The short version
A peptide bond is the link between two amino acids in a chain. Chemically it is an amide: the carbonyl carbon of one amino acid bonded to the nitrogen of the next, formed with the loss of a molecule of water [1]. Every peptide and every protein is a string of these bonds, so their properties decide a great deal about how a peptide behaves: its shape, how long it survives in solution, how it is made, and what can go wrong in manufacture.
This guide covers that chemistry. For the wider picture of what peptides are and how they differ from proteins, start with what are peptides?. Everything we supply is for in-vitro laboratory research only.
Is a peptide bond an amide bond?
Yes. "Peptide bond" is the biochemists' name for an amide bond that happens to join two amino acids through the backbone, from the α-carboxyl of one to the α-amino group of the next. An amide link that joins a side chain instead, for example the ε-amino group of lysine to a carboxyl elsewhere, is usually called an isopeptide bond. The chemistry is the same; the name records where in the molecule the link sits.
| Peptide bond | Isopeptide bond | |
|---|---|---|
| Joins | α-carboxyl to α-amino (backbone) | A side-chain group to another residue |
| Chemical class | Amide | Amide |
| Example | Every link in BPC-157's 15-residue chain | Ubiquitin attached to a lysine side chain |
How the bond forms
Joining two amino acids is a condensation: the carboxyl group loses its –OH, the amino group loses a hydrogen, and the two leave as water. Left to themselves in water, amino acids do not do this; the equilibrium lies on the side of the free acids. Cells drive the reaction on the ribosome using activated amino acids, and chemists drive it by activating the carboxyl group first.
In solid-phase peptide synthesis, the method Merrifield introduced in 1963 and the way almost every research peptide is now made, the growing chain is anchored to a resin bead and built one residue at a time from the C-terminus [2]. Each cycle removes a protecting group from the chain's free end, couples the next protected and activated amino acid, and washes away the excess. After the last residue the peptide is cut from the resin, the side-chain protecting groups come off, and the crude product is purified by HPLC.
Each coupling is very efficient but not perfect. If a coupling falls short, a fraction of chains miss that residue and carry on growing without it. Those "deletion sequences" are one residue lighter and look almost identical to the target on a column, which is why they are the commonest impurities in synthetic peptides and why a certificate needs both HPLC purity and a mass from LC-MS. Our guide to HPLC and LC-MS shows how each shows up.
Why the bond is flat and rigid
The peptide bond is not a simple single bond. The nitrogen's lone pair is shared with the carbonyl group, so the C–N link has partial double-bond character. Pauling and Corey worked this out in the early 1950s, and it is the reason their models of the α-helix and β-sheet worked [3]. Two consequences follow:
- The six atoms around the bond lie in one plane. The carbonyl carbon and oxygen, the nitrogen and its hydrogen, and the two α-carbons on either side cannot twist out of that plane.
- The bond does not rotate freely. The backbone can only turn at the two single bonds on either side of each α-carbon, the angles called φ (phi) and ψ (psi). Ramachandran mapped which combinations of those two angles the atoms allow, and those maps are still how protein structures are checked [4].
The planar unit can sit in two arrangements, trans (the two α-carbons on opposite sides) and cis (on the same side). Trans is strongly favoured for almost every residue. The exception is a bond to proline, whose ring makes cis and trans much closer in energy, so a measurable share of X–Pro bonds sit in the cis form and switch between the two slowly.
How slowly it breaks
A peptide bond in neutral water at room temperature is one of the most stable bonds in biochemistry. Radzicka and Wolfenden measured the uncatalysed rate and estimated a half-life of the order of hundreds of years at 25 °C [5]. In the body, enzymes called proteases cut the same bond in milliseconds, which is why many natural peptides last only minutes in plasma and why so much peptide chemistry is about protecting particular bonds from particular enzymes.
In a vial, outside the body, the backbone bond is rarely what fails first. Peptides degrade mainly through their side chains and a few weak spots in the backbone:
| Weak point | What happens | What it looks like on LC-MS |
|---|---|---|
| Asp–Pro and Asp–Gly bonds | Cleavage or rearrangement, faster in acid | Fragments, or an isomer of the same mass |
| Asparagine (Asn), especially Asn–Gly | Deamidation to aspartate | +1 Da |
| Methionine | Oxidation | +16 Da |
| Cysteine | Disulfide scrambling or oxidation | Depends on the pairing |
| N-terminal glutamine | Cyclisation to pyroglutamate | −17 Da |
That is why some research peptides carry deliberate substitutions. CJC-1295 without DAC replaces the asparagine and methionine of native GHRH(1–29) for exactly these reasons; our comparison of sermorelin and tesamorelin sets out the native GHRH sequences for comparison.
What it means for storage
Because water drives every one of the reactions in the table, a lyophilised (freeze-dried) peptide kept dry and cold is far more stable than the same peptide in solution. The practical rules follow from the chemistry: keep vials sealed at 2 to 8 °C or −20 °C, let them reach room temperature before opening so moisture does not condense on the powder, and once reconstituted, keep the solution cold and use or freeze it in aliquots. Storage and stability covers this in detail.
What it means for the certificate
Each point above maps onto a line of a certificate of analysis. HPLC purity tells you how much of the material is the full-length chain rather than deletion sequences and degradation products. The LC-MS mass confirms the chain is the right length with the right residues, and the small mass shifts in the table above are how oxidation or deamidation show up. A certificate that gives a purity figure without a mass cannot rule out a peptide one residue short.
Every compound we supply has a monograph with its sequence and expected mass, and the peptide mass calculator works out the mass of any sequence you paste in.
References
- International Union of Pure and Applied Chemistry. "Peptides." Compendium of Chemical Terminology (the Gold Book). goldbook.iupac.org.
- Merrifield RB. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society. 1963;85(14):2149–2154. doi:10.1021/ja00897a025.
- Pauling L, Corey RB, Branson HR. The structure of proteins: two hydrogen-bonded helical configurations of the polypeptide chain. Proceedings of the National Academy of Sciences. 1951;37(4):205–211. doi:10.1073/pnas.37.4.205.
- Ramachandran GN, Ramakrishnan C, Sasisekharan V. Stereochemistry of polypeptide chain configurations. Journal of Molecular Biology. 1963;7:95–99. doi:10.1016/S0022-2836(63)80023-6.
- Radzicka A, Wolfenden R. Rates of uncatalyzed peptide bond hydrolysis in neutral solution and the transition state affinities of proteases. Journal of the American Chemical Society. 1996;118(26):6105–6109. doi:10.1021/ja954077c.
This monograph summarises the published literature for laboratory researchers. It is not medical advice, and nothing on this site is for human or veterinary use.