Automated synthesis of backbone protected peptides†

Solid phase peptide synthesis (SPPS) is a powerful technology for the chemical synthesis of peptides and small proteins. However, access to many targets is often complicated and sometimes precluded by the occurrence of so-called difficult sequences. When encountered during SPPS difficult sequences are associated with a collapse of the swollen resin volume, incomplete acylation and in the case of Fmoc/tBu synthesis, incomplete deprotection steps, extending over several residues. As the cause of difficult sequences is intermolecular chain association, double coupling provides no improvement. Pioneering work by Sheppard and co-workers demonstrated that introducing proline into an aggregating peptide sequence, before the onset of aggregation prevented interchain association by removal of hydrogen bonding. By extension, they also demonstrated that reversible alkylation of the peptide backbone suppressed interchain association and was a general solution to the difficult sequence problem. Reversible substitution of the amide bond was called ‘backbone protection’ when introduced by Weygand and co-workers because of similarities to the protecting group strategies then in development. However, it is only rarely necessary to protect the amide bond itself. Amongst the many backbone protection groups available the most widely used are the commercially available pseudoprolines (c-Pro 1 Scheme 1). Pseudoprolines have revolutionized Fmoc/tBu synthesis by enabling the synthesis of previously intractable peptides, unobtainable even by in situ neutralisation Boc protocols. Their key advantage is that they can be introduced with great convenience as dipeptide building blocks and have been successfully applied to enable synthesis of difficult sequences and long peptides. However, their use is unfortunately limited to those sequences containing conveniently positioned X-Ser or X-Thr. Backbone amide protection was first investigated (Dmb 2 Scheme 1) for its dramatic effect on peptide solubility. The effect of Dmb 2 on improving peptide solubility was explored in detail by Narita and co-workers. Current opinion considers that the poor solubility observed for many peptide sequences in solution reappears on the solid phase as difficult peptides and backbone protection acts in both cases by disrupting structure formation. A fully solvated peptide-resin should give the best coupling kinetics and evidence from NMR and I.R. studies on aggregating peptides on solid phase supports this model. Many novel backbone protection strategies have been developed but the increased steric hindrance that accompanies the introduction of a secondary amine into a sequence prohibits quantitative coupling using standard conditions (except with glycine) and has limited the wider adoption of these new backbone protection strategies. A solution to overcome this obstacle harnessed an intramolecular acyl transfer. This was achieved by the use of 2-hydroxy-4-methoxybenzyl (Hmb) 3 which can be considered as a simple modification of Dmb 2 (Scheme 1). Acylation of the exposed 2-hydroxy position gave a phenyl ester, positioned for acyl migration through a constrained, six-membered ring. This procedure gave quantitative coupling under favourable conditions. However, the kinetics of acyl transfer were slow and variable between residues. Additionally, the optimised non-standard conditions used, consisting of a symmetric anhydride in dichloromethane, were difficult to automate. Ideally, for practical convenience the coupling onto the secondary amine