Skip to content

Cleavage

Peptide Release from Resin

Purpose

  1. Release the peptide from the solid support
  2. Remove side-chain protecting groups (global deprotection)

Standard Cleavage Cocktail

TFA-Based Cocktail

Component Function Typical %
Trifluoroacetic acid (TFA) Acidolysis of ester/amide bonds 90–95%
TIS (triisopropylsilane) Carbocation scavenger 2–5%
Water Polar carbocation scavenger 2–5%
EDT or DTT For Met, Cys, Trp containing peptides 0–2%

Recipe for Standard Peptides

TFA (95%) + TIS (2.5%) + H₂O (2.5%)

Recipe for Cys/Met/Trp-Containing Peptides

TFA (94%) + TIS (2%) + H₂O (2%) + EDT (1%) + Phenol (1%)

Procedure

Step-by-Step

  1. Prepare cocktail fresh (do not store TFA cocktails)
  2. Cool to 0 °C in ice bath
  3. Add to resin (10–20 mL/g of resin)
  4. Stir 1.5–4 hours at room temperature (time depends on sequence)
  5. Filter to remove resin beads (glass frit or PE frit)
  6. Wash resin with small amount of neat TFA
  7. Precipitate in cold diethyl ether or MTBE (10:1 ratio ether:filtrate)
  8. Centrifuge or filter to collect precipitate
  9. Wash × 2–3 with cold ether
  10. Dry under nitrogen stream or vacuum

Cleavage Time Guidelines

Protecting Groups Time
Standard (no special groups) 1.5–2 h
Arg(Pbf) present 2.5–3 h
Trp(Boc) present 2–3 h
Cysteine (Trt) present 2–3 h
Cys(Acm) present Keep ≤2 h (Acm is stable)

Precipitation

Cold Ether Method

  • Ether: diethyl ether or MTBE
  • Temperature: 0 °C (ice bath)
  • Ratio: 8–10 volumes ether per volume cleavage solution
  • Time: 5–15 minutes precipitation
  • Result: White to off-white solid

Centrifugation (Small Scale)

  • 3000–5000 × g for 5 minutes
  • Decant supernatant
  • Resuspend in fresh cold ether
  • Repeat × 2

Filtration (Larger Scale)

  • Use glass frit filter (M or F porosity)
  • Wash filter cake with ether
  • Suction dry

What Safety Precautions Are Needed for Cleavage?

Hazard Risk Mitigation
TFA Corrosive, toxic Fume hood, gloves, goggles
TIS Flammable No open flames
Ether Highly flammable, peroxide former Use fresh, no sparks
EDT Strong odor, toxic Fume hood, minimize use
  • Peptide Release from Resin — Review the section above for key parameters, methods, and quality criteria.
  • Standard Cleavage Cocktail — Review the section above for key parameters, methods, and quality criteria.
  • Cleavage Time Guidelines — Review the section above for key parameters, methods, and quality criteria.
  • What Safety Precautions Are Needed for Cleavage? — See the section for detailed parameters and specifications.

Key Takeaways

  • Peptide Release from Resin — Review the section above for key parameters, methods, and quality criteria.
  • Standard Cleavage Cocktail — Review the section above for key parameters, methods, and quality criteria.
  • Cleavage Time Guidelines — Review the section above for key parameters, methods, and quality criteria.
  • Precipitation — Review the section above for key parameters, methods, and quality criteria.

🔗 Related: SPPS Process | Deprotection | Purification | TFA