ABSTRACT
The chemical synthesis of peptides was performed by Merrifield in 1963 and represents the original application of solid-phase synthesis of organic molecules [79]. Whereas very large peptides and proteins are often synthesized by biochemical methods (e.g., overexpression in Escherichia coli. [80]), synthesis on solid support is frequently used when peptides (typically lower than 50 amino acids) or peptides containing unnatural amino acids are desired (Fig. 15). (Peptides containing unnatural amino acids can be expressed using Schultz’s modified tRNA technique [81]. However, for short peptides or in cases where a significant amount of peptide is desired, chemical synthesis is still preferred.) While there have been a number of different routes developed for supportbound peptide synthesis, two techniques are used most frequently. One of these is the 9fluorenylmethoxycarbonyl (Fmoc)—based approach, using amino acids that are protected at the back-
bone nitrogen with the base-labile Fmoc group and at side-chain functional groups with acid-labile protecting groups. In the Fmoc-amino acid approach, side chain nitrogens are protected using t-butyloxycarbonyl (t-Boc) groups. Amino acids are coupled to the amino terminus of the growing peptide on solid support via use of an activating agent to boost the reactivity of the reacting carboxylate [82,83]. After each coupling step, the amino terminus is prepared for the next reaction by removal of the Fmoc protecting group. When completed, the peptide is removed from support by a strong acid cleavage step, which simultaneously removes all of the acid-labile side-chain protecting groups, resulting in the desired peptide. Coupling conditions and reagents have been refined to provide high efficiency while minimizing racemization of the amino acid stereocenters.
