Solid-Phase Peptide Synthesis (SPPS)
The standard method for chemically manufacturing peptides, building chains one amino acid at a time on a solid resin support. Developed by Robert Merrifield (Nobel Prize 1984), SPPS enables incorporation of non-natural amino acids and chemical modifications not possible with recombinant production.
Technical Context
SPPS workflow: (1) resin selection (Wang resin for C-terminal acids, Rink amide resin for C-terminal amides — resin loading determines scale), (2) first amino acid coupling (loading onto resin via ester or amide bond), (3) iterative cycles of deprotection (removing Fmoc group with 20% piperidine in DMF, ~5-20 minutes) → washing → coupling (activating the next Fmoc-amino acid with coupling reagents such as HBTU/HOBt or HATU/HOAt and adding to the growing chain, ~30-60 minutes) → washing → monitoring (Kaiser test or TNBS test for free amines to confirm coupling completion), (4) final deprotection, (5) cleavage from resin (TFA cocktail — typically 95% TFA with scavengers such as triisopropylsilane and water — removes side chain protecting groups simultaneously), (6) precipitation and crude peptide isolation, (7) purification by preparative RP-HPLC, (8) lyophilisation. Automated peptide synthesisers perform steps 2-3 with minimal human intervention. SPPS is practical for peptides up to approximately 50 amino acids; longer sequences may require segment condensation (native chemical ligation or fragment coupling strategies).