SOC.,1968 90 3857. Amino-acids and Peptides 90" for 0-5 hr. regenerates the active hormone in good yield. '" Preparation of new analogues of oxytocin,15* vasopre~sin,'~~ and angiotensin16' are reported. Val'-angiotensin 11 containing an isosteric analogue of histidine P-(pyrazoly1)-L-alanine has high pressor and myotropic activities indicating that activity is independent of the acid-base properties of the irnidazole ring. OH Both swine kidneys and human urine contain a new angiotensinase which breaks the Pro-Phe bond of angiotensin.The resultant heptapeptide is bio- logically inactive.162 Thin-film dialysis and 0.r.d. studies have shown that a remarkable increase in the size of the angiotensin I1 molecule occurs which coincides with the ionisation of the tyrosine phenolic H-Leu-Leu-Val-Tyr-OMe and H-Leu-Leu-Val-Phe-OMe have been found to be competi-tive inhibitors of the renin-angiotensin system. Insulin. Pro-insulin the biosynthe tic precursor of insulin has been isolated from a porcine insulin preparation. The A and B chains are linked (27) with a loop of thirty-three amino-acids (28). Trypsin cleaves the bond between the A chain and the loop correctly but frees the B chain one amino-acid too far down liberating desalanine insulin. "'Cod insulin is found to differ in sequence from ox insulin at sixteen positions.The B chain is one amino-acid short at the C-terminus and has one extra residue at the N-terminus.166 Insulin 15' V. J. Hruby D. Yamashiro and V. du Vigneaud J. Amer. Chem SOC. 1968,90,7106; D. Yama-shiro and V. du Vigneaud. ibid. p. 487. H. Takashima V. du Vigneaud and R B. Merrifield J. Amer. Chem SOC. 1968 90 1323; M. Bodansky and R J. Bath Chem Comm,1968,766; M. Manning T. C. Wiu J. W. M. Baxter and W. H. Sawyer Experientiu 1968,24,660;E Klieger ibid. 1968,24,13;Zh.D. Bespalova,V. F. Martynov and M. k Titov Zhur. obshchei Khim. 1968,38 1684; 0.k Kaurov I. M. Lushchitskaya and V. F. Martynov ibid. pp. 720 and 724; H. Nesvadba K. JoSt J. Rudinger and F. Sorm Coll. Czech. Chem.Comm. 1968,33 2918; k Chimiak K Eisler K JoSt and J. Rudinger ibid. p. 2918. J. Meienhofer and Y. Sano J. Amer. Chem SOC.,1968,90,2996. 160 M. C. Khosla N. C. Chaturvedi R R Smeby and F. M. Bumpus Biochemistry 1968,7 3417. K. Hofmann R Andreatta and H.Bohm J. Amer. Chem SOC. 1968,90,6207. 16' H. Y.T.Yang E. G. Erdos and T. S. Chiang Nature 1968,218 1224. 163 M. T. Franze de Fernhdez A. E.Delius and A. C. Paladini Biochem Biophys. Actu 1968,154 223. 164 T. Kokaby E. Ueda S. Fujimoto K. Howada k Kato H. Akutsu and S. Yamamura Nature 1968,217,456. 16' R E. Chance R M. E Ellis and W. M. Bromer Science 1968,161 165. 166 K. B. M. Reid P. T. Grant and A. Youngson Biochem J. 1968 110 289. 528 P. M. Hardy containing cystathionine in place of the two cysteine residues involved in the intra-A chain disulphide bridge ('carba' insulin) has been synthesised.Hypo- glycaemic activity is of the same order as that of synthetic insulin. 167 Cyclic Peptides.-The use of mixed anhydrides for peptide cyclisation has been investigated,'68 and the method has been applied in the synthesis of antamanide (25). Two papers discuss improved dioxopiperazine syntheses. Free dipeptides or their hydrobromides cyclise in high yield when heated in phenol to just below its b.p.;16' dipeptide ester formate salts when boiled in neutral solvents such as toluene also give good yields of sterically pure dioxopiperazines.''O Cyclisation of dipeptide methyl esters by treatment with ammonia in methanol on the other hand has been shown to cause 5-40 % racemisation.''O In connection with studies on the antibiotic albo- mycin a series of cyclic hexapeptides containing only ornithine and serine residues have been prepared and their complexes with iron have been studied.The yields on cyclisation were found to be very dependent on the configuration of the amino-acids present. Cyclisations were carried out by the aide method. ' ' Studies on the transannular interactions of 1-(hydroxyalkanoyl)piperazine-2,5-diones172 and medium sized cyclodepsipeptides2q are reported. Trans- annular amide-amide interactions have been examined by mass spectro- metry. '73 Aromatic o-mercaptoacyl-lactams such as N-(0-mercaptobenzoy1)- 6-valerolactam have been found to form the thiacyclol or the cyclodepsipeptide (29) spontaneously.'74 The L-and D-forms of P-(0-t-butylsery1oxy)propionic acid cyclodimerise in a straightforward way on cyclisation but the DL-compound yields both the meso form and a racemic mixture of the D,D-and LA-forms. The rneso-form has been synthesised unambiguously. '' 16' K. JoSf J. Rudinger H. Klostermeyer and I€ Zahn 2.Naturjbrsch. 1968,23b 1059. Th. Wieland F. Jiirgen and H. Faulstich Annalen 1968,713,201. 16' K. D.Kopple and H. C. Ghorazian J. Org. Chem. 1968,33,862 D. E Nitecki B. Halpern and J. W. Westley J. Org. Chem. 1968,33,864. N. A.Poddubnaya and A. M. El-Haggar,Zhur. obshchei Khim. 1968,38,450;N. A. Poddubnaya A. M. El-Haggar I. N. Skvortsova and G. L Balandini ibid. p. 732 V. K. Antonov A. M Schkrob and M M.Shemyakin,Zhur. obshchei Khim. 1968,38,2225. 173 V. V. Denisov V. A. Ruchkov N. S. Vulfson T. E. Agadzhanyan V. K. Antonov and M. M. Shemyakin Zhur. obshchei Khim. 1968,38,770. 174 M. Rothe and R Steinberger Angew. Chem Internat. Edn 1968,7,884. C. H. Hassall and J. 0.Thomzs J. Chem SOC.(0,1968 1495. Ammo-acids and Peptides 529 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 (28) Leu-Gly-G1 y-Leu-Gln -Ala-Leu-Ala-Leu-Glu-Gl y-Pro-Pro-Gln-Lys-Arg 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Two groups of workers have independently concluded that the structure of the peptide alkaloid ceanothine B should be revised to (30).'76i177 Complete hydrolysis of dihydroceanothine B yields tyramine,177 and not o-aminoethyl- phenol as would be expected from the structure originally proposed.'78 Mass spectral evidence also points to (30).176Elucidation of the structure of viomycin first isolated in 1958 has proved difficult.Acid hydrolysis yields the amino- acids L-serine L-aP-diaminopropionic acid L-P-lysine and a new basic amino-acid viomycidine (ratio 2 :1 :1 :l) together with carbon dioxide ammonia urea and traces of glycine. The structure (31) is now proposed for viomycin although it is possible that the p-lysyl and one of the seryl units may be reversed in position. Viomycidine appears to be an artefact derived from the guanidine unit;'79 its structure (32) has been confirmed by mass spectrometry. The dehydroamino-acid unit proposed which breaks down on acid hydrolysis agrees with the spectral properties of viomycin and other degradative evidence.179 0 0 II 176 F. M. Klein and H. Rapaport J. Amer. Chem Soc. 1968,90 3576. 177 R E. Semis and A I. Kosak J. Amer. Chem Soc. 1968,90,4179. 17' E. W. Warnhoe S. K Pradham and J. C. N.Ma Canad. J. Chem. 1968,43,2594. B. W. Bycroft D. Cameron L. R Croft A Hassanali-Walji k W. Johnson,and T. Webb Tetrahedron Letters 1968. 5901 ;see also T. Kitagawa Y. Sawada and T. Miura ibid. p. 109. J. C. Floyd J. A. Bertrand and L R Dyer Chem Comm.,1968,998. 530 P.M. Hardy Gramicidin S18' and Gly5G1y1O-gramicidin S18* have been synthesised by a solid-phase method and retrogramicidin S' and all-L-gramicidin S'84 by conventional methods. The latter was cyclised with o-phenylene chlorophosphite and cyclopenta- and cyclodeca-peptides were obtained in a 2:3 ratio.The conformation adopted by gramicidin S in solution continues to attract attention. A preliminary n.m.r. study has been interpreted to support a compact model previously proposed on 0.r.d. evidence,' but more detailed work'86 is thought to show a conformation fairly similar to that adopted in the crystal,' 87 i.e. an all-trans-amide antiparallel pleated sheet The phthalimide group is a promising marker for signalling the proximity of the phenyl side- chains of phenylalanine residues by n.m.r. Absorption occurs in a 'window' region at ca. 8 p.p.m.188 The proximity of the D-phenylalanine residues in diphthaloyl gramicidin S as shown by this method agrees with the results of the detailed n.m.r.study reported above. 18' H The structure of telomycin first isolated in 1958 has now been elucidated (33). This antibiotic contains P-methyltryptophan cis-and trans-3-hydroxy- prolines and erythro-0-hydroxyleucine. A dehydrotryptophan (A-Trp) is postulated in order to account for the spectral properties. About half a residue of tryptophan is formed on alkaline hydrolysis. The threonine residue joining the peptide side chain to the ring is linked to cis-3-hydroxyproline through its hydroxy-group as a lactone.lgO A re-examination of the structure of circulin H. Klostermeyer Chem Ber. 1968,101 2823. lE2 J. Halstrom and H. Klostermeyer Annulen 1968,715 208. lE3 M.Waki and N. Izumiya Tetrahedron Letters 1968 3083; Bull. Chem SOC.Japan 1968 41 1909.lE4 M. Rothe and F. Eisenbeiss Angew. Chem Internut. Edn 1968,7 883. A. M. Liquori and F. Conti Nature 1968,217,635. lB6 A. Stern W. A. Gibbons and L. C. Craig Proc. Nut. Acad. Sci. U.S.A. 1968,61 134. IE7 D. C. Hodgkin and B. M. Oughton Biochem J. 1957 65 752; ct also R Schwyzer CIBA Foundation Symposium on Amino-acids and Peptida with Antimetabolic Acitivity 1958 p. 171. IE8 R Schwyzer and U. Ludescher Biochemistry 1968,7,2514. lE9 R Schwyzer and U. Ludescher Biochemistry 1968,7 2519. 190 J. C. Sheehan D. Mania S. Nakamura J. A Stock and K. Maeda J. Amer. Chem Soc. 1968 90.462 Amino-acids and Peptides 531 B shows it to be a cyclic heptapeptide and not a cyclic decapeptide as previously suggested. It now differs from circulin A only in having an N-terminal iso- octanoyl group in place of a (+)-6-methyloctanoyl residue.lgl all-^-and all-D-enniatin B stereoisomers have been synthesised together with enantio-enniatin B.lg2Although it has not yet been obtained homogeneous a tentative proposal of the structure of the antifungal antibiotic stendomycin has been put forward (34).It contains seven or eight D-residues and a new ‘cyclic arginine’ amino-acid (35).’‘9 lg3 Further extensive work on the synthesis of the actino- mycin series has been published.194 The fact that in peptide antibiotics all the isoleucine and all the threonine residues belong to the L-series while all the do-isomers of these belong to the D-series is thought to support the view that all the D-amino-acids of microbial peptides originate from L-amino- acids.lg5 (33) cis-3-Hypd-Trp-P-rvle-Trp-erythro-3-Hyleu / / / / Ser-Thr-Thr-Ala-Gl y-trans-3-Hyp H-AspOH (34)Fatty acid-Pr~N-Me-Thr-Gly-Val-ullo-Ile-d-But-allo-Thr-Val-Va1 ” t do-Ile-Ser-do-Thr B= ,CH2\ IHN ICH Me-N=C CH- CH-CO2H I Me NH (39 Important advances have been made this year in the successive selective linkage of cysteine residues via disulphide bridges.An insulin model has been synthesised by making three disulphide linkages in a stepwise and specific fashion by use of the sulphenyl thiocyanate coupling method. This method does not involve an intermediate thiol and avoids the possibility of thiol- disulphide interchange. Peptide (36) was prepared from the corresponding t-butyl ester by use of boron trifluoride in acetic acid to effect cleavage and 19’ K.Hayashi Y. Suketa and T.Suzuki Experientia 1968,24,657. 19’ G. Losse and R Hartmut Chem. Ber. 1968,101 1532 193 I. Muramatsu and M. Bodansky J. Antibiotics (Japan) 1968 21 68. 194 H. Brockmann and H. Lackner Chem Ber. 1968,101 1321 and 2231; H. Brockmann and P. Boldt ibid. p. 1940; H. Brockmann and F. Seela Tetrahedron Letters 1968 161 ; J. Meienhofer Experientia 1968,24 776. 19’ M. Bodansky and D. Perlman Nature 1968,218 291. 532 P.M. Hardy the resulting peptide acid was coupled with a tripeptide to yield (37). The S-trityl group of this model A chain (37) reacted with the sulphenyl thiocyanate of a model B chain to eve the 9-12 disulphide bridge (38).Treatment of (38) with trifluoroacetic acid in the presence of thiocyanogen removed the benzhy- dry1 groups and formed the final 2-16 third disulphide link. Partial cleavage of the t-butyl ester also occurred and fission was completed by treatment with f Z-Cys-Cys-Gly-Phe-Gly-ys-Phe-Gly-OH + H-Cys-Gly-Val-OBu' 1 1234 5 6 1 I SBzh (74 % yield) STr dicyclo hexylcarbodi-imide (37) cs I I Z-Cys-Cys-Gly-P he-Gly-C ys-Phe-Gly-Cys-Gly-Val-OBu' model A chain 1 (23 4 5 6 7 8 191011 SBzh STr ZISCN r (76 % yield) ys-Gly-Gly-Gly-ys-Gly-OBu' model B chain 12 13 14 15 16 17 I I HO-Gly-~y~-Gly-Gly-Gly-by~-Z model B chain 17 16 15 14 13 12 Amino-acids and Peptides boron trifluoride in acetic acid to give the insulin model (39).During this series of reactions no evidence of disulphide interchange was detected.2s* The B chain of insulin has been synthesised efficiently without using S-protec- tion by using polydisulphide intermediates and finally converting into the bis-S-sulphonate.2' Protected peptides containing S-trityl cysteine residues can also be converted directly into cystine peptides by treatment with iodine in methanol. This method has been used to prepare the 1-7 disulphide link in the synthesis of human calcitonin. lg7 Peptide Conformations.-0.r.d. studies on all the diastereoisomers of tri-and tetra-leucines show that with respect to the peptide chain and its immediate substituents the conformations adopted in solution closely resemble those of the corresponding alanine stereoisomers.* Theoretical calculations of the mean square dipole moments of polypeptide chains when applied to fourteen diastereoisomeric oligomers of ala~~ine'~~ show good agreement with the values previously obtained experimentally.200 'H N.m.r. spectra of oligo- glycines indicate that in aqueous solution they are not constrained to a small number of preferred conformations,20' while similar studies on DL and LL-phenylalanylvalines suggest that their conformations are determined primarily by nonbonded interactions and secondarily by some weighting of those conformations that bring the opposite charges of the dipolar form somewhat nearer.2o2 The i.r. spectra of the pentamer hexamer and octamer of t-pentyl- oxycarbonyl-L-proline are similar to those of polyproline-11 and it is concluded that these oligomers possess a left-handed three-fold screw axis helical struc- t~re.~'~ An n.m.r.study of cyclo(G1y-Phe) shows that in trifluoroacetic acid the phenyl group is positioned above the dioxopiperazine ring which is itself buckled into a boat form so that the phenyl group occupies a 'flagpole' type orientation.2" Similar conformations have been deduced for cyclo(G1y- Trp)2" and a series of dioxopiperazines containing one tyrosine residue.204 Experiments on N-methylated dioxopiperazines which are more soluble in less polar solvents show that this attractive interaction between an aromatic residue and the amide bonds persists in solvents of lower dielectric constant.'" Copolymers of P-benzyl and P-ethyl-L-aspartate prepared by the partial transesterification of poly( P-L-aspartate) have been found to undergo a transition from a right-handed to a left-handed a-helix when the temperature of a solution in chloroform is raised.Poly-( P-n-propyl-L-aspartate) undergoes a similar transition at 59°.205It is well known that poly(cr-amino-acids) con- ''' R G. Hiskey and R L. Smith J. Amer. Chem SOC. 1968,90,2677. B. Kamber and W. Rittel Helu. Chim Acta 1968 51 2061. D. R Dunstan and P. M. Scopes J. Chem SOC.(C) 1968 1585. P. J. Flory and P. R Schimmel J. Amer. Chem SOC.,1967,89,6807. 'O0 J. Beacham V. T. Ivanov G. W. Kenner and R C. Sheppard Chem Comm. 1965 386. A. Nakamura and 0.Jardetzky Biochemistry 1968,7 1226.'02 V. J. Morlino and R B. Martin J. Phys. Chem. 1968 72 2661. '03 H. Okabayashi T. Isemura and S. Sakakibara Biopolymers 1968,6 307 323. '04 K. D. Kopple and D. H Marr J. Amer. Chem SOC. 1967,89 6193. '05 E. M. Bradbury B. G. Carpenter and H. Goldman Biopolymers 1968,6 837. 534 P.M. Hardy taining branches at the P-carbon atom or oxygen or sulphur atoms in the side- chain at the y-position formP-structures preferentially. Theoretical calculations however now suggest that po1y-L-serine2O6 and poly-~-valine~~~ could exist in the left- and right-handed helical conformations respectively. To test this theory poly-L-valine has been prepared as a block copolymer using poly- D,L-lysine to increase its solubility. 0.r.d. and c.d. evidence showed that about half of the fifteen-residue valine block was in the right-handed a-helical conformation in 98 % aqueous methanol but in water only the intermolecular- p-structure was formed.208 The collagen model poly(L-Pro-L-Pro-Gly) has been synthesised by a solid-phase method with addition of successive tripep- tide units.Material containing twenty units was practically monodisperse and the temperature dependence of the optical rotation was closer to that of natural collagen than that of previously prepared polydisperse material. 209 lo6 K. P. Sarathy and G. N. Ramachandran Biopolymers 1968,6 461. '07 T. Ooi R A. Scott G. Vanderkooi and H. A. Scheraga J. Chem Phys. 1967,46 410. R F. Epand and H. A. Scheraga Biopolymers 1968.6 1551. '09 S. Sakakibara Y.Kishida. Y. Kikuchi R. Sakai and K. Kakiuchi Bull. Chem. SOC. Japan 1968,41 1273.