How Peptides Are Made: Solid-Phase Synthesis and Purification

Building a chain on a resin

Solid-phase peptide synthesis (SPPS) was introduced by Bruce Merrifield in the 1960s and earned him the 1984 Nobel Prize in Chemistry. The first amino acid, the one at the C-terminal end, is attached to tiny insoluble resin beads. The chain is then extended one residue at a time toward the N-terminus, the opposite direction to the way cells build proteins.

Protect, couple, deprotect

Each amino acid is supplied with its reactive groups temporarily blocked so that it bonds only where intended. Every cycle has two steps: remove the temporary protecting group from the end of the growing chain, then couple the next protected amino acid. Washes between steps remove excess reagents. Two protecting-group strategies are widely used, known as Fmoc and Boc.

Cleavage

When the sequence is complete, the peptide is cleaved from the resin and the remaining protecting groups are removed, typically with a strong acid such as trifluoroacetic acid (TFA). The result is a crude mixture.

Purification

The crude product contains the target peptide and by-products. It is usually purified by preparative reversed-phase HPLC. Fractions are collected and analysed, the fractions that meet specification are pooled, and the pool is freeze-dried (see What Is a Lyophilized Peptide).

Why every batch contains small amounts of by-products

• Each coupling step is very efficient but never perfect, so a few chains miss a residue (deletion sequences) or stop early (truncated sequences).
• Protecting groups are occasionally not fully removed.
• A small fraction of amino acids can flip to their mirror-image form during synthesis (racemisation).
• The acid used in cleavage and purification leaves counter-ions, commonly trifluoroacetate, bound to the peptide.

This is why purity is measured and reported, and why a certificate of analysis matters. See How Peptide Purity Is Tested and Common Peptide Impurities.