3.3 Side-chain protecting groups More than half of the amino acids commonly encountered in proteins have side-chains that contain reactive functional groups. In solid phase synthesis, it is usual for all of these potentially reactive groups to be masked because of the rather harsh conditions employed and the need to achieve the highest level of efficiency in all chemical reactions. For routine synthesis, protecting groups that are removed with TFA are usually employed as this allows the peptide to be globally deprotected at the same time as it is cleaved or released from the support. Furthermore, a wide range of groups is also available which can be selectively removed on the solid phase, thus enabling the selective modification of side-chains of individual residues within the peptide chain. These find application in the synthesis of cyclic peptides, phosphopeptides, and biotinylated peptides (see Chapter 6). Table 4 lists the most commonly used side-chain protecting groups, together with the conditions required for their removal; those recommended for routine use are highlighted.3.4 First residue attachment The first step in the process of solid phase peptide synthesis is the attaching, or loading, of the resin linker with the C-terminal amino acid. The satisfactory execution of this process is particular important because, first, the extent of this reaction will determine the yield of the final product and, second, sites on the resin not reacted in this initial process can potentially be acylated in subsequent cycles, leading to the generation of related C-terminally truncated by-products. In the case of resins in which the anchorage point is a hydroxyl group, this process is often accompanied by enantiomerization, owing to the harshness of the conditions applied to effect this esterification. The problem is most serious with histidine and cysteine, and for these residues the use of trityl-based resins is recommended as these are loaded under conditions which do not cause loss of chiral integrity (Chapter 3, Section 5.2 and Chapter 4, Section 3.2.1). Peptides containing proline or N-alkylated amino acids in the C-terminal dipeptide sequence present special problems because of the ease with which these dipeptides cyclize to give the corresponding diketopiperazine (Figure 5). This not only results in a reduction in yield of the desired product, but may also lead to the generation of truncated sequences through subsequent acylation of the regenerated starting resin. This side-reaction is most problematic with supports in which the dipeptide in anchored by a benzyl ester, and for this reason resins in which attachment is via a more hindered trityl ester should be used (Chapter 3, Section 5.2).