The peptide bond: how amino acids join

How peptide bond formation works

The reaction is a condensation: the hydroxyl of one amino acid's carboxyl group and a hydrogen from the next one's amino group leave as water, and a carbon-nitrogen bond remains. In water the reaction is thermodynamically unfavourable, and a carboxylic acid mixed with an amine gives a salt by proton transfer, not an amide.

Peptide formation in the laboratory therefore starts by activating the carboxyl group with a coupling reagent; the formal water ends up in the reagent's by-product, a urea in the case of a carbodiimide. Groups that must not react are blocked with protecting groups, as described in How synthetic peptides are made. In cells, the ribosome forms the same bond.

Why the peptide bond is flat

Resonance shares the nitrogen's lone pair with the carbonyl group, so the C-N bond has partial double-bond character. At roughly 1.33 angstroms it is shorter than a typical C-N single bond (roughly 1.47), and it resists rotation. The six atoms of the amide unit (both alpha carbons, the carbonyl carbon and oxygen, and the nitrogen with its hydrogen) lie in one plane.

In that plane the two alpha carbons sit on opposite sides of the bond (trans) or on the same side (cis). Trans is strongly preferred, because cis brings the two alpha carbons, and the groups they carry, into contact. The partial exception is the bond to a proline nitrogen, which carries a ring carbon instead of a hydrogen: cis is far more common there, though trans still predominates, because the two forms are closer in energy. Three torsion angles per residue describe the chain, and only the last two vary much:

• omega (ω): the peptide bond itself, near 180° (trans) or 0° (cis);
• phi (φ): rotation about the N-Cα bond;
• psi (ψ): rotation about the Cα-C bond.

The peptide backbone and its direction

Joined end to end, residues form a peptide chain. The backbone repeats the unit N-Cα-C, with one side chain on each alpha carbon.

The chain also has a direction. One end keeps a free amino group (the N-terminus), the other a free carboxyl group (the C-terminus), and sequences are written from N-terminus to C-terminus. Each peptide linkage runs one way, from the carbonyl of one residue to the nitrogen of the next, so Gly-Ala and Ala-Gly are different molecules with the same formula.

Counting bonds and adding up mass

A linear peptide of n residues has n − 1 peptide bonds (a head-to-tail cyclic peptide has n). Each residue is an amino acid minus one water, so a peptide's average mass is the sum of its residue masses plus one water (18.015 Da) for the two free ends.

The formula and molecular weight on Solara product pages are PubChem reference values for the compound, not measurements of any Solara lot, and Solara does not publish mass spectra.

How the peptide bond is broken

Hydrolysis, the reverse of condensation, adds a water across the bond. Resonance stabilisation makes it extremely slow at neutral pH and room temperature. Strong acid and heat speed it up; complete acid hydrolysis, classically 6 M hydrochloric acid at about 110 °C for about 24 h, is the first step of amino acid analysis, in which the released amino acids are quantified. Acid hydrolysis destroys tryptophan and deamidates asparagine and glutamine. In dilute acid, bonds next to aspartic acid are more labile than most, Asp-Pro especially.