Merrifield solid-phase peptide synthesis (SPPS) (Nobel Prize in Chemistry, 1984) grows a peptide chain one residue at a time, C-terminus to N-terminus, on an insoluble polystyrene resin bead — so excess reagents are simply filtered and washed away after every step instead of purifying an intermediate in solution.
Repeating Fmoc-SPPS cycle, per residue:
1. Deprotect: Fmoc-N-resin + piperidine -> H2N-resin + dibenzofulvene adduct
2. Activate: Fmoc-AA-OH + HBTU/DIC -> Fmoc-AA-O-activated ester
3. Couple: H2N-resin + activated ester -> Fmoc-AA-C(=O)-NH-resin (new amide bond)
4. Wash: DMF flow removes excess reagent + by-products
... repeat for each residue ...
Cleave: peptide-resin + TFA -> free peptide + resin (+ scavengers)
- Fmoc deprotection — piperidine (base) strips the Fmoc protecting group from the chain's N-terminus, exposing a free amine so the next amino acid has somewhere to attach.
- Activation — HBTU or DIC converts the next Fmoc-amino acid's free carboxylic acid into a reactive ester, since a plain -COOH won't condense with an amine on its own.
- Coupling — the activated amino acid's C-terminus condenses with the resin chain's free N-terminus, forming a new peptide (amide, -C(=O)-NH-) bond and releasing water/byproduct.
- Cleavage — trifluoroacetic acid (TFA) finally cuts the finished peptide off the resin's linker, releasing the free peptide for purification.
- Side-chain colors — grey = nonpolar (Gly, Ala, Val, Leu, Ile, Met, Phe, Trp, Pro), green = polar uncharged (Ser, Thr, Cys, Tyr, Asn, Gln), red = acidic (Asp, Glu), blue = basic (Lys, Arg, His) — the real four-way classification of the 20 standard amino-acid R-groups.
Real-world anchor: this exact chemistry (Fmoc/tBu SPPS) is how essentially all research-scale and most drug peptides — including insulin analogs and GLP-1 agonists — are manufactured today, automated on peptide synthesizers that repeat this cycle unattended.