Convergent peptide synthesis

Abstract

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.