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

Acidolysis of N-Ac-N-alkyl-Xaa N-terminus

Acetylated peptides whose N-terminal residue is N-methylated (Ac-N-Me-Phe-..., common in Arodyn-class designs) undergo a 5-exo-trig acid-catalyzed cleavage of the N-terminal residue. Result: des-N-Ac-N-Me-Xxx peptide.

Affected residue(s): any (when Ac-N-Me-Xxx at N-term)

Why it happens (mechanism)

The acetyl carbonyl oxygen is the nucleophile; under acid, it attacks the proximal amide bond between N-Me-Xxx and Xxx+1, forming a 5-membered ring intermediate that collapses, ejecting the N-terminal Ac-N-Me-Xxx as a small heterocycle and leaving H-Xxx+1- as the truncated product. The N-methyl boosts the cis-amide population, geometrically favoring this.

When it strikes (triggers)

Any peptide with -Ac-N-alkyl-Xaa- at the N-terminus (e.g., Arodyn 2). Cleavage with concentrated TFA, room temperature or above.

How to spot it (MS signature)

Peptide mass missing the N-terminal residue mass + 42 (Ac group). E.g. Ac-N-Me-Phe-Phe-Trp-... → des-Phe-Phe-Trp-...

How to prevent it

Cleave at 4 °C with no scavenger initially; this dramatically slows the process.
Replace N-terminal Ac with methyl carbamate (e.g., Arodyn 3 design): the more electron-withdrawing carbamate can't act as a nucleophile.
If acetyl is required for bioactivity, install it post-cleavage from the unprotected peptide rather than during synthesis.

If it already happened (salvage)

Bond is cleanly cleaved; full-length is not recoverable. Switch to cold cleavage or design change.

Source

Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 1, §1.1.