Trp alkylation by resin linker cations
Old/over-acidified resin linker fragments (Wang, Rink, PAL, MBHA) release benzyl-type cations during TFA cleavage. These attack Trp indole C2. The mass increment depends on which linker decomposed.
- +163 — +163 Da, Rink MBHA-derived
- +265 — +265 Da, PAL-linker derived
- +202 — +202 Da, 4-trifluoroacetoxybenzyl
Why it happens (mechanism)
Wang linker cleaves at site A (intended, releases free C-terminus) but can also cleave at site B (irregular), releasing 4-hydroxybenzyl cation (+106). Rink MBHA can give 2-(4-methylphenoxy)acetamide cation (+163). PAL linker gives 5-(3,5-dimethoxy-4-methylphenoxy)pentanamide (+265). All these cations alkylate Trp indole at C2.
When it strikes (triggers)
Aged peptidyl-resin (pre-decomposed). Concentrated TFA cleavage (>95%). Long cleavage time. Inadequate scavenger.
How to spot it (MS signature)
Specific masses: +106 (Wang/HMPA), +163 (Rink-MBHA), +265 (PAL), +202 (TFA-Wang). Trp side products forming a 'fingerprint' of the linker used.
How to prevent it
- Stepwise cleavage: low-concentration TFA (1-5%) of CTC or 2-Cl-Trt resin → fully protected peptide → separate global deprotection. Linker decomposition is much lower at low TFA.
- Switch from MBHA-anchored Rink/PAL to aminomethyl-anchored versions (more acid-stable).
- Add anisole (5%) as scavenger — competes effectively for benzyl cations.
- Add 1,3-dimethoxybenzene (DMB) for Rink resins specifically — it shifts the linker decomposition equilibrium.
- Use Trp(Boc), as ever.
If it already happened (salvage)
- Not reversible.
Source
Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 3, §3.2.