Trp oxidation (kynurenine, oxindolylalanine, NFK)
The indole of Trp is highly susceptible to oxidative attack — gives oxindolylalanine (+16), kynurenine (+4 after ring rearrangement), or N-formylkynurenine (+32). Often appears as a peak cluster at +16/+4/+32.
- +4 — +4 Da, Trp → Kynurenine (after rearrangement)
- +31.9898 — +32 Da, N-formylkynurenine
Why it happens (mechanism)
The C2-C3 indole double bond is electron-rich; singlet O₂ or peroxide attacks it. Initial product is 2- or 3-oxindolylalanine (+16 Da). Further oxidative cleavage of the ring + rearrangement gives kynurenine (the indole N→formamide, then loss of CO gives +4 net) or N-formylkynurenine (+32).
When it strikes (triggers)
TFA cleavage *without sufficient scavenger*. Long cleavage time. Light. Air. H₂O₂. Photo-oxidation during HPLC under UV.
How to spot it (MS signature)
+15.99 (oxindolylalanine), +3.99 (Kyn), +31.99 (NFK). The whole cluster is a fingerprint of Trp oxidation. MS/MS shows characteristic 2-aminobenzoyl fragment for Kyn.
How to prevent it
- Cleavage cocktail must include a strong reducing scavenger: EDT (2.5%) + thioanisole (5%) + H₂O. The cocktail K (TFA/thioanisole/phenol/EDT/H₂O = 82.5/5/5/2.5/5) is a classic for Trp-rich peptides.
- Use Trp(Boc) building block — the indole N-Boc protection blocks oxidative addition during synthesis (cleaves cleanly during TFA).
- Cleave under inert atmosphere if possible; minimize cleavage duration (<2 h).
- Store the peptide as a powder, in the dark, under Ar.
If it already happened (salvage)
- Not reducible. Re-synthesize with Trp(Boc) + scavenger upgrade.
Source
Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 9, §9.3.