Cyclization Δm -18.0106 severity: moderate

Pyroglutamate formation from N-terminal Glu

N-terminal Glu cyclizes by losing water (instead of NH₃ for Gln) — the α-amine attacks the side-chain carboxylate, dehydrates to a 5-membered lactam. -18 Da.

Affected residue(s): E

Neighbour(s) that trigger it: any (only N-terminal E matters)

Why it happens (mechanism)

Same geometry as the Gln→pyro-Glu route, but the leaving group is now H₂O (since Glu has -COOH instead of -CONH₂). Acid-catalyzed (the carboxyl gets protonated, then NH attacks).

When it strikes (triggers)

N-terminal Glu(OtBu) → after global deprotection, the free Glu side chain + free α-amine = perfect cyclization geometry. Hot acidic conditions push it. CTC-resin cleavage with residual TFA is a common offender.

How to spot it (MS signature)

-18.01 Da. Distinguish from aspartimide (also -18) by location: pyro-Glu only appears at the N-terminus of E-starting peptides; aspartimide at internal Asp.

How to prevent it

Same as pyro-Glu/Gln: use Fmoc-Glu(OtBu) + couple next residue immediately.
Avoid prolonged TFA exposure during cleanup. Lyophilize quickly.
Cold storage, neutral pH.

If it already happened (salvage)

Lactam is irreversible under non-degrading conditions. Re-synthesize.

Source

Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 6, §6.3.