Cleavage and Deprotection

Cleavage and deprotection ends a solid-phase synthesis: the bond anchoring the peptide to the resin is broken and the side-chain protecting groups are removed, in modern schemes simultaneously. In base-labile alpha-protection chemistry this is done with a trifluoroacetic acid cocktail, typically ninety to ninety-five percent acid with the remainder made up of scavengers; the older acid-labile scheme requires anhydrous hydrogen fluoride. The peptide is then precipitated, usually into cold ether, and collected as the crude solid.

The scavengers are not optional. Removing tert-butyl, trityl and sulfonyl groups releases stabilised carbocations that alkylate any nucleophilic residue within reach, and tryptophan, methionine, cysteine and tyrosine are the usual casualties. Cocktails built around thioanisole, ethanedithiol, triisopropylsilane, water and phenol exist to intercept them. Time is the other variable: arginine sulfonyl groups come off slowly, so a cocktail short enough to protect sensitive residues can be too short to finish the job.

That tension is the whole decision. Push the cleavage longer and deprotection completes at the cost of oxidation, alkylation and acid-catalysed side reactions; stop early and partially protected species carry into the crude. Both outcomes appear as distinct masses, so the choice is auditable afterwards, but a cocktail chosen for one peptide is not transferable to another.

The misreading is assuming a correct target mass means the cleavage went cleanly. Partially protected material shows up as a species heavier than target by the mass of whatever group stayed on, but only if the spectrum is examined below the base peak. This step is also why nearly every synthetic peptide arrives as a trifluoroacetate salt, adding weight to the vial that is not peptide.