How Are Peptides Made?

Peptides are short chains of amino acids, but linking amino acids together to make a peptide in the laboratory is not as simple as it sounds. The final material is determined by the order of the amino acids and the chemical conditions used during the synthesis and the purification process.

The most typical method for making research peptides is solid-phase peptide synthesis (SPPS). It allows stepwise construction of a peptide from individual amino acids in a defined sequence. When the chain is completed, it is detached from its solid support. It is purified and tested.

Beginning with the peptide sequence

First, the amino acid sequence must be known before the peptide can be synthesised.

It tells you the sequence of amino acids in the chain. Changing one minute can even lead to a different peptide with different chemical properties.

Researchers can also request modifications, such as swapping the ends of the peptide or some amino acid replacements. These can influence properties such as stability, solubility and behaviour in research systems.

Once the sequence has been checked, synthesis can start.

Solid-phase peptide synthesis

The solid-phase peptide synthesis first attaches the first amino acid on a solid material, called a resin. The resin is a temporary support for the growing chain of the peptide.

Then, more amino acids are added, one at a time. After each reaction the next amino acid is added and the unwanted chemicals are washed away.

You continue doing this until you have the whole sequence built up.

Therefore, a peptide with many amino acids requires many individual cycles of synthesis. This is one reason why longer peptide sequences may be more difficult to produce consistently.

Why do we use protecting groups?

Amino acids have a number of chemical groups that can potentially react. If the groups are not protected, unwanted reactions can occur during the synthesis.

Protecting groups are used to temporarily block reactive parts of an amino acid from reacting. Now you can make the peptide bond you want without having side reactions in other places.

One of the most frequent ways is Fmoc chemistry. Fmoc is short for 9-fluorenylmethoxycarbonyl. The Fmoc group can be cleaved under controlled conditions prior to the addition of the next amino acid.

Each cycle therefore involves deprotecting, adding the next amino acid and washing away the excess.

Connecting Amino Acids

Coupling is the joining together of two amino acids.

During coupling the incoming amino acid is activated. Such that it can react with the growing peptide chain. The new peptide bond is facilitated by special reagents.

Coupling reactions are not 100% efficient. Some small amount of unreacted material may remain, resulting in shortened or incomplete peptide sequences.

This is becoming more important with larger peptides, as small inefficiencies can add up over many cycles of synthesis.

Removal and purification of peptide

The peptide is then released from the resin by cleavage with a chemical process once the complete sequence of the peptide has been assembled. The other protecting groups may be removed at this stage also.

The result is a mixture of crude peptides, not a pure product. It may contain the target peptide with truncated sequences, side products and residual chemicals.

The raw material, therefore, must be purified.

A popular method is high-performance liquid chromatography (HPLC). In HPLC the compounds are separated based on their interactions with the chromatographic system. Then you can collect the fractions with your peptide.

Some peptides are more difficult to purify. Sequence length, chemical modifications, and other properties may affect separation of the target peptide from impurities.

Drying and Testing

Once purified the peptide can be dried by a process known as lyophilisation or freeze drying. In this way water or solvent is evaporated in a controlled manner and a dry preparation of the peptide is obtained.

The final material is then examined to verify what has been made. Mass spectrometry (MS) can be used to confirm the molecular mass of the peptide, and HPLC can be used to provide chromatographic purity information. Depending on the peptide and the research question, other analytical techniques can be employed.

These are important because a peptide sample may look good but contain undesirable components. Analytical testing provides evidence of its identity and composition.

Why peptide synthesis might be tricky

Some peptides are easier to synthesise than others. The longer the sequences, the more difficult this can be, since each synthesis cycle adds a chance for incomplete reactions.

In addition, some sequences have a tendency to aggregate, meaning that the peptide molecules interact with one another, making them more difficult to handle during the synthesis. Certain amino acids or chemical modifications may present further challenges.

Hence, peptide production is a fine balance of chemistry and purification processes.

From amino acids to a peptide for research

The fundamental concept of peptide synthesis is straightforward: assemble a specific amino acid sequence one segment at a time, detach the finished peptide from its support, purify and evaluate the ultimate product.

Solid-phase peptide synthesis has made it possible to synthesise a large number of peptides for laboratory use. However, the quality of the final material is dependent on each stage of the process, from the original sequence and coupling reactions to purification and analytical testing.

That is why terms like peptide purity, molecular weight, sequence, aggregation and degradation are important when dealing with research peptides. Each can be a valuable source of information about the material and what might happen to it in an experimental environment.