Amidation

Amidation is a post-translational modification converting the free carboxyl group (-COOH) at a peptide's C-terminal end into an amide group (-CONH₂). In mammals, this reaction is catalyzed by the bifunctional enzyme PAM (Peptidylglycine Alpha-amidating Monooxygenase), dependent on vitamin C and copper, using a C-terminal glycine as nitrogen donor in two steps (hydroxylation then lysis).

This seemingly minor modification has major biological consequences. It neutralizes the carboxylate negative charge, modifies local conformation, increases resistance to carboxypeptidases and, for many peptides, is strictly essential to biological activity. Approximately 50% of bioactive hormonal peptides and neuropeptides are estimated to be C-terminally amidated.

Examples are numerous and emblematic: oxytocin, vasopressin, substance P, neuropeptide Y, α-MSH, gastrin, CCK, VIP, PACAP, calcitonin, amidorphin, CRH, GRP, bombesin. For each, the non-amidated form loses 90 to 99% of its affinity for the native receptor.

In SPPS peptide synthesis, amidation is obtained by using a Rink amide or MBHA resin instead of a Wang-type resin: upon resin cleavage at synthesis end, the C-terminus is directly released as amide. It is thus a resin choice at synthesis start, not an end modification.

Correctly identifying a peptide's amidation state is critical in research: a peptide advertised as "amidated" must show theoretical molecular mass reduced by one unit compared to the free acid form (the -OH replaced by -NH₂ changes mass by -1 Da). A serious COA explicitly mentions "C-terminal amide" or "C-terminal -OH" and the measured MS mass must match. Confusing both forms can invalidate entire series of comparative pharmacology experiments.

C-terminal amidation is a post-translational modification catalysed by the enzyme peptidylglycine α-amidating monooxygenase (PAM), which uses a C-terminal Gly residue as amide donor after oxidative cleavage. About 50% of native neuropeptides and peptide hormones are C-amidated in vivo: oxytocin, vasopressin, GHRH, calcitonin, gastrin, substance P, NPY, neurotensin. This modification is essential to biological activity of most of these peptides: the C-terminal acid form of oxytocin, for example, is almost totally inactive on its receptor.

In SPPS, C-terminal amidation is obtained directly by resin choice: Rink Amide MBHA or Rink Amide AM for amidated peptides; Wang, 2-chlorotrityl, HMPB for free-acid peptides. After TFA cleavage, Rink resin releases the C-amidated peptide with > 95% yield. ESI-HRMS analysis easily distinguishes amidated vs acid peptide: -1 Da difference (monoisotopic mass). In peptide pharmacology, C-terminal amidation is a common substitution to improve the half-life of synthetic analogues (Ipamorelin, GHRP-2, GHRP-6 and Melanotan II are C-amidated). It is not universal: PT-141, Semax and Selank end in a free carboxylic acid.