Manual multiple synthesis methods

Simultaneous multiple peptide synthesis enables the parallel synthesis of large numbers of peptides. The T-bag (tea-bag) method was developed along with other methods, e.g. pin synthesis, synthesis on paper plates, synthesis on parallel columns, and synthesis on cellulose, as technology to facilitate simultaneous multiple synthesis. Large numbers of peptides, peptidomimetics, and small organic molecules have been prepared using the T-bag method to address different research fields, such as conformational analysis, structure activity analysis, synthesis methodologies, and antibody-antigen interaction studies. Using the T-bag method, more than 150 peptides can be prepared in parallel in flexible amounts, with easily enough material for biological tests and analytical studies. The synthesis of peptides of length of up to 26 amino acid residues has been reported. Moreover, the T-bag technology is easy to apply in practice and requires very little special equipment. T-bags are prepared by containing solid phase resins within polypropylene mesh material. Polypropylene is rather chemically inert as well as fairly thermally stable (to 150°C), allowing a wide range of chemical reactions to be used for solid phase synthesis without affecting the bag material. Polystyrene cross-linked with 1% divinylbenzene, 100-200 mesh, is mainly used as the solid support, but other types of base resin can be used as well, e.g. TentaGel. The size of the resin beads must exceed the size of the pores of the polypropylene mesh material of the T-bags to avoid resin loss during synthesis. Syntheses are carried out manually, using semi automation, or within a multiple peptide synthesizer. The preparation of T-bags for solid phase synthesis, starting with 100 mg resin per bag, is described in Protocol 1. Synthesis using the T-bag method can be performed using either Boc or Fmoc synthetic strategies. For all manipulations, enough solvent should be used to cover the T-bags (about 3-4 ml per bag containing 100 mg of resin). To enable efficient washings and reactions, the reaction vessels (polyethylene bottles) should be shaken vigorously, preferably through the use of a reciprocating shaker.

Instrumentation for automated solid phase peptide synthesis

The concept of solid phase peptide synthesis introduced by Merrifield in 1963 involves elongating a peptide chain on a polymeric support via a two-step repetitive process: removal of the Nα-protecting group and coupling of the next incoming amino acid. A second feature of the solid phase technique is that reagents are added in large excesses which can be removed by simple filtration and washing. Since these operations occur in a single reaction vessel, the entire process is amenable to automation. Essential requirements for a fully automatic synthesizer include a set of solvent and reagent reservoirs, as well as a suitable reaction vessel to contain the solid support and enable mixing with solvents and reagents. Additionally, a system is required for selection of specific solvents and reagents with accurate measurement for delivery to and removal from the reaction vessel, and a programmer to facilitate these automatic operations is necessary. The current commercially available instruments offer a variety of features in terms of their scale (15 mg to 5 kg of resin), chemical compatibility with 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) and tert-butyloxycarbonyl/ benzyl (Boc/Bzl)-based methods, software (reaction monitoring and feedback control), and flexibility (additional washing and multiple activation strategies). In addition, certain instruments are better suited for the synthesis of more complex peptides such as cyclic, phosphorylated, and glycosylated sequences while others possess the ability to assemble a large number of peptide sequences. The selection of an instrument is dependent on the requirements and demands of an individual laboratory. This chapter will describe the features of the currently available systems. As the field of solid phase synthesis evolved, manufacturers designed systems based on the synergy between chemistry and engineering. A key component to an instrument is the handling of amino acids and their subsequent activation to couple to a polymeric support. The goal of an automated system is to duplicate conditions that provide stability to reactive species that might decompose. Standard protocols for automated synthesis incorporate carbodiimide, phosphonium, and aminium/uronium reagents, preformed active esters, and acid fluorides. For further details on coupling methods, see Chapter 3. A second issue related to coupling chemistry is the time required to dissolve an amino acid and store this solution.

Purification of large peptides using Chemoselective tags

In solid phase peptide synthesis (SPPS), deletion sequences are generated at each addition of amino acid due to non-quantitative coupling reactions. Their concentration increases exponentially with the length of the peptide chain, and after many cycles not only do they represent a large proportion of the crude preparation, but they can also exhibit physicochemical characteristics similar to the target sequence. Thus, these deletion-sequence contaminants present major problems for removal, or even detection. In general, purification of synthetic peptides by conventional chromatography is based on hydrophobicity differences (using RP-HPLC) and charge differences (using ion-exchange chromatography). For short sequences, the use of one or both techniques is in general sufficient to obtain a product with high purity. However, on increasing the number of amino acid residues, the peptide secondary and progressively tertiary and quaternary structures begin to play an important role and the conformation of the largest peptides can decisively affect their retention behaviour. Furthermore, very closely related impurities such as deletion sequences lacking one or few residues can be chromatographically indistinguishable from the target sequence. Therefore, purification of large synthetic peptides is a complex and time-consuming task that requires the use of several separation techniques with the inevitable dramatic reduction in yields of the final material. Permanent termination (capping) of unreacted chains using a large excess of an acylating agent after each coupling step prevents the formation of deletion sequences and generates N-truncated peptides. However, even under these more favourable conditions, separation of the target sequence from chromatographically similar N-capped polypeptides requires extensive purification. If the target sequence could be specifically and transiently labelled so that the resulting product were selectively recognized by a specific stationary phase, then separation from impurities should be facilitated. This chapter deals with such an approach and in particular with the purification of large polypeptides, assembled by solid phase strategy, using lipophilic and biotin-based 9-fluorenylmethoxycarbonyl (Fmoc) chromatographic probes. Assuming that the formation of deletion sequences is prevented by capping unreacted chains, a reciprocal strategy can be applied that involves functional protection of all polymer-supported peptide chains that are still growing, with a specially chosen affinity reagent or chromatographic probe.

Basic procedures

A number of excellent descriptions of the techniques related to peptide chain assembly have already been published. These processes are also described in the operator manuals supplied by the peptide synthesis instrument manufacturers. Accordingly, the treatment of the subject presented here has been kept brief in order to provide more space in this volume for those topics not covered in detail in other publications of this type. The protocols have been written as they would be carried out using a manual peptide synthesis vessel. Whilst it is appreciated that most scientists preparing peptides will be using automated peptide synthesizers, it is not possible, given the wide variation in operating procedures, to describe how such methods may be applied to individual instruments. Particular emphasis has been given here to those operations which are typically carried out off-instrument, such as first residue attachment and peptide-resin cleavage. The operations described in this chapter can be carried out in a purpose-built peptide synthesis vessel or in a sintered glass funnel fitted with a three-way stopcock. The operation of the system is extremely simple: solvents are added from the top of the vessel, ensuring any resin adhering to the sides is rinsed down into the resin bed; the resin bed is agitated by setting the tap to position 1 to allow flow of nitrogen to the reaction vessel; solvents and reagents are removed by setting the tap to position 2 to connect the vessel to the vacuum. The use of such vessels has previously been described in detail. Peptide synthesis resins are extremely fragile and the beads, if wrongly handled, can easily fracture, leading to the generation of fines which can block reaction vessel filter-frits and solvent lines. It is particularly important that the correct method is used for mixing the resin and soluble reactants. Polystyrene-based supports are best agitated by bubbling an inert gas through the resin bed, or by shaking or vortexing the reaction vessel. Whilst all of these approaches are employed in commercial synthesizers, gas-bubbling and shaking are the most appropriate for use in manual synthesis.

Basic principles

Construction of a peptide chain on an insoluble solid support has obvious benefits: separation of the intermediate peptides from soluble reagents and solvents can be effected simply by filtration and washing with consequent savings in time and labour over the corresponding operations in solution synthesis; many of the operations are amenable to automation; excess reagents can be employed to help to drive reactions to completion; and physical losses can be minimized as the peptide remains attached to the support throughout the synthesis. This approach does, however, have its attendant limitations. By-products arising from either incomplete reactions, side reactions, or impure reagents will accumulate on the resin during chain assembly and contaminate the final product. The effects on product purity of achieving less than 100% chemical efficiency in every step are illustrated dramatically in Table 1. This has serious implications with regard to product purification as the impurities generated will, by their nature, be very similar to the desired peptide and therefore extremely difficult to remove. Furthermore, the analytical techniques employed for following the progress of reactions in solution are generally not applicable, and recourse must generally be made to simple qualitative colour tests to detect the presence of residual amines on the solid phase. The principles of solid phase synthesis are illustrated in Figure 1. The C-terminal amino acid residue of the target peptide is attached to an insoluble support via its carboxyl group. Any functional groups in amino acid side chains must be masked with permanent protecting groups that are not affected by the reactions conditions employed during peptide chain assembly. The temporary protecting group masking the α-amino group during the initial resin loading is removed. An excess of the second amino acid is introduced, with the carboxy group of this amino acid being activated for amide bond formation through generation of an activated ester or by reaction with a coupling reagent. After coupling, excess reagents are removed by washing and the protecting group removed from the N-terminus of the dipeptide, prior to addition of the third amino acid residue.

Introduction — a retrospective viewpoint

The Chemical Society publication Annual Reports on the Progress of Chemistry for 1963 attempted to inform readers of all the highly significant advances in all the major fields of pure chemistry during that year. Fortunately, the section on peptide chemistry drew attention to a paper by R. B. Merrifield which had just been published in the Journal of the American Chemical Society: A novel approach to peptide synthesis has been the use of a chloromethylated polystyrene polymer as an insoluble but porous solid phase on which the coupling reactions are carried out. Attachment to the polymer constitutes protection of the carboxyl group (as a modified benzyl ester), and the peptide is lengthened from its amino-end by successive carbodiimide couplings. The method has been applied to the synthesis of a tetrapeptide, but incomplete reactions lead to the accumulation of by products. Further development of this interesting method is awaited. I remember thinking at the time that in this paper we had possibly seen both the beginning and the end of the interesting new technique of solid phase peptide synthesis. To many organic chemists, the described result was that anticipated—difficulty in bringing heterogeneous reactions to completion resulting in impure products. Both this and purification problems were expected to worsen as the chain length was increased beyond Merrifield’s tetrapeptide limit. In fact, I probably had at the time an inadequate appreciation of the difference between truly heterogeneous or surface reactions and those in the solvated gel phase. The latter approaches much more closely the solution situation. However, the new technique also flouted many of the basic principles of contemporary organic synthesis which required rigorous isolation, purification, and characterization regimes following each synthetic step. In Merrifield’s new technique, isolation consisted simply of washing the solid resin, there was no other purification of the products of each reaction, and little or no characterization of resin-bound intermediates was attempted. The first two of these are of course the important characteristics which give the method its speed and simplicity and contribute to its efficiency. Small wonder, though, that in many minds there was doubt about the future of the new technique.

Phosphopeptide synthesis

Protein phosphorylation mediated by protein kinases is the principal mechanism by which eukaryotic cellular processes are modulated by external physiological stimuli. Phosphopeptides are essential tools for the study of this process, serving as model substrates for phosphatases, as antigens for the production of antibodies against phosphorylated proteins, and as reference compounds for determining their physical parameters. The development of methods for the production of phosphopeptides has consequently attracted considerable interest over the last few years, and these endeavours have yielded reliable procedures which have now made their synthesis routine. There are two strategies used currently for the preparation of phosphopeptides: the building block approach, in which pre-formed protected phosphoamino acids are incorporated during the course of chain assembly, and the global phosphorylation method, which involves post-synthetic phosphorylation of serine, threonine, or tyrosine side-chain hydroxyl groups on the solid support. The building block procedure is certainly the more straightforward of the two approaches and has now become, owing to the availability of suitably protected phosphoamino acids, the standard method for the routine production of phosphopeptides. For the side-chain protection of phosphotyrosine in Fmoc/tBu-based solid phase synthesis, methyl, benzyl, t-butyl, dialkylamino, and silyl groups have been employed. Of these, benzyl is most useful as it is the most convenient to introduce and is rapidly removed during the TFA-mediated acidolysis step. Only the mono-benzyl ester, Fmoc-Tyr(PO(OBzl)-OH)-OH 1, is available commercially; the dibenzyl ester offers no practical benefit as it undergoes mono-debenzylation in the course of the piperidine-mediated Fmoc deprotection reaction. Also available commercially is Fmoc-Tyr(PO3H2)-OH 2. This derivative, despite having no phosphate protection, appears to work well, particularly in the synthesis of small- to medium-sized phosphopeptides; although formation of the pyrophosphate 3 can be a problem in peptides containing adjacent Tyr(PO3H2) residues. Phosphate triesters of serine and threonine are not compatible with Fmoc/tBu chemistry as they undergo β-elimination when treated with piperidine, resulting in the formation of dehydroalanine and dehydoaminobutyric acid, respectively For this reason, it was long believed that the building block approach could not be used for preparation of peptides containing these amino acids.

Synthesis of modified peptides

Several important hormones such as oxytocin, secretin, and LHRH are known to be peptidyl amides. In addition to these, other peptidyl amides such as indolicidin and the protegrins have been shown to exhibit potent antimicrobial activity. The in vivo production of such compounds is via endogenous enzymatic cleavage of propeptides, making their synthesis by genetic engineering notoriously difficult. Furthermore, to facilitate the survival of synthetic peptidyl amides in vivo, an obvious defence against the action of carboxypeptidases is the N-alkylation of the carboxylic amide terminus. Such secondary amides would be expected to exhibit vastly different solubility and transport properties to primary amides, thus their chemical synthesis is of immense importance. The solid phase synthesis of peptidyl amides and peptidyl N-alkyl amides is centred around two main strategies: 1. Ammonolysis/aminolysis of resin-bound esters. 2. Use of resin-bound primary amines, which may in turn be chemically modified to generate novel secondary amine functionalized linkers for the synthesis of peptidyl N-alkyl amides. Early examples of the use of resin-bound amines for the solid synthesis of peptidyl amides involve the use of linkers such as benzhydrylamine or benzylamine. Following peptide assembly, these linkers require highly acidic (e.g. HF) mediated cleavage, and hence simultaneous removal of acid-labile sidechain protection groups occurs and may cause problems. Systematic modifications of these linker-resins by substitution with electron-donating substituents have resulted in the generation of numerous linker-resins with increased acid sensitivity. Notably, the 4-(2',4'-dimethoxyphenylaminomethyl) phenoxy derivatized (Rink) resin 1, which is cleavable by 95% v/v TFA, and the 5-(2-fluorenylmethoxycarbonylaminornethyl-3,5-dimethoxy)- phenoxyvaleric acid (PAL) linker 2 acidolysed by 75% v/v TFA. Greater acid lability has been achieved using the xanthenyl derivatized resin, 9-(fluorenylmethoxycarbonylamino)xanthen-3-yloxymethyl polystyrene (Sieber amide) resin 3, which besides being cleavable by 1% v/v TFA, holds the added advantage of readily undergoing reductive N-alkylation to afford resin-bound secondary amines for the synthesis of peptidyl N-alkyl amides. In addition to the method detailed below, a number of alternative approaches have recently been reported.

Convergent peptide synthesis

Besides the classical step-by-step synthesis, the convergent solid phase peptide synthesis (CSPPS) was developed for the preparation of complex and difficult peptides and small proteins. According to this method, suitably protected peptide fragments spanning the entire peptide sequence and prepared on the solid phase are condensed, either on a solid support or in solution, to the target peptide. Convergent synthesis is reviewed in recent publications. In this chapter, full experimental details are given for the preparation of complex peptides by applying convergent techniques, using 2-chlorotrityl chloride resin (CLTR) and Fmoc-amino acids. In the step-by-step peptide chain elongation the resin-bound C-terminal amino acid is reacted sequentially with suitably protected and activated amino acids. The peptide is thus elongated steadily towards the N-terminal direction. This is advantageous over the opposite direction where the elongation is performed from the N- to the C-terminus, because in the second case the growing peptide is activated at the C-terminal amino acid, which leads to its extensive racemization. This limits considerably the synthetic possibilities of the method. In convergent synthesis, no directional restrictions exist and the chain elongation can be performed with equal possibility to be successful to any direction. Figure 1 describes schematically the C- to N-terminal synthesis which is the most studied to date. The strategies where the synthesis begins from a central fragment and the peptide chain is extended to both C- and N-terminal directions and from the N-terminal towards the C-terminal can be considered, at the present time, to be in its infancy. In general, protected peptide fragments of any length can be used in the condensation reaction, if they are of satisfactory purity and solubility. Usually, fragments of up to 15 amino acids in length are used, because of their simpler purification by RP-HPLC compared with the longer peptides. The solubility of protected peptide acids is independent of their length. The selection of the correct fragments is very important for the success of convergent synthesis. It is helpful to analyse all available structural information, determined or calculated, for the target peptide. Peptide regions where β-turns are known to occur are readily identified as ‘difficult’ sequences during their synthesis.

Preparation and handling of peptides containing methionine and cysteine

Among the genetically encoded amino acid residues, methionine (Met) and cysteine (Cys) are special because they each contain an atom of sulphur. The present chapter describes how these residues are incorporated into peptides in the context of an Fmoc/tBu solid-phase synthesis strategy, as well as further considerations once the synthetic peptide is released from the support. Of added interest, some manipulations of Cys are advantageously performed at the level of the assembled peptide-resin, prior to cleavage. Many of the aspects discussed here also carry over to the preparation of peptides using a Boc/Bzl strategy. The major problems associated with management of Met reflect the susceptibility of the thioether to alkylation and oxidation. One of the merits of the Fmoc/tBu strategy, in contrast to Boc/Bzl, is that in the former strategy Met is usually introduced without recourse to a protecting group for the thioether side-chain. As documented in this chapter, a proper understanding of acidolytic cleavage conditions and the availability of selective procedures to reverse any inadvertent oxidation are likely to lead to success in obtaining homogeneous peptides containing Met. Management of Cys provides additional significant challenges. For some targets, Cys is required with its side-chain in the free thiol form, whereas for other targets, an even number of Cys residues pair with each other via disulphide linkage(s) to provide cystine residue(s). Disulphide bridges play an important role in the folding and structural stabilization of many natural peptides and proteins, and their artificial introduction into natural or designed peptides is a useful approach to improve biological activities/specificities and stabilities. Furthermore, use of a disulphide bridge is a preferred method to conjugate peptides to protein carriers for increasing the response in immuno-logical studies, to link two separate chains for developing discontinuous epitopes, and to generate active site models. This chapter describes Cys protecting groups, how they are removed to provide either free thiols or disulphides directly, and various strategies and practical considerations to minimize side reactions and maximize formation of the desired products. The thioether side-chain of Met is subject to alkylation and oxidation side reactions, either during the synthetic process or during subsequent handling of the Met-containing peptide.