CHAPTER 1
Peptide Synthesis
BY DONALD T. ELMORE
1 Introduction
As in the previous Report, many reviews have been published. Some relate to several sections of this article whereas others are cognate to particular sections as follows: Section 2.1, Section 2.3, Section 2.4, Section 2.5, Section 2.6, Section 2.7, Section 3.1, Section 3.2, Section 3.3, Section 3.4, Section 3.5, Section 3.6, Section 3.8, Section 3.9 and Section 3.9..
2 Methods
2.1 Amino-group Protection. – It often happens in chemistry that a technique that has been regarded as passé for some years, is slightly modified and resumes its former status in the literature. Protection of the α-amino group by the phthaloyl group is a recent example. The need for rather vigorous conditions in order to effect removal with hydrazine led to discontinuance of its use. An improved method of phthaloylation using monomethyl phthalate would hardly have stayed its abandonment, but the discovery that tetrachlorophthaloyl groups, while stable to piperidine and to acids, are removed with N2H4/CHONMe2 (3:17) at 40°C for 1 hr has offered a fresh orthogonal substituent. Moreover, preparation of tetrachlorophthaloyl amino acids under microwave irradiation is straightforward. Microwave irradiation also permits rapid synthesis of phthaloyl derivatives with no loss of chiral purity, but this development has probably come too late for modern peptide synthesis. New variations in the conditions for removal of the Boc group have been reported. A fast selective method uses 4M-HCl in dioxan. Deprotection is complete in 30 min. at room temperature. The method is selective in the presence of But esters and alkyl esters and also But thioethers but not in the presence of But phenolic ethers. Nitrolytic removal of Boc groups has been reported and Z-groups and But esters are unaffected, but oxidation of susceptible groups is a hazard. Boc groups can be removed from substituted peptides on Wang resin using conc. H2SO4 in dioxan (1:9 v/v) at 8°C for 2 hr. Cleavage of the peptide from the resin is quite limited. Lipidated peptides are often very labile to acids and bases and it is valuable that Boc groups can be removed using trimethylsilyl triflate in the presence of a tertiary base such as lutidine or EtMe2N. The use of silica gel under microwave irradiation for the detachment of acid-labile groups was disappointing. Strong irradiation for long periods was required and this gave unacceptable amounts of byproducts. A precolumn method of preparation of Fmoc-amino acids and -peptides can be carried out with Fmoc-Cl after adsorption on silica gel. Excess reagent is washed away with EtOAc. The Fmoc-derivatives are then eluted for analysis; much less byproduct is present than with earlier protocols and Fmoc-Cl is completely absent. Details for the synthesis on a large scale of Fmoc-4-aminomethyl benzoic acid, Fmoc-trans-4-(aminomethyl)cyclohexane carboxylic acid and Fmoc derivatives of cis-β-amino acids have been described. Fmoc- and Z-derivatives of N-methylserine and N-methylthreonine can be accessed by the general method in Scheme 1. Polymer-bound N-hydroxysuccinimide can be prepared from commercial styrenemaleic anhydride copolymer by reaction with 50% w/v aqueous NH2OH. Treatment of the product with Fmoc-Cl/aq. K2CO3 gives rise to polymer-bound Fmoc-OSu. This is a convenient reagent for the preparation of Fmoc amino acids and the unused and reacted polymer can be filtered off and reused. Fmoc-Amino acids can also be directly synthesised using organo-zinc chemistry. For example, Fmoc-3-iodoalanine-OBut (obtained in 7 steps from L-serine) is converted into the organo-zinc derivative and then into a substituted Phe derivative ready for SPPS after appropriate deprotection (Scheme 2). An interesting method of preparing Fmoc- and Z-derivatives of amino acids uses the chloroformate at neutral pH in presence of activated Zn powder. An improved method for the acidolytic removal of protecting groups from thionopeptides used aqueous CF3CO2H (<80% w/v) for about 2h. This procedure minimises the acid-catalysed cleavage at the peptide bond immediately following the -CSNH- moiety. The (2-nitrofluoren-9-yl)methoxycarbonyl group offers interesting possibilities. Not only does it possess enhanced lability to bases, but it also undergoes photo-chemical cleavage under appropriate conditions. α-Fmoc groups have been employed inter alia for the synthesis of cyclic peptides involving ring closure on a specially synthesised backbone amino acid. This procedure has been recommended for combinatorial synthesis of potential peptide drug candidates. The Alloc group can be used to protect α-amino groups in the synthesis of lipopeptides since Pd(0) rather than acid can be used for deprotection as an alternative to that mentioned above. The same paper recommends the use of But groups for the protection of carboxy and thiol groups. Neutral conditions can be used to release protected amino groups if the 2-nitrobenzyl group is used. A low cathodic potential converts the nitro group into a hydroxyamino group that undergoes ring closure to benzisoxazolone with release of the amino group. The 2-[phenyl-(methyl)sulfonio]ethoxycarbonyl group ensures that, after protection of the amino group, the product will be soluble in water and Leu enkephalin has been synthesised using it. This could be useful in syntheses employing enzyme-catalysed steps and it could also simplify the production of crystals for X-ray diffraction studies. A base-labile group, 2-(2,4-dinitrophenylsulfonyl)ethoxycarbonyl, has been recommended for solid-phase peptide synthesis, but does not appear to offer any special advantages. A novel side reaction was discovered when an O-silyl group was removed from protected pyroglutaminol. The expected product was formed admixed with an isomer in which a Boc group had migrated (Scheme 3). The use of enzymes in peptide synthesis is still limited to a few workers. Immobilised penicillin G acylase in toluene has been used to acylate amino acids, but attempts to effect esterification and trans-esterification failed. It is not surprising that an enzyme will not operate on both ends of an amino acid molecule. If it did, a jumble of polymers would be the outcome. The established PhAcOZ group, which is enzyme-labile, has been used to synthesise acid- and base-labile nucleo-peptides.
2.2 Carboxy-group Protection. – Although trifluoromethyl esters are not much used in peptide synthesis, it may be useful to know that these derivatives of N-acylamino acids are accessible using catalysis by 4-dimethylamino pyridine. 2,2,2-Trichloro-t-butyl (Tcb) esters are of interest because they are deprotected in slightly acidic or neutral conditions in the presence Zn2+ or Cd2+ ions with the super nucleophile cobalt(I) phthalocyanine. Preparation of Tcb esters so far described involve the use of acyl chlorides, but unsymmetrical anhydrides might be a suitable alternative starting material. The esterification of amino acids can be effected in the presence of 'triphosgene' (CCl3OCOOCCl3) in tetrahydrofuran at 55-60°C. Amino acids have been esterified with MeOH under pressure and in the presence of an ultrastable zeolite as catalyst. There has been a further report for the esterification of acylamino acids with 3,4,5-tris(octadecyloxy)-benzyl alcohol before peptide synthesis. The product is purified by size-exclusion chromatography on Sephadex LH-20. Using Fmoc amino acids, subsequent deprotection could be effected using 4M-HCl in EtOAc. This could alternatively be achieved under milder conditions using a lipase for deprotection in the presence of activated charcoal to retain the liberated alcohol with filtration or centrifugation to isolate the product. Treatment of Boc-Ala-OBut with CeCl3.7H2O/NaI removed both protecting groups. If the CeCl3.7H2O were heated in MeCN under reflux, then cooled to room temperature and added to the substrate, the ester group was removed but the Boc group survived. Obviously further work is required to delineate conditions for selective deprotection. The β-CO2H of Asp in peptides prepared by SPPS can be isotopically enriched by hydrolysis with Na17OH in MeOH/CH2Cl2. The peptide is detached from the support using acid.
2.3 Side-chain Protection. – An efficient synthesis of N-Boc-O-cyclohexyl-L-tyrosine has been described. Boc-Tyr-OH was treated with NaH in CHONMe2 and then with 3-bromocyclohexene. The product was hydrogenated over PtO2. The highest yield was obtained when a byproduct from the previous step was not removed because the yield then was almost quantitative. A problem has been encountered when the hydroxyl group of Thr was protected by tosylation. A mixture of O-tosyl- and dehydro-Thr was formed. The ratio of the two products depended strongly on the choice of groups for protecting the amino and carboxy groups. In the case of Fmoc-Thr-OBzl, only the unsaturated product was formed. In order to obtain fluorescent enzyme substrates, α-Fmoc-ε-[(7-methoxycoumarin-4-yl)acetyl]-L-lysine was used as starting material. Strictly speaking, the fluorescent substituent is not a protecting group, since it can not be detached without destroying the peptide. Further examples of this type of fluorescent substrate are cited in Section 3.3. For protection of the thiol group in the side chain of an amino acid, 4-methoxytrityl chloride is appropriate. The S-(4-methoxytrityl) group can be retained if the peptide is detached from the resin using CH3CO2H/CF3CH2OH/CH2Cl2 (1:2:7) for 15 min. at room temperature, but is cleaved with 1-5% CF3CO2H in CH2Cl2/Et3Si (95:5). Allylic protection of cysteine is possible with the S-[N-{2,3,5,6-tetrafluoro-4-(phenylthio)-phenyl}-N-allyloxycarbonyl]-aminomethyl (Fsam) group (1), which can be removed by oxidation with I2 with concomitant formation of disulphide bonds or the allylic moiety can be removed with Pd(PPh3)4 in the presence of an allyl scavenger to give the peptide with free thiol groups.
The side-chain of Met, normally protected as the sulfoxide, can be regenerated by using Bu4NBr in CF3CO2H as a reducing agent. Nucleophilic attack of halide at the S atom of the protonated sulfoxide is proposed. The tetracovalent S intermediate undergoes release of water after further protonation to give a halosulfonium ion and this is the rate-determining step.
Protection of the imidazole ring of His with the Bum group has been reexamimed. Byproduct formation during deprotection could be diminished by scavenging the HCHO formed using MeONH2.
2.4 Disulfide Bond Formation. – There has been a surprising report that a cyclic disulfide is formed from a peptide with cysteine at both termini in degassed water under reduced pressure. Cyclisation occurred even when the amount of oxygen present in the sytem was only 1/16 of that theoretically required. The authors maintain that disulfide formation does not necessarily require an oxidant such as elementary oxygen or iodine. The report last year that exposure to trans-[Pt(en)2Cl2] effects intramolecular disulfide bond formation from peptides containing two cysteinyl residues has been amplified by the discovery that fully reduced α-conotoxin GI and SI are similarly converted into the oxidised form with 3 disulfide bonds in one step at pH values between 3 and 7.
Water-soluble reagents containing Se such as (2) are quite powerful oxidising agents, although these have not been applied to the formation of peptide cyclic disulfides. Based on appropriate model experiments, it was shown that Fmoc-Cys-β-Ala-Cys-OMe, which was immobilised on a resin through a benzyl thioether link involving the side chain of the C-terminal cysteine residue, underwent detachment and intramolecular cyclisation in the presence of N-chlorosuccinimide (Scheme 4). A library of amphipathic bicyclic peptides in which one of the rings contained a disulfide has been assembled. The first cyclic structure was produced by intramolecular thioester ligation while attached to a solid support and the second ring was closed by oxidation with MeSOMe of the side chains of two cysteinyl residues after detachment from the resin.
2.5 Peptide Bond Formation. – Boc2O is commonly used in the unsymmetrical anhydride synthesis of peptides or for protecting -OH or -NH- groups. Imidazole or trifluoroethanol are convenient scavengers to react with excess reagent. Pentafluoro-, 2,4,5-trichloro- and pentachloro-phenyl esters of Fmoc amino acids are conveniently prepared using a 2-phase system in which 3% NaHCO3 solution is the aqueous phase. Good yields are obtained in 2-3 h. Pentafluorophenyl 4-nitrobenzenesulfonate is a useful reagent for peptide coupling and HOBt strongly promotes synthesis. 4-NO2-C6H4-SO2OBt is a probable intermediate. No loss of chiral purity was detected when Boc amino acids were coupled but not surprisingly there was extensive loss when Bz amino acids were used. N-Protected amino acid bromides have not previously been used for peptide synthesis because they are unstable and difficult to purify. They have now been obtained from N-protected amino acids using 1-bromo-N,N-2-trimethyl-1-propenylamine under mild and neutral conditions. They are useful for coupling very hindered amino acids and are used in the presence of collidine. Carbodiimides have not been completely abandoned as coupling agents. Sparingly soluble protected peptides have been coupled in CHCl3/PhOH using EtN = C = N(CH2)3NMe2 in the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt) with no loss of chiral purity. Similarly, Z-Gly-Phe-OH and H-Phe-OBzl were coupled in the same solvent but using PriN = C = NPri with a combination of HOOBt and its Bu4N+ salt as additive. There has been another paper extolling the value of Cu(II) derivatives for retaining chiral purity during SPPS. The use of the Cu(II) derivatives of HOBt and HOAt seem to be particularly effective. Another group has adduced further supporting evidence. For example, the use of HOBt and CuCl2 minimises loss of chiral purity when PyBOP is the coupling agent. Again, CuCl2 and HOAt eliminates enantiomerisation in segment coupling reactions. It really is quite surprising that this technique is not routinely used in the light of reports in recent years. It has been reported that HOBt, HOOBt and HOSu react with CH2Cl2 and ClCH2CH2Cl to form respectively di- and mono-substituted derivatives. In the light of the successful use of CHCl3/PhOH mentioned above, it would be useful to determine if CHCl3 reacts with the above additives. Carpino has synthesised the 4,5- and 5,6-benzo derivatives of HOAt. Both can be converted into coupling reagents with (Me2N)C+QPF6-. The 4,5-derivative gives a uronium derivative (3) whereas the 5,6-derivative gives a guanidinium derivative (4). Although neither possesses any special advantage over HOAt, the former caused coupling faster than the latter. Li and Xu have described a battery of onium-type peptide coupling agents. Some of these (e.g. BOMI, BDMP, BEP, BEMT) have been reported by these workers before and have been mentioned in vols. 32 and 33 of this series of publications. Li and Xu have summarised their conclusions about the potential value of some of these and earlier reagents. Reagents based on HOBt and incorporating phosphonium and uronium structural components (e.g. BOP, HBTU) are regarded as satisfactory for syntheses involving coded amino acids. For segment coupling, however, where loss of chiral purity may occur, reagents based on HOBt and incorporating immonium structural components (e.g. BOMI, BDMP) would be a better choice, because of their ability to limit loss of chiral purity and on account of their reactivity. Although reagents based on HOAt are acknowledged as the strongest candidates for the synthesis of hindered peptides, Li and Xu cite the disadvantage of high expense of reagent. They suggest, however, that 2-halopyridinium (e.g. BEP,5) and 2-halothiazolium (e.g BEMT,6) reagents are also suitable and can be synthesised from cheaper starting materials. The original paper should be consulted for a list of structures of other potential coupling reagents. Prop-2-ynyl triphenylphosphonium bromide is another onium-type coupling agent that has been used to synthesise a number of small peptides. Microwave radiation has been used to promote the synthesis of Aib peptides using PyBOP or HBTU in CHONMe2 as solvent. A one-pot method for deprotecting Alloc amines and coupling to N-Boc or N-Fmoc amino acids uses Pd(PPh3)4 and 1,8-diazabicyclo[2,2,2]-cyclooctane (DABCO) as scavenger and a carbodiimide to effect coupling involves a reaction time of 10-20 min. Although derivatives of 1,3,5-triazine have been used as coupling agents in the past, there has been a further development in this area. When racemic N-protected amino acids were used in peptide synthesis in the presence of a chiral tertiary amine such as strychnine, brucine or sparteine and 2-chloro-4,6-dimethoxy-1,3,5-triazine, the coupling proceeded enantio-selectively. Kagan enantioselectivity parameters were derived and were in the range 1.6-195. Strychnine gave the best results (s = 98-195). The authors postulated that chiral triazinyl-ammonium chlorides were formed as intermediates. Another coupling method merits further assessment. Instead of using N-protected amino acids, α-azido acids, prepared by Wong's Cu2+-catalysed diazo transfer method, were the starting material. There was negligible loss of chiral purity and peptides that are prone to formation of diketopiperazines were obtained in good yield by a solid-phase method.
