Peptide bond

A peptide bond is a specialised amide-type covalent bond that links two consecutive α-amino acids in peptides and proteins. It forms between the C¹ (carboxyl carbon) of one amino acid and the N² (amide nitrogen) of the next, creating the fundamental structural connection along polypeptide chains. To distinguish it from alternative amide linkages between amino acids, it may also be termed a eupeptide bond, in contrast to an isopeptide bond which forms between side-chain functional groups.
Within biological systems, peptide bonds serve as the backbone of proteins, providing both structural integrity and conformational constraints essential for three-dimensional folding and biological function.

Synthesis

Formation of a peptide bond occurs through a condensation (dehydration) reaction between two amino acids. In this process:

• The carboxyl group (–COOH) of one amino acid loses an –OH.
• The amino group (–NH₂) of the second amino acid loses an H.
• The released atoms combine to form water (H₂O).
• The remaining fragments join to create the amide linkage –CONH–.

The resulting two-residue molecule is known as a dipeptide. This reaction is energetically unfavourable, requiring an input of energy that, in living organisms, derives from ATP-dependent biochemical processes. Ribosomes catalyse peptide bond formation during protein synthesis via a mechanism that differs in detail from simple dehydration synthesis, whereas various specialised enzymes generate non-ribosomal peptides.
An illustrative example is the biosynthesis of glutathione, in which glutamate–cysteine ligase first forms an isopeptide bond, followed by glutathione synthetase creating the conventional peptide linkage.

Degradation

Peptide bonds can undergo hydrolysis, the reverse of condensation, through the addition of water. Hydrolysis releases approximately 8.16 kJ mol⁻¹ (about 2 kcal mol⁻¹) of Gibbs free energy. The uncatalysed reaction is extremely slow under ambient conditions, with estimated half-lives of 350–600 years per peptide bond at 25 °C.
In biological systems, hydrolysis is carried out efficiently by peptidases and proteases. Some evidence suggests that conformational strain during protein folding can promote non-enzymatic hydrolysis of vulnerable peptide bonds via ground-state destabilisation, although this is considered uncommon.

Spectral Properties

Peptide bonds absorb ultraviolet light in the 190–230 nm range. This susceptibility to short-wavelength UV radiation reflects transitions associated with the amide functional group and contributes to the photochemical vulnerability of proteins.

Cis–trans Isomerism

The peptide bond exhibits partial double-bond character due to resonance delocalisation of the nitrogen lone pair into the carbonyl group. This confers:

• Planarity of the amide unit.
• Restricted rotation around the carbon–nitrogen bond.
• Two possible conformations: cis (ω = 0°) and trans (ω = 180°).

In unfolded polypeptides, peptide bonds may isomerise between these states. In folded proteins, however, only one isomer is typically favoured. The trans isomer predominates overwhelmingly (approximately 1000:1), due to reduced steric clashes. The exception involves X–Pro (proline) peptide bonds, where the cis:trans ratio is closer to 1:3, reflecting the comparable steric environment of proline’s cyclic structure.
Isomerisation is slow, with timescales around 20 seconds at room temperature, and requires overcoming a high activation energy (~80 kJ mol⁻¹) due to the need to disrupt the partial double bond. Enzymes known as peptidyl-prolyl isomerases (PPIases) catalyse the interconversion by stabilising conformations that favour single-bond character or by hydrogen-bonding to the nitrogen of the X–Pro bond. Because protein folding typically occurs much faster (10–100 ms), non-native cis isomers can inhibit folding until corrected.

Chemical Reactivity

Owing to resonance stabilisation, peptide bonds are relatively unreactive under physiological conditions, even less so than esters. Nevertheless, they can undergo nucleophilic attack on the carbonyl carbon, forming a tetrahedral intermediate characteristic of amide chemistry. This mechanism underlies:

• Proteolysis, mediated by proteases with catalytic residues that activate attacking groups.
• N→O (and related) acyl-exchange reactions, seen in intein chemistry.
• Formation of cyclol derivatives, where the attacking group is a thiol (thiacyclol), hydroxyl (oxacyclol), or amine (azacyclol).

These reactions illustrate that despite their stability, peptide bonds remain chemically versatile under the right catalytic conditions.

Biological and Structural Roles

Peptide bonds are fundamental to the architecture of all proteins and many biologically active peptides. Their planarity and partial double-bond character restrict conformational freedom, giving rise to defined angles (φ and ψ) that shape protein secondary structures such as α-helices and β-sheets. The uniformity and stability of peptide linkages also enable proteins to maintain precise three-dimensional structures essential for catalytic activity, signalling, transport, and structural support.