Cleavage

Cleavage designates the final step of solid-phase peptide synthesis: release of the complete peptide chain from the support resin and simultaneous elimination of side chain protecting groups. This seemingly simple step concentrates several chemical challenges that condition the quality and purity of the crude peptide obtained before purification.

In Fmoc chemistry, cleavage is performed by treatment with an acid cocktail based on concentrated TFA (trifluoroacetic acid), typically 95% with 5% additional solvents. Side chains protected by acid-labile groups (tBu on Ser/Thr/Tyr, Trt on Cys/His/Asn/Gln, Boc on Lys, Pbf on Arg) are eliminated in parallel by the same acidic treatment. TFA protonates the link between peptide and resin (typically a benzyl ester or amide), causing release into solution.

Cleavage cocktails systematically integrate "scavengers": thiol compounds (EDT, mercaptoethanol) or aromatics (anisole, thioanisole, TIS for triisopropylsilane), sometimes with added water. Their role is to capture carbocations generated by departure of protecting groups, which can otherwise alkylate nucleophilic side chains (Trp, Met, Cys, Tyr) and generate impurities difficult to separate. Cocktail choice depends on amino acid composition: "Reagent K" cocktail (TFA/phenol/H2O/thioanisole/EDT) is standard for Met/Trp/Cys-rich sequences; a simpler TFA/TIS/H2O mixture suffices for less sensitive sequences.

The Boc chemistry cleavage cocktail is radically different: it employs liquid gaseous HF at -5°C for 1 to 2 hours, in the presence of scavengers adapted to the strong acid pathway. The extreme toxicity of HF and the corrosion it generates have progressively excluded this chemistry from modern industrial productions in favor of more benign Fmoc conditions, reserving HF for specialized laboratories.

After cleavage, the crude peptide in TFA solution is precipitated by addition of cold diethyl ether, washed several times to eliminate scavengers and byproducts, dried, then purified by preparative reverse-phase HPLC. Post-purification yield depends on the quality of initial synthesis and chromatographic separability of impurities. Hence the value of independent testing: on lab-peptides-france.com, our published Janoshik reports give the purity and content actually measured.