14 Biological Chemistry Part (iii) Peptides and Proteins P. M. HARDY Department of Chemistry University of Exeter Stocker Road Exeter EX4 4QD 1 Introduction This section reviews work published in the period 1977-1978 and abuts the section with the same title in Annual Reports (B) for 1976. Some earlier references are given where topics have developed over a number of years and have not been hitherto discussed in earlier Reports. In view of the number of papers published in the span of time considered the choice of material largely reflects the idiosyncracies of the reporter and current growth areas rather than any even coverage of the field. Peptide synthesis is covered more comprehensively than peptide structures; struc- ture determination is omitted as are detailed structures of peptides containing more than about 30 residues.Only a few selected aspects of proteins have been included. It seems appropriate at this point to draw the attention of readers to a recent authoritative review of many aspects of peptide synthesis in the Bakerian lecture 'Towards synthesis of proteins' by the late Professor G. W. Kenner of Liverpool University (Proc.Roy. Suc. 1977 B197,237-253). This account is written with the same clarity and insight that has characterized the many papers on peptide chemistry that he has written over the years. 2 Peptide Synthesis Protecting Groups.-Amino Protection. Despite all the new protecting groups developed in recent years the t-butoxycarbonyl (Boc) group remains the most popular for a-amino masking.Several new reagents are now available for its introduction and these circumvent problems inherent in earlier methods; i.e. the corresponding chloroformate is unstable while the azide is also subject to thermal decomposition is shock-sensitive and the vapour causes severe headaches. (Several reports have been published of the violent detonation of t-butyl azidoformate. ') Two crystalline reagents are now available commercially for attaching the Boc group i.e. t-butoxycarbonylpyrocarbonate (1)2and 2-(t-butoxycarbonylimino)-2-phenyl-acetronitrile (2).3Other acylating agents of a similar type which may be used include t-butyl S-(4,6-dimethylpyrimid-2-yl)thiocarbonate (3)4 and the water-soluble ' e.g. P. Feyer Angew. Chem. Internat. Edn.1977 16 115. L. Moroder A. Hallett E. Wunsch 0.Keller and G. Wersin 2.physiul. Chem. 1976,357 1651. M.Itoh D.Hagiwara andT. Kamiya Bull. Chem. SOC.Japan 1977,50,718. T.Nagasawa K. Kuroiwa K. Narita and Y. Isowa Bull. Chem. SOC.Japan 1973,46 1269. 370 Biologica1 Chemistry 37 1 materials 1-t-butoxycarbonyl-3-ethylimidazoliumfluoroborate (4)5 and N'-t-butoxycarbonyl- 1,2,4-triazole (5).6 CN BU~-O-CO-O-CO-O-BU~ Bu'O-CO-N=C (1) (2) Me 0 II {~-S-C-OBu' 0 II Bu'O-C-N BF,-!\+ N-Etw 0 The t-butoxycarbonyl group of course has long been the parent from which a number of acid-sensitive urethane N-protecting groups have been developed to give a range of derivatives with sometimes rather subtle variations in reactivity. Two recent examples of fine tuning of this sort illustrate the point.The 1-(1-adamanty1)- 1-methylethoxycarbonyl (Adpoc) group prepared from 1-adamantanecarboxylic acid (Scheme 1)is cleaved by acid lo3times faster than the Boc group making it CO,H C-OH I Me liii Me 0 Adpoc-OPh Reagents i PC1,-EtOH; ii MeMgI; iii PhOCOC1-py; iv H,NCHR'CO;[NRt]' Scheme 1 possible to use the Adpoc group selectively in conjunction with Boc but unlike the a,a-dimethylbenzyloxycarbonyl-typeprotecting groups is also stable to hydro- genolysis.' Purification of certain fragment peptides with Boc N-protecting groups by gel filtration in 50% aqueous acetic acid has been found to lead to substantial loss of the protecting group. This observation stimulated the search for an acid-labile protecting group more stable under these conditions.The 1-methyl-cyclobutyloxycarbonyl group (B)(McBoc) has been found to meet these require- ments. After 48 h in 50% aqueous acetic acid more than 99% of McBoc-phenyl- alanine remains unchanged whereas under the same conditions amino-acid was ' E. Guibb-Jampel,G. Braun M. Wakselman and M. Vilkas Synthetic Comm. 1973,3,111. 'G.Brown Tetrahedron Letters 1973,469. H. Kalbacher and W. Voelter Angew. Chem. Internat. Edn. 1978.17 944. 372 P.M. Hardy liberated from Boc-phenylalanine to the extent of 10-15%. Complete removal of McBoc can be effected when required with trifluoroacetic acid at 25 "C. Use of this group for N-protection therefore enables the favourable solvent properties of 50% aqueous acetic acid to be utilized when working with large peptides.8 One of the advantages of the Boc group is its removal to give only volatile by-products.A base-sensitive protecting group the 2-trimethylsilylethoxycarbonyl (Teoc) has now been explored and found to give an analogous stream of gaseous by-products on cleavage. Introduction of this group is best done with the cor- responding azidoformate and removal by reaction with fluoride ion in e.g. acetoni-trile (Scheme 2). The reaction requires 4-5 h at 50 "C with Et,NF in this solvent. The RNHC02CH2CH2SiMe3 *RNH2+C02+CH2=CH2 +Me3SiF Reagents i F-MeCN; ii H20 Scheme 2 Teoc group is stable towards catalytic hydrogenation and common basic reagents but is rapidly decomposed by trifluoroacetic acid.This clean acid-induced cleavage of urethane derived from a primary alcohol is attributed to carbon-metal U-T conjugation involving the p-silyl function.' In a similar way 2-trimethylsilyl (Tmse) esters have been used for carboxyl group protection. These esters are prepared from N-protected amino-acids and are normally not crystallizable. If used in conjunction with N-benzyloxycarbonyl groups the latter may be selectively removed by hydro- genolysis but methanol as solvent should be avoided to prevent transesterification. Hydrogen chloride in organic solvents does attack the Tmse group but sufficiently slowly to allow selective removal of a Boc group. Their removal otherwise parallels that of the Teoc group but it should be noted that cleavage with fluoride ion causes disproportionation of asymmetric disulphides and partial cleavage of aspartyl P-t- butyl esters.loQ The removal of N-benzyloxycarbonyl groups using catalytic transfer hydro- genation has attracted attention in more than one laboratory.If cyclohexene is used as the hydrogen donor in the presence of 10%palladium-charcoal in alcohol under reflux reaction is complete within 1.5-2 h." However 1,4-cyclohexadiene is a more effective donor and its use enables the cleavage to be effected at room temperature. Benzyl ester and tyrosine benzyl ether groups are also removed under * S. F. Brady R. Hirschmann and D. F. Veber J. Org. Chem. 1977,42,43. L. A. Carpino J. H. Tsao H. Ringsdorf E. Fell and G. Hettrich J.C.S. Chem. Comm. 1978 358. Peptides Proceedings of the Fifth American Peptide Symposium University of California San Diego June 20-24th 1977 ed.M. Goodman and J. Meienhofer John Wiley and Sons 1977; (a)P. Sieber R. H. Andreatta K. Eisler B. Kamber B. Riniker and H. Rink p. 543; (6)M. Ueki S. Ikeda and F. Tonegawa p. 546; (c)Y. S. Klausner T. H. Meiri and E. Schneider p. 536. A. E. Jackson and R. A. W. Johnstone Synthesis 1977 685; G. M. Anantharamaiah and K. M. Sivanandaiah J. C. S. Perkin I 1977 490. Biological Chemistry these conditions; serine and threonine benzyl ethers the W"-benzyl group of histidine and the Ng-nitro-group of arginine are better removed in the presence of the more active palladium-black catalyst.I2 A number of P-haloalkoxycarbonyl N-protecting groups have been investigated in recent years but all show some degree of base lability.It has now been found that the 2,2,2-trichloro-t-butyloxycarbonyl group (Tcboc; 7) is much more stable under these conditions. Its potential is illustrated by the observation that it is retained intact during alkaline hydrolysis of methyl esters and also on acidic cleavage of t-butyl esters. Tcboc-amino-acids can be prepared from the stable and distillable chloroformate and are highly crystalline. Removal may be brought about with the 'supernucleophile' cobalt(1) phthalocyanine anion in methanol or acetonitrile [see Ann. Reports (B) 1976 73 3441 or with zinc in glacial acetic acid.I3 The acid lability of the P-N bond has again been utilized in a protecting group.Dime thylphosp hinothioyl (Mpt) chloride prepared from te tramet hylp hosphine disulphide (Scheme 3) may be used to convert amino-acid esters into their Mpt Me 0 I c1~c-c-o-cII I Me (7) ss S S II I1 II MeMgBr + S=PC13 -+ Me2P-PMe2 --bMe2P' -&Me2P-NHCHR1CO2R2 'c1 Reagents i SO,Cl,; ii NH2CHR'C02R2 Scheme 3 derivatives; alkaline hydrolysis furnishes the corresponding acids. This type of protection is recommended for use with tryptophan. A tripeptide of tryptophan was synthesized by the solid-phase method in 86% yield using Mpt-L-tryptophan and 0.2M HC1/0.2 M triphenylphosphine in dichlorornethane for deprotection (30 min 25 "C). The anti-oxidant activity of the triphenylphosphine obviated the need for addition of any scavenger.Coupling was mediated by the oxidation-reduction method [see Ann. Reports (B),1969 66 503].'0h Although the bulk of this section on protection has been largely devoted to variations on old themes novel types of protection are still being explored and reported. The 1,2,4-dithiazolidine-3,5-dione heterocyclic system has been used as the basis of a new type of N-protecting group. Ethylethoxythiocarbonyl derivatives of amino-acid esters react in anhydrous solution with chlorocarbonyl sulphenyl chloride to give an adduct which undergoes ring closure to generate a dithiasuccinoyl (Dts)-protected amino-ester (8; Scheme 4). Direct preparation from the ethyl- ethoxythiocarbonyl-amino-acid is not possible. The Dts group is stable to acid I2 A. M. Felix E. P.Heimer T. L. Lambros C. Tzougraki and J. Meienhofer J. Org. Chem. 1978 43 4194. l3 H. Eckert M. Lid. and I. Ugi Angew. Chem. Inteemat. Edn. 1978,17,361. 374 P.M. Hardy 0 c3s-c-II c1 -+ 0 II ,c-CI S I 0 S Et-O \ + / C=NH-l CI 3Et -4 J 0 I1 SAC\ I N-R S.c' I\ 0 Scheme 4 (enabling the ester group to be removed) and photolysis above 330nm but is removed by mild reductive procedures. Cleavage with thiol for example (Scheme 9,generates the free amine; this reaction is markedly accelerated by addition of tertiary amines. Prolonged treatment with bases such as the a-amino-group of 0 II SC\ I N-R' +2R2SH -* H2N-R'+2COS+R2SSR2 S.p/ L II 0 Scheme 5 amino-acid esters does not cause detectable cleavage but aliphatic amines or strong aqueous alkali yield the mixed urea or free parent amine.14 The inactivation of the peptide hormone oxytocin on allowing it to stand in contact with acetone first observed in 1965,15has stimulated an investigation into the use of the interaction products of peptides and acetone in peptide synthesis.The reaction involves the formation of an imidazolidinone ring system from the N-terminal dipeptide unit (Scheme 6). Free dipeptides in many instances can be converted into R'HC-co I\ ~ HZNCHR' CONHCHR2COR3 HN ,N-CHRZCOR3 + P Me2C0 /"\ Me Me Scheme 6 l4 G. Barony and R. B. Merrifield J. Amer. Chem. SOC.,1977,99,7363. Is D. Yamashiro H. L. Aanning and V. Du Vigneaud Proc. Nat. Acad. Sci. U.S.A.,1965,54 166.Bio logica 1 Chemistry their NN'-isopropylidene derivatives by stirring in the cold or under reflux with acetone. Such N-protected modified dipeptides may be coupled using NN'-dicy- clohexylcarbodi-imide without risk of racemization even in the absence of additives as oxazolone formation is precluded. Deprotection may be effected by heating the neutral aqueous solution at 60-100 "C for a few hours. A heptapeptide has been prepared by successive addition of three dipeptide units to an amino-acid ester but the method lacks generality as some derivatives are difficult to prepare pure and some are too unstable towards hydrolysis. Free dipeptides are also not readily available starting materials.'6 Carboxyl Protection. Although protection of carboxjrl groups perhaps receives rather less attention than amino-groups each year still brings its crop of novelties and improvements.The preparation of esters of Boc-amino-acids from the caesium salt of the corresponding acid and chloromethylated poly(styrene -1YOdivinylbenzene) (solid-phase resin) was found in 1973 to proceed rapidly and quantitatively; the method avoids the production of quaternary ammonium salts and the presence of residual chloromethyl group^.^' This use of caesium salts has now been applied to the synthesis of N-protected amino-acid and peptide esters for use in solution-phase peptide synthesis." Methyl esters and more usefully benzyl esters can be prepared efficiently in this way. Boc-Phe-Phe-OH showed no racemization within the experimental error of the method (Manning and Moore test)." The use of caesium salts is applicable to other types of ester although Z-Ala-OBu' (Z = benzyloxy-carbonyl) could only be prepared from 2-Ala-OH and 2-bromo-2-methylpropane in 14% yield.18 A novel type of carboxyl protection recently described ingeniously utilizes 2-oxymethyleneanthraquinone (Maq) esters.Simple amino-acids and peptides give highly crystalline Maq esters which are readily soluble in organic solvents but stable to the conventional operations of peptide synthesis and catalytic hydrogenation. The ester group can be removed either by treatment with sodium dithionite photolysis (350 nm) in isopropanol containing N-methylmorpholine treatment with 9-hydr- oxyanthrone in DMF containing triethylamine or with polystyrene functionalized with 9,lO-dihydroxy-anthracene residues.The reduction of the quinone gives a highly unstable intermediate (9) which decomposes to a xylylidene derivative (10)as shown in Scheme 7. This latter apparently tautomerizes sufficiently rapidly to obviate the need for the addition of trapping agents. The Maq group has been successfully used in the synthesis of methionine enkephalin (a pentapeptide) and a heptapeptide angiotensin analogue.2o The use of polyethylene glycol (PEG)in peptide synthesis originated in 1971" and has been pursued in a number of papers since that time notably in the past year. This linear polymer was introduced as a solubilizing carboxyl-protecting group in contrast to the solid-phase crosslinked polymer.Its advantage lies in the ease with which the high mol. wt. growing peptide chain may be purified from low mol. wt. reagents as l6 P. M. Hardy and D. J. Samworth,J.C.S. Perkin I 1977 1954. B. F. Gisin Helv. Chim. Acta. 1973,56 1476. S.S.Wang B. F. Gisin D. P. Winter R. Makofske 1. D. Kulska C. Tsougraki and J. Meienhofer J. Org. Chem. 1977,42,1286. l9 J. M. Manning and S. Moore J. Biol. Chem. 1968,243 5591. 2o D.S.Kemp and J. Renek Tetrahedron Letters 1977 1031. *' M.Mutter H. Hagenrnaier and E. Bayer Angew. Chem. Internat. Edn. 1971. 10,811. 376 P.M. Hardy 0 required either by ultrafiltration or by differences in solubility in organic solvents.” Neutral amino-acids may be attached directly to polyethylene glycol without N-protection in the presence of toluene-p-sulphonic acid in benzene by heating under reflux for 2-3 days the condensate returning to the pot through a bed of molecular sieve to remove water.Arginine and histidine can be introduced in this way in the presence of an additional amount of the acid.22 However one disad- vantage of the PEG approach has been the relatively low yield of final peptide often obtained on cleavage from the polymer because of the rather drastic conditions required to split the ester linkage. Interpolation of a photosensitive moiety between the polymer and the peptide chain is one way in which this drawback can be overcome Use of 3-nitro-4-bromomethylbenzoyl-PEG (11)has enabled a tetra- peptide to be built up and removed from the polymer in 98% yield compared with the 69% cleavage from the polystyrene support used in solid phase synthesis.(11) Peptides attached to the photosensitized PEG are soluble in dichloromethane and DMF but insoluble in ether and ethyl a~etate.’~ Cleavage from the polymer requires photolysis (>350nm) in methanol under N2for 10-20 h; in this way four different N-protected heptapeptides were obtained with an average yield of 91Yo.24 Reagents bound to PEG as opposed to the growing peptide chain have some advantages in peptide synthesis. A comparison between the reaction rates of 22 C. S. Pande and J. D. Glass Tetrahedron Letters 1978,4745. 23 F. S. Tjoeng W. Staines S. St.-Pierre and R. S. Hodges Biochim. Biophys. Acta. 1977 490 489. 24 F. S. Tjoeng E.K. Tong and R. S. Hodges J. Org. Chem. 1978,43,4190. Biological Chemistry PEG-bound and low molecular weight active ester derivatives in the peptide bond- forming step indicated that the soluble polymeric group did not appreciably influence the speed of reaction. Excess polymeric active ester reagent can be simply and quantitatively removed by precipitation with negligible loss of the peptide product which remains in solution. In order to attach active ester groups to PEG it was first converted to the corresponding cvw-diamine (Scheme 8)which could then be coupled lii,iii Reagents i TsCI; ii K phthalimide;iii N,H Scheme 8 directly with appropriate carboxylic acids such as (12) and (13) with NN'-dicyclo- hexylcarbodi-imide to give polymeric derivatives of the well-known o-nitrophenyl and 1-hydroxybenzotriazole active esters respectively.A carbodi-imide has been prepared bound to PEG (Scheme 9) to overcome the insolubility of the correspond- ing urea which can be difficult to separate from desired peptide after a carbodi- imide-mediated coupling. The resulting PEG-urea is soluble in dichloromethane and can be recycled back to carbodi-imide.2s i.ii PEG-NH:! -PEG-N=C=N-CHMe Reagents i Me,CHNCO; ii TsCI NEt,. Scheme 9 Another reagent (14)for deblocking the fluoren-9-ylmethoxycarbonyl (Fmoc) group [cf. Ann. Reports (B) 1974 71 5061 has been developed (Scheme 10) to overcome the problem which can occur of separating the adduct (15) formed when piperidine is used to remove the Fmoc group (Scheme 11).On stirring Fmoc- tryptophan with the resin (14)in dichloromethane containing some water to prevent precipitation of the amino-acid on the polymer a 90% yield of deprotected material was obtained on evaporation of the solvent all liberated dibenzofulvene (16) having been scavenged by the polymer.26 25 M. Mutter Tetrahedron Letters 1978 2839 2843. 26 L. A. Carpino,J. R. Williams and A. topusihski J.C.S. Chem. Comm. 1978,450. 378 P. M. Hardy T Q i,ii,iii n CH,CI CH,N NH U chloromethylated polystyrene -1‘/o divinylbenzene n Reagents i HN NBoc -EtNPri; ii HCl; iii NEt, w Scheme 10 CH ,OCONHR CH Reagent i HN3 Scheme 11 Side-chain Protection. Removal of S-protecting groups by heavy metal salts such as silver or mercury is well known e.g.in the cleavage of S-trityl S-acetamidomethyl and S-ethylcarbamoyl groups from cysteine peptides. Recent work however has shown that a number of other S-protecting groups are also susceptible to fission by mercuric acetate in trifluoroacetic acid or mercuric trifluoroacetate in aqueous acetic acid. These include the S-p-methoxybenzyl group hitherto removed by anhydrous strong acids such as HF and the S-t-butyl and adamantyl derivatives; the S-benzyl group however is quite stable towards these reaction conditions. Removal of the S-p-methoxybenzyl group in this way has been exemplified in syntheses of biologic- ally active oxytocin and somatostatin each of which contains two cysteine residues2’ When the phenolic group of tyrosine is protected as its benzyl ether there is the tendency for rearrangement to 3-alkyltyrosine to occur during cleavage of protecting groups with hydrogen fluoride.The introduction of electron-withdrawing substi- tuents such as m-bromobenzyltyrosine28 or 2,6-dichlorobenzyltyrosine29improved selectivity during removal of Boc groups with trifluoroacetic acid and reduced the 3-alkylation side-reaction to about 5%. However even this level is not tolerable for solid-phase synthesis of large peptides containing many tyrosine residues. A study of four secondary alkyl ethers has shown 0-cyclohexyltyrosine to undergo minimal 27 0.Nishimura C. Kitada and M. Fujino Chem. and Pharm. Bull. (Japan),1978,26 1576. 28 D.Yamashiro and C. H. Li J.Org. Chem. 1973,38,591. 29 B.W.Erickson and R. B. Merrifield J. Amer. Chem. SOC.,1973 95,3750. Biological Chemistry 379 rearrangement (0.3%) on cleavage with HF yet on treatment with 50% CF,CO,H-CH,Cl under the conditions for Boc cleavage in solid-phase synthesis only 0.006°/~ loss of alkyl group occurs. It is therefore not as stable to this reagent as the 0-2,6-dichlorobenzyl group but in the solid-phase synthesis of ribonuclease A which contains 6 tyrosine residues it should give a product in which 97.2% of the molecules would have all six tyrosine-protecting groups intact.30 In order to try and prevent the racemization sometimes observed when N-a-alkoxycarbonylhistidine derivatives are coupled specific blockage of the 7r-nitrogen of the imidazole ring has been investigated.Both the N(T)-and N(r)-phenacyl derivatives of 2-L-His-OH were prepared (Scheme 12) and their couplings with ZNHYHC0,Me ZNHYHC0,Me ZNHYHC0,H -" i ii N kN> kN> k:, N H I I CH,COPh CH,COPh liv (17) ZNHCHC0,Me ZN HCHCO,Me kN\;CH2coPhZN HCHCO,H -k;"2coph I I vi iii N H k N ! N v N ~ CPh 3 CPh (18) Reagents i AgNO,; ii PhCOCH,Br-Me,SO; iii NaOH; iv Ph,CCl-CH,CI,; v PhCOCH,Br-Et,O; vi AcOH-H,O 100 "C. 10 min Scheme 12 L-prolinamide using NN'-dicyclohexylcarbodi-imideunder conditions designed to exacerbate the danger of racemization were compared. Material protected on the 7-nitrogen (17) yielded 35% D-isomer but that from the 7r-nitrogen phenacyl derivative (18) was stereochemically homogeneous within experimental error.Similar results have been obtained with the corresponding Boc derivatives. The Nim-phenacyl group is stable to HBr-HAc,CF3C02H or brief exposure to hydro- genolysis conditions but may be removed by Zn dust in acetic acid.31 Coupling of H-Pro-Ser(Bzl)-His-Arg(NO2)-I1e-Ser-OMe with the 1-succinimidyl ester of Boc- Asp( P-Bzl) in the presence of 1-hydroxybenzotriazole has been found to cause significant 0-acylation. This was traced to catalysis of the alcoholysis of the active ester by the imidazole group of the histidine side-chain.' Such a situation can only be completely prevented by further protection of the side-chains involved and the danger of such 0-acylation must obviously be borne in mind when considering the degree of side-chain protection to use in the synthesis of peptides containing these residues.30 M. Engelhard and R. B. Merrifield J. Amer. Chem. SOC.,1978 100,3559. 31 J. H. Jones and W. I. Ramage J.C.S. Chem. Comm. 1978,472. 32 M. Bodanszky M. L. Fink Y. S. Klausner S. Natarajan K. Tatemoto A. E. Yiotakis and A. Bodanszky J. Org. Chem. 1977 42 149. 380 P.M. Hardy Formation of the Peptide Bond.-A new coupling agent for peptide synthesis described as a ‘push-pull’ acetylene (19) has been found to form peptide bonds efficiently with little racemization and it can be used in aqueous solution. Addition of this compound to a solution of N-protected amino-acid or peptide gives a primary non-isolable adduct (20) which rearranges to a crystalline enol ester (21).This active derivative couples selectively with amino-components (Scheme 13). The R:N H Me H RiN-CrCCOMe +R2C02H + \/c=c \/c=c (19) [R2C02/ ‘COM] R2C0,/ ‘CONRi (20) (21) b3NH2 Me H \c=c / R =Me2or -NMe U (22) + RVON HR Scheme 13 NN-dimethylacetoacetamide(22) formed as a by-product is readily soluble in both water and ether. Tyrosine 4-hydroxyproline cysteine histidine and asparagine can all be linked without protection of their side-chain functional groups. Themethod has been tested successfully up to the tetrapeptide level. The acetylene (19) can be prepared from the corresponding olefin by a simple bromination-dehydrobromina-tion sequence (Scheme 14).33*34 The cyclo-adduct 1,3,4-trimethyl-A3-phospholen-I pii Me R~N-CEC-COM~ Reagents i Br,; ii NEt,; iii KOBu‘ Scheme 14 1,l-dichloride (23) of 2,3-dimethylbutadiene and methylphosphorus dichloride has also been examined as a coupling agent.Although the highly sensitive Young test3’ gave 47% racemization coupling of dipeptides with C-terminal valine leucine or 33 M. Neuenschwander H.-P.Falwin and U. Lienhard Helu. Chim. Acta. 1978,61 1609 2437. 34 H.-J. Gais Angew. Chem. Internat. Edn. 1978,17,597. ’’M.W. Williams and G. T. Young,J. Chem. Suc. 1963 881. Biological Chemistry (23) phenylalanine residues to amino-acid esters was apparently racemization-free yields being 86-90°/0. Reactions were carried out in the presence of N-methyl- imida~ole.~~ Of the established coupling agents for peptide synthesis NN’-dicyclohexyl- carbodi-imide (DCC) of course remains by far the most widely used.The use of 1-hydroxybenzotriazole in conjunction with DCC to depress racemization in its coupling of peptides3’ has proved immensely useful. A recent paper however shows that a by-product may be formed by interaction of these compounds. On allowing them to stand in dichloromethane (20“C,24h) 31% of the 1,3-diazetidine (24) was A number of unsymmetrical carbodi-imides have been compared with DCC in respect of their tendency to cause racemization when used to couple peptides in the absence of additives. According to the Young test DCC is worst in this respect and N-ethyl-N’-phenylcarbodi-imidebest (25).The latter compound was also found to cause less N-acylurea formation in the coupling of 2,4-dinitrophenylsulphenylglycineto valine methyl C6H1 lN=C=NC6HI >PhCH,N=C=NEt >p-MeC,H,N=C=NEt >PhN=C=NEt (25) The building up of peptide bonds by means of active esters is still a very popular method and structural variants continue to attract attention.N-Protected amino- acid esters of 3-hydroxyquinazolin-4(3H)-one (26)have been shown to be useful in 0 (26) 36 E. Vilkas M. Vilkas and J. Sainton Tetrahedron Letters 1978 3922. 37 W.Konig and R. Geiger Chem. Ber. 1970 103 788. 38 H.-D. Jakubke and C. Klessen J.prukt. Chem. 1977,319 159. 39 H.Ito N. Takamatsu and I. Ichikizaki Chem. Letters 1977,539. 382 P. M. Hardy this respect and their practicality demonstrated in the stepwise synthesis of the C-terminal hexapeptide of substance P.On coupling CF,CO-Pro-Val-OH to H-Pro-OMe by this method only 2.5% of the L-D-L-tripeptide was formed.40 Introduction of a sulphonic acid group into the well-known o-nitrophenyl ester gives a water-soluble derivative. These o-nitro-p-sulphophenyl esters are best prepared from N-protected amino-acids by the symmetrical anhydride method and are coupled at pH 8-8.5. Acylation is somewhat faster than with the parent o-nitrophenyl ester and 1-hydroxybenzotriazole is an efficient catalyst in aqueous as well as organic solution. The desired product of reaction normally precipitates out. The tetrapeptide Z-Lys(&-Boc)-Pro-Val-Gly-OEt was synthesized by this method even though the lysine active ester was not water-soluble; the suspension soon dissolved and the acylation was rapid."' Another method which involves a largely aqueous reaction medium is the use of enzymes for coupling.There has been a recent resurgence of interest in this approach which was originally investigated back in 193741and has not since been systematically explored using modern protecting groups. Hydrolysis of protected product peptides is low due to their water insolubility and inhibition of secondary hydrolysis of the product if soluble is inhibited by the presence of a high concen- tration of the amino-component nucleophile. Specificity for synthesis is of course very similar to the leaving group specificity in hydrolysis. As acceptor nucleophiles however free amino-acids and their esters are inadequate.Peptide bond formation by enzymes has the great advantage of being highly stereospecific and therefore of potential particularly in fragment condensations. Proteinases of all types can be effective in appropriate cases.42 The rather specific enzyme a-chymotrypsin can be used to couple peptides with C-terminal Phe Tyr or Trp residues and since esterase activity is also present esters can be used as donors. The best result obtained was in the coupling of Z-Ala-Phe-OMe to H-Phe-Leu-OH. A 95% yield of product was obtained in 5 min. in water containing 35% DMF to solubilize the starting ester. By contrast the worst result (43%) was obtained in the reaction of Z-Ala-Phe-OMe with H-G~Y-NH~.~~ More generally useful of course are the proteinases of rather broader specificity.Of three metalloproteinases studied that from Bacillus subtilis var. amyloliquefaciens (Prolisin A) has proved the most promising. Even crude enzyme preparations in the presence of the serine proteinase inhibitor from potatoes to prevent the amidase action of impurities proved reasonably successful. This enzyme may be used to link amino-components with hydrophobic N-terminal amino-acid side-chains. The use of some organic solvents in modest proportion often increased yields; e.g. in the case of Z-Leu-Gln-OH and H-Leu-Val-NH2 dioxan (16%)improved the yield of tetrapeptide from 71 to 87.3%. The use of Boc for N-protection however gave in general poor results; only 20% of the pentapep- tide was obtained in the coupling corresponding to that mentioned above.44 40 H.D.Jakubke C. Klessen and K. Neubert J. prukt. Chem. 1977,319,640. M. Bergmann and H. Fraenkel-Conrat J. Biol. Chem. 1937,119,707. 42 Y. Isowa M. Ohmori T. Ichikawa H. Kurita M. Sato and K. Mori Bull. Chem. SOC.Japan 1977,50 2762. 43 K. Morihara and T. Oka Biochem. J. 1977,163 531. 44 Y. Isowa T. Ichikawa and M. Ohmori Bull. Chem. SOC.Japan 1978 51 271. Biological Chemistry 383 New Peptide Structures.-The myotropic substance proctolin isolated from the cockroach Periplanata americana has been found to be the linear pentapeptide H-Arg-Tyr-Leu-Pro-Thr-OH. This compound is of neural origin occurring in nerves of the viscera and evoking contraction of the visceral musculature at concen- trations down to M.It is thought to function as an excitatory neuromuscular transmitter and probably occurs in a wide variety of insects. The structure has been confirmed by synthesis.45 The most basic angiotensin (27) so far known has been H-Asn-Arg-Val-Tyr-Val-His-Pro-Phe-His-Leu-OH (27) isolated from the goosefish. The basicity is a consequence of the masking of the side-chain of the N-terminal aspartic acid residue as the amide. This appears to be the first natural peptide characterized in which asparagine is N-terminal. Apart from the side-chain amidation of the aspartic acid this decapeptide sequence is identical to that of bovine angiotension I.46 Like all other natural tachykinins the dodecapeptide kassinin (28) derived from the skin of the African amphibian Kassina senegalensis possesses the characteristic H-Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-Val-Gly-Leu-Met-NH~ 5 10 (28) Gly-Leu-Met-NH2 C-terminal tripeptide sequence but the amino-acids at the other end of the molecule differ sharply from known members of the series Like substance P kassinin exhibits a free amino-group instead of the more usual pyroglutamyl re~idue.~'The peptide has been synthesized using methionine as its sulphoxide to prevent alkylation of this residue on stripping off protecting groups with methanesulphonic acid.As a final step methionine was regenerated from its sulphoxide by treatment with 2-mer~aptoethanol.~~ A peptide of the same size has been found in the skin of the frog Rana rugosa. This compound named granuliberin (29) has a potent mast-cell-degranulating activity and belongs to a new family of active peptides in amphibian skin.The N-terminus is hydrophobic and the C-terminus basic and hydr~philic.~~ H-Phe-Gly-Phe-Leu-Pro-Ile-Tyr-Arg-Arg-Pro-Ala-Ser-NH* 5 10 (29) Details of the structure determination of three new peptides from the venom of the common European honeybee Apis mellifera have been reported. Melittin F proved to be a fragment of the known peptide melittin lacking the N-terminal heptapeptide of the latter. Mast-cell-degranulating peptide (30)shows a similarity to the neuro- toxic venom component apamin both containing a Cys-Asn-Cys-Lys sequence. The third novel peptide secapin (3l),is structurally unrelated to the other basic peptides " A.N. Starratt and B. E. Brown Canad. J. Chem. 1977,55,4238. 46 T.Hayoshi T. Nakayarna,T. Nakajima and H. Sakabe Chem.and Pharm. Bull. (Jupan),1978,26,215. 47 A. Anastasi P. Montecucchi V. Erspamer. and J. Visser Experientiu 1977,33 857. 48 H. Yajima T. Sasaki H. Ogawa N. Fujii T. Segawa and Y. Nakata Chem. and Phurm. Bull. (Japan), 1978,26,1231. 49 T. Nakajirna and Y. Yasuhara Chem. and Pharm. Bull. (Japan) 1977,25,2464. w S S H-Ile-Lys-Cys-Asn-Cys-Lys-Arg-His-Val-I1e-Lys-Pro-His-Ile-Cys-Arg-Lys-Ile-Cys-Gl I I y-Lys-Asn-NH2 P SI 5 10 15 s I 2o (30) S S I I H-Tyr-Ile-Ile-Asp-Val-Pro-Pro-Arg-Cys-Pro-Pro-Gly-Ser-Lys-Phe-Ile-Lys-Asn-Arg-Cys-Arg-Val-Pro-Val-OH 5 10 15 20 (31) Human H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro- Val-Gly-Lys-Lys-Arg-Arg-Pro-Val- Ostrich _--_----Arg---Dogfish _------Met -Arg --Ile-10 20 Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala- Phe-Pro-Leu-Glu-Phe-OH --_ -Val-Gln-Glu -Thr-Ser -Gly ------Ser-Phe-Glu-Asp -Ser-Val -Asn-Met-Gly-Pro -Leu 30 Figure 1 (-means same as above) Antiamoebin Ac-Phe-Aib-Aib-Aib-Iva-Gly-Leu-Aib-Aib-Hyp-Gln-Iva-Hyp-Aib-Pro-Phol Emericin I11 --Val -----Phol Emericin IV --Val ----Ala-Phol 5 10 15 Figure 2 [Iva =L-isovaline=(S)-a-ethylalanine;Hyp =L-4-hydroxyproline] Biological Chemistry 385 present in bee venom.It contains only one disulphide bridge and a Pro-Pro sequence is repeated twice. This peptide is not toxic and the quantity present in the venom is very ~ariable.~' The first avian corticotropin (ACTH)sequence has been elucidated.Material from the ostrich Struthi0 camelus (Figure 1)shows the invari- ance in the N-terminal dodecapeptide sequence hitherto observed but like dogfish (Squalusacanthias) ACTH shows rather more differences in the rest of the molecule than is seen between mammalian species.'l In 1968 the antibiotic antiamoebin was reported to be a cyclic peptide linked to phenylalaninol (Ph~l).~ A recent reinvestigation of this molecule however shows it to be a linear peptide (Figure 2)much richer in a-aminoisobutyric acid (Aib). It thus falls into a class of peptide antibiotics containing Phol and Aib residues as well as other amino-acids for which the name peptaibophols has been Two antibiotics from Emericillopsis microspora emericins I11 and IV (Figure 2) show a close similarity to antiam~ebin.~~ In all three peptides the presence of the glutamine residue was indicated by the formation of ornithine after dehydration (ethylene chlorophosphite-triethyl phosphite 100"C 24h) reduction (Na-NH,-MeOH) and hydrolysis (6 M HCI 110"C 24h).53*54 CH2CH=CHMe I CHMe2 CHMe I I CH2 Me CHMe2 MeCHOH Et Me I II II I I MeN-CH-CO -N-CH-C-N-CH-CO-N-CH-C-N-CH2 II I I' 'co 0 y 0 I I co MeCHCH2CH I I I NMe NMe H 0 s\ I I II OC-CH-NH-CO-CH-N-CO-CH-N-C-CH-N-CO-CHCH2CHMe2 I I II I Me Me CH2 Me CHMe2 I CHMe2 (32) Cyclosporin A a cyclic undecapeptide metabolite (32)of the fungus Trichoderma polysporum has been found to have a remarkable immunosuppressive activity.55 Its antilymphocytic action has been shown by its greater effectiveness than any other drug in prolonging the survival of heart grafts in pigs.56 This hydrophobic peptide contains the novel amino-acid (2S 3R 4R)-(6E)-methylamino-3-hydroxy-4-methyl-oct-6-enoic acid and its sequence was established by Edman degradation after an acid-induced NO-acyl migration (Scheme 15).The crystal structure of an iodo-derivative has been determined and shows a conformation which is partly 50 J. Gauldie J. M. Hanson R. A. Shipolini and C. A. Vernon European J. Biochem. 1978,83,405. 51 C. H. Li D. Chung W. Oelofsen and R. J. NaudC Biochem. Biophys. Res. Comm. 1978,81,900. 52 M. G. Voidya P. V. Deshmukh and S.N. Chari Hindustan Antibiot. Bull. 1968 11 81. R. C. Pandey H. Meng J. C. Cook and K. L. Rinehart J. Amer. Chem. SOC. 1977,99,5203. 53 54 R. C. Pandey J. C. Cook and K. L. Rinehart J. Amer. Chem. SOC. 1977,99 5205. A. Ruegger M. Kulm H. Lichti H.-R. Loosli R. Huguenin C. Quiquerez and A. von Wartburg Helv. 55 Chim. Acta. 1976,59 1075. 56 A. J. Kostakis D. J. G. White and R. Y. Calne IRCSMed. Sci. Libr. Compend. 1977,5 243. 386 P.M. Hardy Reagents i,MeS0,H-MeOH; ii reflux in dioxan Scheme 15 &pleated sheet and partly open 100p.~~*~’ Cyclosporins B C and D which occur in the same culture contain respectively L-Ala L-Thr and L-Val in place of L-Q-aminobutyric acid. 58 The complete structure of another cyclic peptide antibiotic which is a potent inhibitor of bacterial protein synthesis has also now been determined.Berninamycin A (33) contains five residues of dehydroalanine out of its ten ring amino-acid residues as well as two 5-methyloxazole units and the hitherto unreported thia- zolonaphthyridinium chromophore. The sequence was assigned on the basis of products obtained on trifluoroacetolysis of the antibiotic; two fragments were I NH oc \ JH2 I C C HN’ *CH~ I Me 1 CH I /\ &\ MeCH CO-NH-C ’H I OH N& Me@,N ?Me “-(C-NH HI \ HN-CO H2C co ‘CH’ I Me-C-OH I Me (33) ’’T. J. Petcher H.-P. Weber and A. Ruegger Helv. Ckim. Acta. 1976 59 1480. 58 R. Traber M. Kuhn H. R.Loosli W. Packe and A. von Wartburg Helv. Chim. Acta. 1977,60 1247 1568. Biologica1 Chemistry isolated [the larger one after partial reduction of dehydroalanine residues; arrows in (33)show the break points] which comprise the whole amino-acid sequence.59 Aspects of Protein Structure.-The first synthesis of a functional polypeptide from a chemically synthesized gene was achieved in 1977. Tke gene for somatostatin a tetradecapeptide hormone which inhibits the secretion of a number of other hormones was fused to the Escherichia coli P-galactosidase gene on the plasmid pBR322. After transformation into E. coli the chimaeric plasmid directed the synthesis of a protein containing P-galactosidase linked to somatostatin. In order to enable somatostatin to be separated from the enzyme a methionine codon was included prior to the amino-terminal end of the hormone.Treatment of the protein product with cyanogen bromide specifically cleaved the peptide chain at methionine to yield active somatostatin.60 The use of recombinant DNA methods in this way is only limited by the difficulty of synthesizing the DNA corresponding to the required peptide. It is clear that the idea of using bacteria to synthesize foreign peptides by hiding them in a natural bacterial protein has great potential as an alternative to classical organic synthesis for the production of peptides and proteins of medical interest. Insulin in particular is an attractive target being actively pursued. However it must be remembered that only analogues containing natural amino-acids are accessible by this method.The form in which proteins are initially synthesized by the ribosome continues to be very actively explored and some interesting work has been reported over the past two years in the field. The exotoxin of Pseudomonas aeruginosa has enzyme activity catalysing the transfer of the adenosine diphosphate ribose portion of nicotinamide adenine dinucleotide to eukaryotic elongation factor Z. The exotoxin is synthesized as a pro-enzyme but conversion into the active form does not unusually enough require proteolysis. Diphtheria toxin which catalyses the same reaction does require peptide bond cieavage for activation. In the case of the Pseudomonas toxin activation results from simultaneous treatment with a protein denaturant and a chemical able to split disulphide bonds.It is concluded that such treatment induces a conformational change that exposes the previously buried active site no peptide fragments at all being released.61 Most of the secretory proteins so far examined have been found to be synthesized with an amino-terminal extension of from 15-30 amino-acid residues which is rich in hydrophobic residues. This 'signal' sequence is thought to interact with a specific membrane receptor which directs this 'recognized' protein through the cell membrane. The signal sequence is normally removed before the protein assembly is coinplete but its existence can be shown by using cell-free protein-synthesizing systems lacking membranes and associated proteases in the presence of the appro- priate mRNA.In this way pre-lysozyme was found to have an additional 18residues (34) at its N-terminus 16 of them being hydrophobic.62 There is a 75% identity in the eight amino-acids preceding the cleavage site of pre-lysozyme and another '' J. M. Liesch and K. L. Rinehart J. Amer. Chem. Soc. 1977 99 1645. 6" K. Itakura T. Hirose R. Crea A. D. Riggs M. L. Heyneher F. Bolivar and M. W. Boyer Science 1977 198,1056. '' S. H. Leppla 0.C. Martin and L. A. Muehl Biochem. Biophys. Res. Comm. 1978 81 532 R. D. Palmiter J. Gagnan L. H. Ericsson and K. A. Walsh J. Biol. Chem. 1977 252 6386. 388 P. M. Hardy Y h G x Biologica1 Chemistry egg-white protein pre-ov~mucoid.~~ Pre-lipoprotein from E.coli itself a membrane protein has a twenty-amino-acid N-terminal extension (35);64 like prelysozyme removal of this sequence requires specific cleavage of a C-terminal glycine and in both cases the initiator amino-acid methionine (which is probably N-formylated) has not been lost. Egg-white ovalbumin in contrast to other proteins synthesized in the same cells appears to lack a transient hydrophobic leader sequence. When the growing peptide chain is about 20 residues long the N-terminal initiator methionine is lost and the new N-terminal glycine acetylated when the peptide is 44 residues long. It may be that a signal sequence is located elsewhere in the molecule but the amino-terminal end of ovalbumin shows only a weak resemblance to other hydrophobic signal sequences.One speculation considers the possibility that the transacetylase is itself the receptor.65 Parathyroid hormone (PTH) a single-chain polypeptide regulating the level of calcium in the extracellular fluid is on the other hand formed as a precursor which then undergoes two successive proteolytic cleavages. Like other secretory proteins PTH is synthesized specifically on polyribosomes bound to membranes and conversion of the pre-protein in cell-free systems can be brought about by the addition of microsomal membranes; normally in the endoplasmic reticulum a 25-amino-acid N-terminal fragment is lost within one minute of synthesis and the resulting prohormone takes 15 minutes to be transported to the Golgi apparatus where an N-terminal hexapeptide is removed to give PTH itself .66 Insulin is another protein in which two cleavages occur the second one in this case excizing the central portion of the molecule to leave two separate but disulphide- bridged chains.Partial sequences of pre-proinsulins from the rat6' and two fishes the angler fish and the sea raven,68 have been determined (Figure 3). There is consider- able homology at least with respect to the leucine residues. Since the cleavage site for the conversion of nascent fish pre-proinsulin into nascent proinsulin is recognized correctly by dog pancreas microsomal enzyme and the signal peptide is also recognized by the corresponding microsomal membrane it is thought that mechanisms and information for the transfer of secretory proteins are highly conserved during Two unnatural disulphide bond isomers of human insulin (36) and (37) have been synthe~ized~~ using the strategy earlier developed s-s w s-s s-s I 7 19 7 19 (36) (37) 63 S.N. Thibodeau J. Gagnan and R. D. Palrniter Fed. Proc. 1977,36 656. 64 S. Inouye S. Wang J. Schizawa S. Halegona and M. Inouye Proc. Nut. Acad. Sci. U.S.A.,1977 74 1004. " R. D. Palrniter J. Gagnan and K. A. Walsh Proc. Nut. Acad. Sci. U.S.A.,1978 75 94. J. F.Habener M. Rosenblatt B. Kemp. H. M. Kronenberg A. Rich and J. T. Potts,Proc. Nut. Acad. Sci. U.S.A. 1978 75 2616. 67 S. J. Chan P. Keirn andD. F. Steiner Proc. Nut. Acad. Sci. U.S.A.,1976,73 1964. D.Shields and G. Blobel Proc. Nut. Acad. Sci. U.S.A. 1977,74 2059.''P. Sieber E. Eisler B. Karnber B. Riniker W. Rittel F. Marki and M. de Gasparo J. physiol. Chem. 1978,359,113. 390 P.M. Hardy for the total synthesis of human insulin (38) itself.70 Their biological activities are similar and qualitatively indistinguishable from insulin and for several tests range from 13 to 37% compared to insulin. Chromatographically they can easily be distinguished from insulin but they are significantly less stable and partially iso- merize to insulin. Their perhaps unexpectedly high biological activities would seem to indicate that their tertiary structures may resemble that of insulin in essential features. It is unlikely that their activity is due to enzyme-catalysed isomerization to insulin during biological testing.69 The key intermediate in this synthesis of insulin and its isomers was the compound (39) (side-chain-protecting groups are omitted S for clarity but are all t-butyl esters or ethers).This could be coupled with the BOG(1-1 3) sequence containing a preformed intramolecular disulphide bridge (6-7 7-1 1 or 6-1 1) and an S-acetamidomethyl (Acm) cysteine. Treatment with iodine generated the third disulphide bridge from the S-Acm-cysteines and all protecting groups were stripped off with trifluoroacetic The structure of a 70-residue protein growth factor from human plasma (IGF-1) has now been determined. The molecule contains 3 disulphide crosslinks and displays obvious homology to proinsulin positions 1-29 resembling insulin B chain and 46-62 resembIing insulin A chain.The connecting peptide 30-41 is much smaller than the 30-35 fragment found in proinsulin and there is no homology between them. The C-terminal octapeptide sequence is also not found in the insulins. All the cysteine and glycine and most of the non-polar core residues of the insulin monomer are conserved indicating that its conformation may resemble that of insulin. It has been suggested that duplication of the gene of the common ancestor of IGF-1 and proinsulin occurred before the time of appearance of the vertebrates7* Somatomedin B is a polypeptide found in plasma which is thought to mediate the action of growth hormone. Material from human plasma has now been sequenced and the 44-residue compound shows a relationship to protease inhibitors and phospholipase rather than any known growth factors.Residues 5-8 for example Cys-Lys-Gly-Arg resemble those of basic trypsin inhibitor (Cys-Lys-Ala- Arg). Somatomedin B inhibits trypsin but not plasmin thrombin or kallikrein and its precise biological role remains to be elucidated.’’ 70 P. Sieber B. Kamber A. Hartmann A. Johl B. Riniker and W. Rittel Helv. Chim.Acta. 1977,60,27. ” E. Rinderknecht and R. E. Humbel J. Bid. Chem. 1978,253,2763. 72 L. Frykland and H. Sieverteson F.E.B.S.Letters 1978,87 55.