Fragmentation / deletion Δm loss of C-terminal residue severity: low-moderate

C-terminal N-Me-Xaa acidolysis

Peptides ending in -N-Me-Xaa-OH (free acid form; not amide) suffer slow acidolytic loss of the C-terminal residue under TFA. The free C-terminal carboxyl attacks the cis-amide bond between N-Me-Xaa and the preceding residue.

Affected residue(s): any (when N-Me-Xaa-OH at C-term)

Why it happens (mechanism)

The N-methyl on Xaa boosts the cis-amide population at the Xaa-(Xaa-1) bond. Under acid, the C-terminal -COOH is the nucleophile (its O attacks the cis-amide carbonyl), forming a 5-membered intermediate that rearranges to an anhydride. Hydrolysis of the anhydride detaches the C-terminal N-Me-Xaa as an isolated amino acid.

When it strikes (triggers)

Free-acid C-terminus + N-Me-Xaa at C-terminus + TFA cleavage. Hot acid, long cleavage time. Especially relevant for N-methyl-rich drug-class peptides (Tide-class, MeAA-rich macrocycles).

How to spot it (MS signature)

Truncated peptide, C-terminal residue mass missing. Slow side reaction (typically <10% in normal cleavage), but can dominate if cleavage is hot or extended.

How to prevent it

Use a C-terminal amide (Rink amide / Sieber resin → -CONH₂). Amide ends fully suppress this side reaction (the amide-N can't attack the cis-amide).
Cool TFA cleavage, ≤2 h.
Use CTC resin + dilute TFA → fully protected peptide → separate side-chain deprotection. Each acid step is brief.

If it already happened (salvage)

Bond is broken; re-synthesize with amide C-terminus.

Source

Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 1, §1.6.