How peptides are made

Almost every synthetic peptide today is built using a method that's more than 60 years old: solid-phase peptide synthesis (SPPS). Here's how a sequence on paper becomes a white powder in a vial.

1. Anchoring to a resin

In 1963 Bruce Merrifield had the idea of attaching the first amino acid to an insoluble polymer bead, the resin. Because the growing chain stays stuck to the bead, excess reagents and by-products can simply be washed away after each step. There's no need to purify the product every time. The idea earned him the 1984 Nobel Prize in Chemistry.

2. The coupling cycle

Chains are built from the C-terminus towards the N-terminus, one residue at a time. Each new amino acid carries a temporary protecting group on its amino end, most commonly Fmoc (fluorenylmethyloxycarbonyl), so it can only react in the intended way. Every cycle has the same three steps:

1. Deprotect: remove the Fmoc group from the end of the chain, usually with piperidine.
2. Couple: add the next Fmoc-protected amino acid together with an activating reagent, forming the new peptide bond.
3. Wash: rinse away everything that didn't attach.

Side chains carry their own permanent protecting groups, which survive every cycle and only come off at the end. Modern automated synthesisers, some using microwave heating to speed up couplings, run these cycles unattended.

3. Cleavage

Once the sequence is complete, the peptide is cut free from the resin and its side-chain protecting groups are removed in one step. This usually uses strong acid, typically trifluoroacetic acid (TFA), with "scavenger" additives that mop up reactive fragments. This is why many synthetic peptides end up as TFA salts.

4. Why the crude product isn't pure

Even at 99% efficiency per step, small errors add up over a long chain. The crude product typically contains:

• Deletion sequences: chains missing a residue where a coupling failed
• Truncated sequences: chains that stopped growing early
• Incompletely deprotected chains that still carry a protecting group
• Side-reaction products such as oxidised methionine or racemised residues

5. Purification by preparative HPLC

The crude mixture is separated by reverse-phase high-performance liquid chromatography. Molecules travel through a column at different speeds depending on how hydrophobic they are. Fractions containing the target peptide are collected, checked analytically, and pooled.

6. Freeze-drying (lyophilisation)

The pooled fractions are frozen and placed under vacuum so the water sublimes directly from ice to vapour. What's left is a dry, fluffy powder that is far more stable for storage than the peptide in solution.

Other routes

For long peptides and some commercial products, chemists also use liquid-phase synthesis, fragment ligation (joining shorter purified pieces) or recombinant expression in microorganisms. Recombinant expression is how insulin is produced at scale, for example.

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Sources and further reading

1. Merrifield RB. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J Am Chem Soc 1963;85:2149–2154. doi:10.1021/ja00897a025
2. Carpino LA, Han GY. 9-Fluorenylmethoxycarbonyl function, a new base-sensitive amino-protecting group. J Am Chem Soc 1970;92:5748–5749. doi:10.1021/ja00722a043
3. Isidro-Llobet A, Álvarez M, Albericio F. Amino acid-protecting groups. Chem Rev 2009;109:2455–2504. doi:10.1021/cr800323s · PMID: 19364121
4. Mant CT et al. HPLC analysis and purification of peptides. Methods Mol Biol 2007;386:3–55. doi:10.1007/978-1-59745-430-8_1 · PMID: 18604941
5. Dawson PE, Muir TW, Clark-Lewis I, Kent SBH. Synthesis of proteins by native chemical ligation. Science 1994;266:776–779. doi:10.1126/science.7973629 · PMID: 7973629
6. The Nobel Prize in Chemistry 1984 (Merrifield). www.nobelprize.org/prizes/chemistry/1984/summary