Fmoc chemistry (revision 8)

Fmoc chemistry is the protecting-group scheme that dominates modern solid-phase peptide synthesis. The temporary N-terminal group, fluorenylmethyloxycarbonyl, is removed by base; the side-chain protecting groups are removed by acid. The two are orthogonal, so each can be removed without disturbing the other.^[1]

Its predecessor, Boc chemistry, used acid for both and therefore required a much stronger acid for final cleavage — hydrogen fluoride — with the handling requirements that implies. Boc chemistry was the scheme of the original solid-phase method.^[2] Orthogonality is what made peptide synthesis a routine operation.^[1]

Deprotection is monitored spectrophotometrically: the dibenzofulvene released when Fmoc is removed absorbs strongly, so the extent of each deprotection can be followed in real time. This is one of the few in-process checks available in stepwise synthesis.^[1]

Fmoc is removed with a solution of piperidine in dimethylformamide, which abstracts the fluorenyl proton and triggers elimination. The released dibenzofulvene is trapped by excess piperidine to prevent it alkylating the peptide.^[1]

The resin is then washed and the next Fmoc-protected amino acid coupled using an activating reagent. See Peptide coupling reagent. The cycle repeats once per residue.

Side-chain protection uses acid-labile groups: tert-butyl for hydroxyl and carboxyl side chains, trityl for cysteine and asparagine, and sulfonyl-type groups for arginine. All are removed in the final acidic cleavage. See Resin cleavage.^[3]