The crucial link in peptide synthesis is the peptides bond formed by condensation of the alpha-amino group of one amino acid and carboxyl group of another amino acid. Amino acids contain a central carbon atom surrounded by hydrogen, an amino group, a carboxyl group, and a side chain group, designated as R. This R group is what defines each individual amino acid and determines the physical and chemical properties of the amino acid. Certain R groups can interfere with the formation of the peptide bond, and, therefore, it is necessary to orthogonally protect the functional side chains of certain amino acids. Different protecting groups are available depending on the type of chemistry used and the application.
Synthetic peptides differ from natural protein synthesis on ribosomes in that, the synthesis is conducted from the C to N terminuses. In solid phase peptide synthesis, the first amino acid is covalently linked to a solid support with the alpha amino group protected by either an Fmoc or t-Boc moiety. Treatment with a deprotection agent (piperidine for Fmoc, TFA for Boc) frees the alpha amino group in preparation for coupling the next amino acid in the sequence. The next amino acid is activated by one of several reagents and a peptide bond is formed.
This freshly coupled amino acid is protected on its alpha amino group as well, and in this way prevents double addition of amino acids into the sequence. This stepwise addition continues until the desired peptide length is obtained. After the last amino acid has been added, it is necessary to perform one additional deprotection step to remove the last moiety on the N terminal amino acid. Once the synthesis has been completed, the peptide must be removed from the solid support. This is accomplished by adding an appropriate acid (TFA for Fmoc, HF for Boc) as well as scavengers to collect protected functional groups.