Solid-Phase Peptide Synthesis: How Research Peptides Are Made
Nearly all synthetic research peptides are produced by solid-phase peptide synthesis (SPPS), a method introduced by Bruce Merrifield in the early 1960s and recognised with the 1984 Nobel Prize in Chemistry1. Understanding it explains where impurities come from.
The core idea
Rather than building a peptide in solution, SPPS anchors the growing chain to an insoluble polymer resin. Because the product stays bound to the solid support, excess reagents and by-products can be washed away at every step simply by filtration. This turns a difficult purification problem into a rinsing operation and makes automation possible.
The cycle
The chain is assembled from the C-terminus toward the N-terminus, one residue at a time. Each cycle has two operations: deprotection, in which a temporary protecting group is removed from the N-terminal amine, and coupling, in which the next protected amino acid is activated and joined to form a peptide bond. Modern practice predominantly uses the Fmoc/tBu strategy2, where the base-labile Fmoc group is removed with piperidine and side chains carry acid-labile protection. The cycle repeats for every residue in the sequence.
Where impurities originate
No coupling reaction is perfectly quantitative. When a coupling fails on a fraction of chains, the result is a deletion sequence — a peptide missing one internal residue. Because a deletion product differs from the target by a single amino acid, it is chemically similar and can be difficult to separate chromatographically. Incomplete deprotection produces related truncations. Aggregation of the growing chain on the resin, common in hydrophobic sequences, makes reagents less accessible and increases these failures.
Cleavage and isolation
Once assembly is complete, a strong acid — typically trifluoroacetic acid with scavengers — simultaneously cleaves the peptide from the resin and removes side-chain protecting groups. The crude peptide is precipitated, then purified by preparative HPLC and lyophilised. The purity of the finished lot is therefore a product of both synthesis efficiency and purification rigour.
References
- Merrifield RB. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J Am Chem Soc. 1963;85(14):2149–2154. doi:10.1021/ja00897a025
- Carpino LA, Han GY. The 9-fluorenylmethoxycarbonyl amino-protecting group. J Org Chem. 1972;37(22):3404–3409. doi:10.1021/jo00795a005
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