How peptides are made (SPPS)

Why a solid support solves the problem

A peptide is a chain of amino acids in a precise order. Building that chain by hand in a solution is difficult, because after each coupling you have to separate the intermediate from the leftover reagent. That costs time and loss at every step. The breakthrough of SPPS was a simple idea: attach the growing peptide to an insoluble bead, a resin. Then you can simply rinse away the excess reagents while the peptide stays put.

Robert Bruce Merrifield devised this approach, for which he received the Nobel Prize in Chemistry in 1984. Since then SPPS has been the standard method for making peptides in laboratories. It made the process reliable enough to automate with machines.

The repeated cycle step by step

SPPS is a loop you repeat as many times as there are amino acids. Each amino acid enters the process with a temporary protecting cap on its couplable end, so it does not react at the wrong moment. The cycle has two core steps that alternate.

First the deprotection, in which the protecting cap is removed from the top amino acid of the chain, creating a free end. Then the coupling, in which the next amino acid, activated with a helper substance, is attached to that free end. After that everything is rinsed clean and the cycle starts again for the next amino acid.

Protection and activation

The secret of a clean synthesis lies in protection. Amino acids have several spots that can react, and you want to couple at only one spot at a time. So each amino acid carries protecting groups on the spots that must be left alone. The most used strategy is called Fmoc, named after the protecting group that is removed after each step.

Activation is the other half. An amino acid does not couple fast enough on its own, so its couplable side is chemically prompted with an activation reagent. That speeds up the reaction and makes the coupling succeed to a high degree. The higher the success rate per step, the purer the final product.

Cleaving and purifying

Once the full sequence is built, the peptide has to come off the resin and all permanent protecting groups have to come off. This happens in a cleavage step, often with a strong acid. What remains is the crude peptide, a mixture of the desired product and small amounts of by-products formed during the many steps.

Then purification follows, usually with HPLC. That separates the desired peptide from the by-products. After purification the peptide is checked, among other things with mass spectrometry to confirm the molecular weight and with HPLC to measure the purity. Those results go on the certificate of analysis.

Limits and longer chains

SPPS works excellently for short to medium chains. At every coupling a tiny bit is lost, and those losses accumulate. For very long peptides or proteins the yield therefore drops and purification becomes harder. For those cases other routes exist, such as linking shorter pieces together or biological production with cells.

For most research peptides on the market, however, SPPS remains the workhorse method. It is predictable, easy to automate, and produces material whose identity can be confirmed with standard tests.