MTBE-induced tert-butylation

MTBE (used to precipitate cleaved peptide from TFA) decomposes under residual strong acid (TFMSA, hot TFA) → isobutene → tert-butyl cation. Adds onto Trp/Tyr/Cys/Met just like global-deprotection tBu cation. +56 Da, but happens at workup.

Why it happens (mechanism)

(CH₃)₃C-O-CH₃ + strong acid → (CH₃)₂C=CH₂ (isobutene) + CH₃OH. Isobutene + nucleophile (Trp indole, Tyr OH, Cys S, Met S) → tBu adduct (+56 Da). MTBE has been chosen over diethyl ether for safety, but is more vulnerable to this side reaction.

When it strikes (triggers)

TFMSA-containing cleavage cocktail + MTBE precipitation. Hot precipitation. Trp/Tyr/Cys/Met-rich peptides are most affected.

How to spot it (MS signature)

+56 Da. Same Δmass as Trp tBu (Ch 3.1) but the source is different (MTBE during workup vs. tBu cation during cleavage). Distinguish by which step introduced it (run a small HPLC of the pre-precipitation peptide; if +56 only after MTBE step, it's MTBE-induced).

How to prevent it

• Use diethyl ether for precipitation when alkylation-prone residues (Trp/Tyr/Cys/Met) are present, especially with TFMSA-containing cleavage.
• Avoid heat at the precipitation step.
• Reduce the need for TFMSA — modern TFA/scavenger cocktails handle most cases.

If it already happened (salvage)

• Not reversible.

Source

Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 14, §14.6.