Alkylation Δm +56.0626 severity: moderate

Trp tert-butylation

tert-Butyl cations released from cleaved tBu/OtBu/Boc protecting groups alkylate the indole C2 position. +56 Da. Especially common in long peptides with many tBu protections.

Affected residue(s): W

Why it happens (mechanism)

TFA cleaves tBu groups, releasing tert-butyl cation (or tert-butyl trifluoroacetate). The indole C2 of unprotected Trp is electron-rich and acts as a nucleophile, attacking the cation: gets a tBu group at indole 2-position. +56.06 Da.

When it strikes (triggers)

TFA cleavage of long Trp-containing peptides with abundant tBu/OtBu/Boc groups. No scavenger in cocktail. High concentration of TFA (e.g., 95% vs. 88%). Long cleavage time.

How to spot it (MS signature)

+56.06 Da. Combined with other Trp adducts (+ 172 EDT, +106 OH-Bzl, etc.), gives a characteristic Trp-modified peak cluster.

How to prevent it

Use a scavenger that competes for tBu cation: phenol or anisole at 5%. EDT also helps but is less specific for this.
Use Trp(Boc) building block — the N-Boc on indole physically blocks alkylation, then comes off during TFA in the unmodified form.
Reduce TFA % from 95% to 88-90% (with H₂O making up the rest) for Trp-containing peptides.
Lower temperature (0-4 °C) and short time.

If it already happened (salvage)

Not reversible. Avoid by Trp(Boc) + proper scavenger.

Source

Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 3, §3.1.