Cys-EDT and Cys-EDT-tBu adducts
EDT thiol adds across Cys disulfide or to a Cys cation, sometimes with a tert-butyl group on the second EDT thiol. +92 or +148 Da.
- +76 — +76 Da, Cys-EDT (single bond)
- +148 — +148 Da, Cys-EDT-tBu
Why it happens (mechanism)
EDT (HS-CH₂-CH₂-SH) under TFA loses one proton, attacks Cys S(III) intermediate (e.g., Cys sulfenyl cation in the Acm-cleavage process), forming a mixed disulfide -S-S-CH₂CH₂-SH. The pendant SH can then alkylate with a tBu cation in the cleavage cocktail to give -S-S-CH₂CH₂-S-tBu (+148). Or remain as -SH (+76 / +92).
When it strikes (triggers)
TFA cleavage with EDT scavenger and abundant tBu cations (long peptide, lots of tBu groups). Cys(Acm) cleavage performed in TFA (instead of I₂).
How to spot it (MS signature)
+92 or +148 Da on Cys. Distinguishable by mass; both unique to EDT-containing cocktails.
How to prevent it
- Reduce EDT to ≤2.5% in cocktail.
- Add a competing scavenger (DMS, thioanisole) to entrap tBu cations.
- Cleave Cys(Acm) with I₂ in MeOH/H₂O, not in TFA — completely orthogonal pathway.
- Lower temperature.
If it already happened (salvage)
- Mixed disulfide can be reduced by TCEP/DTT, regenerating Cys-SH. The tBu version (+148) — only the disulfide bond is reducible; the tBu group on the EDT side stays as an adduct on the peptide unless workup of EDT-derived fragment is done.
Source
Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 3, §3.7.