Rearrangement Δm 0 (isomer) severity: moderate

Acid-catalyzed N→O acyl migration

Same mass as target. Under acid, the backbone amide N→O migrates onto the Ser/Thr side-chain hydroxyl, forming a depsi-peptide (ester). Normally reverts at neutral/basic pH but kinetically can stick.

Affected residue(s): S T

Why it happens (mechanism)

Acid protonates the amide carbonyl; Ser/Thr side-chain OH attacks intramolecularly, giving a 5-membered ortho-amide intermediate that collapses with the OH replacing the NH. The peptide now has an ester (depsi) linkage instead of amide at -Xxx-Ser- position.

When it strikes (triggers)

Long TFA cleavage (>3 h) of Ser/Thr-rich peptides. Hot acid. Acidic HPLC purification of fully deprotected peptides. Note: this is the same chemistry intentionally used in O-acyl isodipeptide strategy — the difference is timing.

How to spot it (MS signature)

Same mass. Distinguished by Edman degradation (ester is hydrolyzed differently) or by NMR (Ser/Thr CH-O signals shift). On RP-HPLC, depsi peptide elutes earlier (more polar).

How to prevent it

Cleave at room temperature, ≤2 h.
Lyophilize from neutral pH; restore the depsi peptide to amide form by dissolving in NaHCO₃ buffer (pH 8) for 30 min — the O→N migration is base-catalyzed and equilibrates back.
Avoid prolonged acidic HPLC.

If it already happened (salvage)

Reversible: pH 8 buffer at RT for 30-60 min equilibrates back to the amide form.

Source

Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 4, §4.1.