7 Solid Phase Peptide Synthesis
7.1 General principles
The revolutionary principle behind solid phase peptide synthesis (SPPS) as conceived by Merrifield in 1959 is that if the peptide is bound to an insoluble support then any unreacted reagents left at the end of any synthetic step can be removed by a simple wash procedure, greatly decreasing the time required for synthesis. What is more, the arrangement is amenable to automation. This is only valid, however, if the individual synthetic steps occur with essentially quantitative yields. This latter requirement entails rigorous testing of the chemistry and tends to result in the use of a limited range of tried and tested methodologies.
The two most commonly used schemes are the original Merrifield method and that proposed by Sheppard in 1975. While the protection schemes used are different, they have the same basic concepts, a resin support, the use of an excess of reagents and building the peptide in a C®N terminal direction.
The resin supports used are designed to swell in commonly used solvents, expanding to many times their original size. Thus, the reactions occur not on the surface of a rigid particle, but within the support in a solvated gel which permits easy access to the growing peptide chain.
Reagents are used in excess, this allows the reactions to proceed to completion in the minimum time and results in faster synthesis of peptides with high purity. The couplings proceed in a C®N terminal direction to allow the use of racemisation limiting amine protection for the activated species.
7.2 Merrifield synthesis
This methodology is characterised by the use of tert-butyl based temporary a-amino protection and benzyl, or substituted benzyl, groups for permanent side chain protection. There are over one hundred different substituted resins suitable for peptide synthesis generally based on polystyrene and polyethylene glycol. These resins allow introduction of an amino acid through either substitution, condensation or addition reactions. The traditional resin used for Merrifield synthesis was a chloromethylphenyl substituted resin. The first amino acid was attached to the resin through substitution of the chloride by the caesium salt of the BOC-amino acid, generating an equivalent to a benzyl ester (figure).
Deprotection of the temporary BOC group uses a 20-50% solution of trifluoroacetic acid (TFA) in dichloromethane and has to be followed by neutralisation of the resulting ammonium salt with a hindered tertiary base. Final cleavage from the resin as well as deprotection of benzyl based side chain protecting groups is achieved using strong acids, usually liquid hydrogen fluoride or trifluoromethane sulphonic acid. Such procedures require specialised apparatus and the highly acidic conditions catalyse several possible rearrangements.
7.3 Fmoc Polyamide Synthesis
The fundamental differences between the Fmoc polyamide strategy when compared to the Merrifield approach are that the reactions are carried out under continuous flow and that the conditions for a-amino deprotection and cleavage from the resin are far more mild. This arises from the adoption of the base labile Fmoc protecting group for a-amino protection. The side chains are generally protected with tert-butyl based groups which, in common with the linkage to the resin, can be cleaved by TFA in the presence of scavengers.
As discussed above, a large number of resins are available. Traditionally, resins with 4-hydroxymethylphenoxy substitution were used. These allowed were esterified with the anhydride of the first amino acid. As a result of the mesomerically electron donating para oxygen atom stabilising the resultant carbocation, cleavage of the peptide from the resin occurs under more mild acid conditions, typically using trifluoroacetic acid with scavengers (figure).
The use of continuous flow means that the reagents are passed through a reaction chamber containing the resin supported peptide. Having passed through this chamber, the reagents can be recirculated back into the chamber again or taken to a waste collection bottle. This allows the resin to be washed clean of excess reagents and unwanted reaction products, which in turn helps drive deprotection steps to completion following Le Chatelier's principle. In addition, the solution can be passed through a u.v. detector and monitored at a suitable wavelength for the Fmoc chromophore.
A typical cycle consists of:
1. Deprotection of the preceding residue with piperidine.
2. Wash to remove any remaining reagents from 1.
3. Acylation in a recirculatory mode.
4. Wash to remove excess reagents.
A typical uv trace indicates these steps.
In this way, the cycle can be qualitatively monitored. Such monitoring can be used in conjunction with automation. For example, a slow deprotection step suggests inaccessibility of the peptide amino terminal, indicating that the next coupling step may require a longer acylation time. If this is detected then the cycle can be interrupted for later manual intervention.
8 Summary
Since the turn of the century, there has been a constant drive to develop new and improved strategies for peptide synthesis. The areas of protection, deprotection, activation and coupling have all received attention. The field of solid phase peptide synthesis has rapidly grown since its inception by Merrifield but is dominated by use of BOC/benzyl and Fmoc/tert-butyl protection schemes.
