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.
Automated solid-phase peptide synthesis to obtain therapeutic peptides
