-Asp-Pro- acidolysis
The Asp-Pro amide bond is exceptionally labile to acid — cleaves cleanly under TFA, producing two fragments. Sequence-specific weak point.
Why it happens (mechanism)
Acid protonates the Asp side-chain carboxyl, which then participates in cleavage of the proximal Asp-Pro amide via a 5-exo-trig anchimeric assistance. Pro's tertiary nitrogen is a good leaving group at this geometry. Result: cleavage between D and P, two halves.
When it strikes (triggers)
Any -D-P- internal sequence under TFA cleavage. Aggravated by: hot TFA, long cleavage time (>2 h), high TFA concentration. Concentrated HF is even worse.
How to spot it (MS signature)
Two fragment peaks instead of the expected full-length peptide. The N-terminal fragment ends in -Asp; C-terminal starts with Pro-. Total mass conserved when summed (+18 Da accounting for water).
How to prevent it
- Minimize TFA cleavage time (≤1.5 h, room temp) for any -DP- containing peptide.
- Use cooled cleavage cocktail (0–4 °C).
- Switch to CTC-resin → mild-TFA cleavage of the protected peptide → separate side-chain deprotection in two steps. Each acid exposure is brief.
- Avoid HF cleavage if -DP- is present.
- If feasible at design stage, replace D with E (no analogous E-P fragility) or P with N-Me-Ala.
If it already happened (salvage)
- Not salvageable post-cleavage — the bond is broken. Re-synthesize with milder acid protocol.
Source
Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 1, §1.4.