Cyclization Δm -17.0265 severity: high

Pyroglutamate formation from N-terminal Gln

N-terminal Gln spontaneously cyclizes — the free α-amine attacks the side-chain amide carbonyl, ejects NH₃, gives a 5-membered pyroglutamate (lactam). Loses 17 Da and the N-terminus becomes blocked.

Affected residue(s): Q

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

Why it happens (mechanism)

Once the α-amine of N-terminal Gln is liberated (after Fmoc removal, or in solution), the lone pair attacks the side-chain amide C=O at a 5-exo-trig favorable geometry. Tetrahedral intermediate collapses, NH₃ leaves, and the lactam is locked. Acid- and base-catalyzed both work.

When it strikes (triggers)

Any peptide with N-terminal Gln that sits on the resin (or in solution) for any time after Fmoc removal. Aggravated by: heat, polar protic solvents, residual base. Even a few hours of storage can produce significant pyro-Glu.

How to spot it (MS signature)

-17.03 Da. Tip-off: a peak 17 Da below main, often co-elutes very close on RP-HPLC. The peptide is also resistant to N-terminal Edman sequencing.

How to prevent it

Use Fmoc-Gln(Trt)-OH, never the free-side-chain Gln building block. Trt blocks the cyclizing amide.
Couple the next residue immediately after Fmoc removal — minimize the time the free α-amine sits on the resin.
If the target has N-terminal Glu/Gln, consider incorporating pyroglutamate intentionally (Fmoc-pyroGlu-OH building block) and accepting that as the spec.
For solution-phase or post-cleavage handling, store cold and dry; avoid basic buffers.

If it already happened (salvage)

Cannot be reverted — the lactam ring is too stable. If you find pyro-Glu in your batch, the path forward is design-time fix (Trt protection, fast subsequent coupling, or pyro-Glu-as-spec).

Source

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