How peptides are made

A peptide is a chain of amino acids joined by peptide bonds. By convention its sequence is written from the amino end, the N-terminus, to the carboxyl end, the C-terminus. Most synthetic peptides today are made by solid-phase peptide synthesis, or SPPS, which builds the chain one amino acid at a time while it is anchored to an insoluble resin. Knowing how that works explains the impurities a certificate reports.

The idea: build the chain on a bead

Forming a single peptide bond is routine chemistry; joining many amino acids in exactly the right order, without side reactions, is the hard part. In the early 1960s R. Bruce Merrifield solved the handling problem by attaching the first amino acid to an insoluble solid. The growing chain stays on the solid while excess reagents are simply washed away after each step, and the process can be automated. The work earned him the 1984 Nobel Prize in Chemistry.

The solid is a resin: small polymer beads carrying a chemical handle, the linker, that holds the growing chain and later releases it. Synthesis runs backwards compared with how the sequence is written. The chain is anchored by its C-terminus, so the last amino acid in the written sequence is the first one attached, and the chain grows toward its N-terminus.

Fmoc chemistry: protect, deprotect, couple

Amino acids have reactive groups at both ends, and many have reactive side chains too. To add exactly one amino acid at a time, every group that should not react must be temporarily blocked with a protecting group.

• The amino end of each incoming amino acid is blocked with Fmoc (9-fluorenylmethoxycarbonyl). Fmoc comes off with a mild base, usually a secondary amine in an organic solvent such as dimethylformamide (DMF).
• Reactive side chains carry protecting groups that the base leaves alone and strong acid removes, such as tert-butyl and trityl groups. Because the two kinds of protection come off under different conditions, one can be removed without touching the other.

Each amino acid is then added by the same cycle:

1. Deprotection. Base removes the Fmoc group from the end of the chain, exposing a free amine.
2. Wash. The base and the released fragments are washed away.
3. Coupling. The next Fmoc-protected amino acid is activated, so that its carboxyl group reacts with the free amine and forms a new peptide bond. Common activators include carbodiimides such as DIC, used with an additive such as Oxyma, and uronium-type reagents.
4. Wash. Excess reagent is washed away.
5. Capping, an optional step. Chains that failed to couple can be capped with an acetylating reagent, which stops them from growing any further.

Cleavage

When the last amino acid is in place, the chain is released with strong acid, usually trifluoroacetic acid (TFA). The same step removes the side-chain protecting groups. Scavengers, such as water and triisopropylsilane, are added to trap the reactive fragments that the protecting groups release, so those fragments do not reattach to the peptide. What comes out is the crude peptide: the target chain plus everything that went slightly wrong along the way.

Why impurities are unavoidable

Each coupling is very efficient but never perfect. If a step reaches 99%, then 1% of the chains miss that amino acid. If those chains are not capped, they keep growing and end up as deletion sequences: full-looking chains missing one residue. Across many steps the losses compound, because the share of chains that are complete is the per-step efficiency multiplied by itself once for every coupling.

Take an invented 16-residue chain, Peptide A. After the first residue is attached, it needs 15 couplings. At 99.0% per coupling, and if nothing else goes wrong, 0.99 to the power of 15 is 0.860, so 86.0% of the chains are full length. At 99.5% it is 92.8%; at 98.0% it is 73.9%. A 31-residue chain at 99.0% per step keeps only 74.0%.

Other impurities come from side reactions:

• a protecting group that was not fully removed, leaving a heavier chain;
• aspartimide formation, a ring-closing side reaction at aspartic acid residues triggered by the base, often described as the most serious side reaction in Fmoc chemistry;
• racemization (strictly, epimerization at one residue), in which an amino acid flips into its mirror-image form, which has the same mass;
• oxidation of sensitive residues.

Impurities like these closely resemble the target, which is exactly why they are hard to separate from it.

Purification by preparative HPLC

The crude peptide is purified by reversed-phase HPLC run at preparative scale: the same principle as the analytical method in HPLC and how to read a chromatogram, with a larger column and a much larger load. The liquid leaving the column is collected in fractions. The fractions are checked by analytical HPLC, often together with mass spectrometry, and those that meet the purity target are combined and freeze-dried (see Why research peptides ship as a powder).

Purification trades yield for purity. Collecting only the cleanest fractions raises the purity and lowers the amount collected. Because the mobile phase usually contains TFA, the purified peptide typically ends up as a TFA salt. Replacing that counterion with acetate or chloride is a separate exchange step; Purity, net content, net peptide content and salt forms explains what that does to the powder’s mass.

What this means when you read a lab report

• A purity figure is the end point of two things: how well the synthesis went, and how hard the purification worked.
• Deletion sequences appear in mass spectrometry as masses one residue lighter than expected, and in HPLC as peaks close to the main one; see Mass spectrometry: identity, not purity.
• Racemized chains have the right mass, so a mass match cannot rule them out.
• The salt form follows from purification and from any exchange step.