The first application of 11 towards the solid phase synthesis of duocarmycin analogues: coupling to resin bound amino acids
3.2 Sold phase peptide synthesis
The introduction of solid phase synthesis in 1963140 significantly improved the synthetic accessibility of peptides. Since its inception the technique has evolved considerably, and has found far reaching applications beyond simply the construction of polyamide targets.141 However, it is still within the field of peptide and oligonucleotide synthesis that this method dominates. When first introduced, the method was received with unveiled hostility by many within the peptide synthesis community.142 It certainly represented a paradigm shift from the classical solution approach, and early work was not altogether convincing. However, many initial challenges were soon overcome, and the method was validated with results that only the most harden critic could deny as impressive.143 It is now arguably the most common method for the synthesis of peptides on a laboratory scale. The importance of this technique saw its inventor Bruce Merrifield later recognised with the 1984 Nobel prize in chemistry.144
The basic concept is elegant in its simplicity.145 Synthetic transformations are performed on a substrate which is covalently anchored to an insoluble support. The solid supported product is isolated by filtration, allowing unbound side products and reagents to be simply washed away. This operational simplicity allows for the use of large excesses of reagents to drive reactions to completion. As multiple sequential reactions can be performed without the need for intervening purification, this technique can significantly reduce the time required to perform multistep syntheses. Furthermore, the use of reaction vessels designed to allow in situ filtration, under pressure, or by the application of a vacuum, mean multistep syntheses can effectivity be performed as one pot processes. This can be particularly advantageous when working on a small scale, where in solution phase chemistry the additive attrition of material during the work up and purification of individual reactions can limit the effective starting scale of a long synthesis. The final product is isolated by cleavage from the solid support.
The need for orthogonal cleavage conditions, and a cleavage product that introduces a desired structural motif can limit the scope of solid phase synthesis. The repetitive nature of peptide synthesis and the relative simplicity of the synthetic strategy means it is an ideal area for application of solid phase methodology. It is therefore unsurprising that peptides were the first target of solid phase synthesis, and represent its most common application. The operational simplicity of solid phase peptide synthesis means automation
113 of the process was quickly introduced, and indeed peptide targets are now routinely synthesised by commercially available automated peptide synthesisers.146
In solid phase peptide synthesis the target peptide is constructed in a stepwise fashion via the sequential coupling of amino acid residues by amide bond formation. The first amino acid is anchored to the solid support through its carboxylic acid. Each subsequent amino acid is coupled with its amine protected. This prevents unintended homocoupling in solution. Subsequent deprotection of the amine after coupling, allows the next amino acid to be coupled, and so on, until the desired peptide is complete. Orthogonal side chain protection is also required to prevent the formation of solid supported impurities. Side chain protection is typical chosen that will be cleaved under the same conditions used to release the completed peptide from the solid support. Linkage to the solid support is usually, but not exclusively, designed to provide a free carboxylic acid after cleavage.
Solid supports typically take the form of cross-linked polymeric resins. Polystyrene based resins are perhaps the most commonly used.145 The term solid phase synthesis is arguably a misnomer, as reactions do not take place at the surface of a truly heterogeneous system. Solvation, in fact, leads to swelling of the resin, and the formation of a gel like matrix in which the reactions take place. Studies have shown that this environment provides similar access to reagents, as if the individual polymers were free in solution.147
The classical Merrifield approach utilises a Boc and benzyl-protecting strategy. The N-terminal amino group of the growing peptide is protected by a Boc group. This is cleaved prior to coupling by treatment with TFA. The side chains of trifunctional amino acids are protected by benzyl derivatives, and the peptide anchored to the resin through a benzyl ester. Treatment with anhydrous hydrofluoric acid yields the cleaved unprotected peptide.
In skilled hands this method has proved an extremely powerful technique. However, it does suffer from several drawbacks. Firstly, the protection strategy is not truly orthogonal, and relies on differing acid sensitivity between the Boc and benzyl-protecting groups. As such some undesired loss of benzyl protection is often observed, which can result in the accumulation of resin bound impurities. The strongly acidic conditions required for cleavage can also limit the success of syntheses incorporating sensitive residues.
However, perhaps the biggest deterrent to the use of this method, is the hazardous nature of hydrofluoric acid. This reagent is notoriously toxic, and requires demanding safety protocols. It is also incompatible with common laboratory equipment.148
114 Although still in use, the Merrifield approach has been largely superseded by the development of Fmoc solid phase peptide synthesis,149 which allows the use of milder deprotection and cleavage conditions.150 This technique utilises the base sensitive Fmoc group for N-terminal protection. As such, side chain protection and linkage to the resin can be effected by protecting groups and linkers that are cleavable under mildly acidic conditions.
Fmoc deprotection is typically achieved by treatment with piperidine (scheme 3.1). The Fmoc group is base sensitive due to the acidic nature of the proton at the bridgehead of the fluorenyl group. Acidity is a result of the aromatic stabilisation of the carbanion formed by deprotonation. In the Fmoc group, deprotonation results in the intramolecular attack of the anion at the neighbouring partially-positive carbon. Here the carbamic acid anion serves as a leaving group, and subsequently decomposes releasing CO2 and the desired amine. A large excess of piperidine is typically used to scavenge the reactive dibenzofulvene by-product.
Many resins differing in both their linking strategy and polymeric construction have been developed for use in Fmoc solid phase synthesis. As will be seen, this would prove extremely useful in optimising the solid phase synthesis of duocarmycin analogues using the new Fmoc-protected alkylation subunit 11.
Scheme 3.1 Mechanism of Fmoc-deprotection by treatment with piperidine.
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