How a peptide is made: solid-phase synthesis

A research peptide is not made in a vat but on a bead of synthetic resin, one amino acid at a time. Once you know the process, you also know where the impurities come from that later show up on the certificate of analysis.

The chain grows on a solid bead

In 1963 Robert Bruce Merrifield presented a method that reshaped peptide chemistry: the far end of the future chain is chemically bound to an insoluble resin bead, and everything else happens on that bead. The idea, now known as solid-phase peptide synthesis, earned him the Nobel Prize in Chemistry in 1984.

The advantage lies in the washing. Because the growing peptide is anchored to the resin while the reagents are in solution, everything left over can simply be rinsed away after each step. There is no need for a full purification after every single addition, and with fifteen links that is exactly the effort that would make the method unworkable.

Each cycle has three steps

Deprotect: the temporary protecting group on the free end of the chain is removed, so that anything can couple there at all.
Couple: the next amino acid is added in excess, activated by a reagent, and binds to the exposed end.
Wash: anything not attached to the resin is rinsed out. Then the next cycle begins.

For a peptide of fifteen links this sequence of steps runs fourteen times. A sequence of forty links needs thirty-nine cycles, and each one is an opportunity for an error.

Protecting groups fix the order

An amino acid has more than one reactive site. Without protection it would bind at any position, and a defined sequence would turn into a mixture. That is why every building block carries protecting groups: a temporary one at the end of the chain, removed before each cycle, and permanent ones on the side chains, which stay until the very end.

The usual approach today is the Fmoc strategy. The temporary group is fluorenylmethoxycarbonyl, abbreviated to Fmoc, and it comes off with a mild base. The side-chain protecting groups, by contrast, need acid. Because the two respond to different conditions, they can be removed independently of each other, and that is the whole trick.

Where the impurities come from

No cycle runs to completion. Whatever is left over ends up in the crude product:

Deletion sequences: if an amino acid fails to couple on one bead, the chain carries on growing without it. The result is a molecule missing exactly one link, close to the target in mass and therefore not always easy to separate.
Leftover protecting groups: if a side-chain group stays attached, the molecule is heavier than expected. Mass spectrometry picks this up reliably.
Difficult stretches: some sequences fold up on the resin and make the end of the chain hard for reagents to reach. The yield of these stretches drops markedly.

Even at ninety-nine per cent per cycle, after fourteen cycles the arithmetic leaves roughly eighty-seven per cent target molecule. That remainder is the reason purification is needed at all.

Cleavage and purification

At the end the peptide is released from the resin. This is usually done with trifluoroacetic acid, which removes the side-chain protecting groups at the same time. The crude peptide is then precipitated in cold ether and separated by preparative HPLC: the same technique as in the later analysis, only on a larger scale and with the aim of collecting the clean fraction.

Something of the acid remains. The peptide is then present as a salt, usually a trifluoroacetate, and this counterion adds to the weight. It is the main reason why the peptide content of a lot is lower than its purity.

Finally, freeze-drying

The clean fraction goes into the vial in solution and is freeze-dried. What arrives is a dry cake with a lot number and a test report that belongs to it. How to read that report is explained in a separate article.

For the catalogue this means that a short molecule such as GHK-Cu, with three links, goes through two couplings, while BPC-157, with fifteen links, goes through fourteen. The number of steps explains a good part of the price difference between two substances.

Questions

Is a synthetic peptide the same as one that occurs naturally?

With the same sequence, yes: the molecule does not know where it came from. Differences arise only from impurities left by the synthesis and from the counterion with which the peptide is present as a salt.

Why does a certificate of analysis rarely show one hundred per cent?

Because every coupling step leaves a residue of incomplete chains, and purification does not remove all of them. That is why purity is measured lot by lot. UKPeps states no general minimum purity and publishes the measured value for each lot in the certificate library.

What does the abbreviation Fmoc on a data sheet mean?

Fmoc stands for fluorenylmethoxycarbonyl, the temporary protecting group used in the synthesis strategy that is standard today. It is no longer present on the finished peptide; the term describes the manufacturing process.