How Peptides are made

What does bodybuilding, anti-ageing cream and Bleomycin (a cancer drug) have in common? Peptides of course! Peptides are large molecules that are vital to life. If you were to take a protein and break it into smaller pieces, each piece would be called a peptide. Just like proteins, peptides are made of amino acids linked together in a chain-like structure. Whenever you ingest a protein, your body breaks it down to its individual amino acids. It then puts those amino acids back together in a different order to make whatever peptide or protein your body needs. Insulin, for instance, is a peptide that is 51 amino acids long. Your body synthesizes insulin from the amino acids it gets from the proteins you eat.

Peptides and small proteins can be synthesized in a lab as well. Peptide synthesis is a huge market in the pharmaceutical and skin care industry. They’re also used, somewhat shakily, as a steroid substitute by serious athletes and bodybuilders. we’re going to go over the basic steps of how to join amino acids together to make a peptide. The chemistry of peptide synthesis is complex and well beyond the scope of explaining. But the basic steps of making a peptide are not as difficult as you might think.


There are 20 amino acids that occur in nature. They all have the same core — an amine group (NH2) attached to a carboxylic acid (COOH) via a single carbon atom, called an alpha carbon.  The thing that gives each amino acid its uniqueness is the functional group called the R group or R side chain, that is also attached to the alpha carbon. When building peptides, the amine group of one amino acid gets attached to the carboxylic acid of the other. The R functional group determines the overall shape, structure and properties of the peptide, as it will form bonds and cause the peptide to fold in on itself. By convention, the NH2 side is drawn and written on the left and the COOH side is on the right. The NH2 side is called the N-Terminus and the COOH side is called the C-Terminus.

In order to make peptides, you start with amino acids obviously. By themselves, they have the consistency of white power and are usually stored in plastic containers. Amino acids used in peptide synthesis need to have some sort of protection on the amine group to prevent unwanted reactions. This is accomplished with a molecule the most common of which is called FMOC. This is important to note because before attaching two amino acids together, we need to remove the FMOC protecting group from the amine side of one, but leave it on the other. Leaving the FMOC on one is necessary to prevent the same amino acid from coupling with itself. For instance, if I wanted to couple A to N, how do I prevent A-A couplings from occurring? Keeping A protected with FMOC ensures we only get an A-N outcome.

The R functional groups need protection as well, but will not be covered in this basic introduction.


Solid Phase Peptide Synthesis (SPPS) was developed by a chemist named Bruce Merrifield in 1963. His technique has become the standard for making peptides in the pharmaceutical industry. It works by attaching each peptide chain to tiny polystyrene beads. This allows you to keep your peptide inside a vessel to do your chemistry. A filter in the vessel will retain the resin (with the peptide attached)  while allowing solutions to be drained out.

The basic process starts with a preloaded resin, meaning the first amino acid in your sequence is already attached to your resin. Then successive FMOC removal/coupling cycles are done to make the peptide chain. The overall process goes like this:

  1. Remove the FMOC protecting group from the amine side of the amino acid.
  2. Add the next amino acid in the chain and coupling activation reagents.
  3. Repeat step’s 1 and 2 until the sequence is complete.
  4. Cleave the peptide from the resin.

Remember that the amine side of each amino acid is protected with an FMOC group. And recall that coupling amino acids together is done by attaching the NH2 side of one amino acid to the COOH side of the other. So when you remove the FMOC group and add another amino acid, the COOH side of the new amino acid gets coupled to the NH2 side of the amino acid you just deprotected.

Let’s walk through the steps of making the peptide sequence NH2-HACK-COOH. Couplings are done from C-Terminus to N-Terminus, so we start with Lysine (K) and then a couple on Cystine (C,) then Alanine (A) and finally Histidine (H) We’ll want to start with the Lysine (K) preloaded onto our resin.

The first thing that has to be done is to remove the FMOC protecting group that’s on the first amino acid in our sequence. This is done with a deprotection solution. Generally, a 20% piperidine in DMF solution is used, but piperidine is becoming increasingly more difficult to get because of its use in the making of street drugs. Alternatively, many labs now use a 5% piperazine solution in NMP. DMF is usually the main solvent used in the peptide synthesis process because it’s much cheaper than NMP, but they both can be interchanged.

We’ll need a vessel with a filter so we can submerge the resin with the deprotection solution, and then drain the solution out while leaving our resin behind. There are numerous ways to do this, but I’d recommend something like this.

A measured amount of resin is added to the vessel, then the deprotection solution. You’ll need to do the math, as the amount of resin will dictate the number of coupling reagents needed and final product. Let it sit for a while, then drain. If you want to check your deprotection step, FMOC has a UV absorbance at 302 nanometers.

Once you’ve done the deprotection, you need to wash several times with DMF. Any piperazine left in the vessel will destroy the coupling attempt.

This process has taken us from FMOC-K-resin to NH2-K-resin.


Amino Acid 

Now it’s time to go from NH2-K-resin to FMOC-CK-resin. To do this, the COOH side of C has to be coupled to the newly deprotected NH2 side of K. The COOH has to be activated before this can happen. There are several ways to do this, and every chemist has their favourite For simplicity, I’d stick to the onium salt method. You’ll need two things — the hydrated form of HOBt and DIEA.

Solute these in DMF, as well as your amino acid, and add the proper amounts to the vessel. Again, you’ll need to do the math to know how much of each to add. Note that the ratios need to be accurate for the amount of resin, and use 3 to 5 fold excess to ensure a complete coupling.

The coupling will take a few hours. If you can use an inert gas to agitate, it will help.


That’s about it really. Once you have your FMOC-CK-resin, the next step is to remove the FMOC from C and couple in Alanine (A). The process repeats over and over until your sequence is completed. Once done, you will need to remove the peptide from the resin with a process called cleavage.

The cleavage process uses a powerful acid called TFA to separate the peptide from the resin. It will also take off any R functional protecting groups. Warning – Do not handle TFA without proper protection. You’ll also need scavengers to catch any R protecting groups that come off during the cleavage process.


Once the peptide is cleaved from the resin, it will be in the cleavage solution. You can precipitate the peptide with very cold ether and a centrifuge. Afterwards, you’ll need access to some type of mass spectrometer to analyze your crude peptide and make sure they’re no mistakes. There likely will be, and then starts the trial and error process. Many universities will analyze it for you for a small fee.


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