Peptide Synthesis
How laboratory peptides are assembled step by step on a solid support, and where the impurities in crude material typically originate.
Published 14 Sept 2026 · Updated 14 Sept 2026
Solid-phase synthesis
Most research peptides are produced by solid-phase peptide synthesis (SPPS). The growing chain is anchored to an insoluble polymer bead, and reagents are added in solution and then washed away. Because purification between steps is reduced to simple filtration and washing, the cycle is fast and automatable.
The repeating cycle
Each added residue follows the same logic:
- Deprotection — remove the temporary protecting group from the chain terminus to expose a free amine.
- Coupling — activate the carboxyl group of the incoming amino acid and allow it to form the new peptide bond.
- Washing — remove excess reagents and by-products before the next cycle.
Repeating this cycle builds the chain one residue at a time from the C-terminus toward the N-terminus.
Why protecting groups are needed
Amino acids carry more than one reactive group. Without protection, they would react in unintended ways and produce branched or polymerised products. Two strategies dominate:
- Fmoc chemistry — the temporary N-terminal group is removed under mildly basic conditions, while side-chain protecting groups and the resin linkage are cleaved at the end under acid.
- Boc chemistry — the temporary group is removed with acid, with final cleavage typically under strong acid.
Orthogonality — being able to remove one class of group without disturbing another — is the principle that makes stepwise assembly possible.
Cleavage and crude material
When assembly is complete, the peptide is cleaved from the resin and the side-chain protecting groups are removed, usually with an acid cocktail containing scavengers to capture reactive fragments. What results is crude peptide: the desired sequence along with a distribution of process-related impurities.
Where impurities come from
Common sources include incomplete coupling leaving chains missing one residue (deletion sequences), chains that stopped growing early (truncated sequences), incompletely removed protecting groups, and side reactions on sensitive residues. Longer and more hydrophobic sequences generally accumulate more of this population, which is why crude purity alone is rarely sufficient for quantitative work.